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1
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Arragain B, Pelosse M, Huard K, Cusack S. Structure of the tilapia lake virus nucleoprotein bound to RNA. Nucleic Acids Res 2025; 53:gkaf112. [PMID: 39995042 PMCID: PMC11850232 DOI: 10.1093/nar/gkaf112] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2024] [Revised: 01/27/2025] [Accepted: 02/06/2025] [Indexed: 02/26/2025] Open
Abstract
Tilapia Lake virus (TiLV) belongs to the Amnoonviridae family within the Articulavirales order of segmented negative-strand RNA viruses and is highly diverged from more familiar orthomyxoviruses, such as influenza. The viral nucleoprotein (NP), a key component of the replication machinery, packages the viral genome into protective ribonucleoprotein particles. Here we describe the electron cryo-microscopy (cryo-EM) structure of TiLV-NP bound to RNA within in vitro reconstituted, small ring-like, pseudo-symmetrical oligomers. Although TiLV-NP is considerably smaller than its influenza counterpart and unrelated in sequence, it maintains the same topology and domain organisation. This comprises a head and body domain between which is a positively charged groove, where single-stranded RNA binds. In addition, an oligomerisation loop inserts into a hydrophobic pocket in the neighbouring NP, the flexible hinges of which allow variable orientation of adjacent NPs. Focused cryo-EM maps unambiguously define the 5' to 3' direction of the bound RNA, confirmed by double stranded, A-form RNA regions that extrude out from some of the NP-NP interfaces. This is the first fully resolved description of how single-stranded and stem-loop RNA binds to an articulaviral NP assembly. Superposition with orthomyxoviral NPs suggest that the mode of RNA binding is likely similar across the Articulavirales order.
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Affiliation(s)
- Benoît Arragain
- European Molecular Biology Laboratory, EMBL Grenoble, 71 Avenue des Martyrs, CS 90181, 38042, Grenoble Cedex 9, France
| | - Martin Pelosse
- European Molecular Biology Laboratory, EMBL Grenoble, 71 Avenue des Martyrs, CS 90181, 38042, Grenoble Cedex 9, France
| | - Karine Huard
- European Molecular Biology Laboratory, EMBL Grenoble, 71 Avenue des Martyrs, CS 90181, 38042, Grenoble Cedex 9, France
| | - Stephen Cusack
- European Molecular Biology Laboratory, EMBL Grenoble, 71 Avenue des Martyrs, CS 90181, 38042, Grenoble Cedex 9, France
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2
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Chenavier F, Zarkadas E, Freslon LL, Stelfox A, Schoehn G, Ruigrok RH, Ballandras-Colas A, Crépin T. Influenza a virus antiparallel helical nucleocapsid-like pseudo-atomic structure. Nucleic Acids Res 2025; 53:gkae1211. [PMID: 39673795 PMCID: PMC11797009 DOI: 10.1093/nar/gkae1211] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/21/2024] [Revised: 10/29/2024] [Accepted: 11/21/2024] [Indexed: 12/16/2024] Open
Abstract
Influenza A viruses are responsible for human seasonal epidemics and severe animal pandemics with a risk of zoonotic transmission to humans. The viral segmented RNA genome is encapsidated by nucleoproteins (NP) and attached to the heterotrimeric polymerase, forming the viral ribonucleoproteins (vRNPs). Flexible helical vRNPs are central for viral transcription and replication. In this study, we present an advanced biological tool, the antiparallel helical RNP-like complex, assembled from recombinant N-terminally truncated NP and short synthetic RNA. The 3.0 Å cryo-electron microscopy structure details for the first time the whole RNA pathway across NP as well as NP-NP interactions that drive the antiparallel helical assembly accommodating major and minor grooves. Our findings show that the surface of the protein can harbour several conformations of the RNA, confirming that the number of nucleobases that binds to NP is not fixed, but ranges probably between 20 and 24. Taking all together, our data provide details to further understand the genome encapsidation and explain the inherent flexibility of influenza A virus vRNPs.
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Affiliation(s)
- Florian Chenavier
- Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
| | - Eleftherios Zarkadas
- Univ. Grenoble Alpes, CNRS, CEA, EMBL, ISBG, 71 avenue des Martyrs, F-38000 Grenoble, France
| | - Lily-Lorette Freslon
- Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
| | - Alice J Stelfox
- Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
| | - Guy Schoehn
- Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
| | - Rob W H Ruigrok
- Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
| | | | - Thibaut Crépin
- Univ. Grenoble Alpes, CNRS, CEA, IBS, 71 avenue des Martyrs, F-38000 Grenoble, France
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3
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Cao L, She Z, Zhao Y, Cheng C, Li Y, Xu T, Mao H, Zhang Y, Hui X, Lin X, Wang T, Sun X, Huang K, Zhao L, Jin M. Inhibition of RAN attenuates influenza a virus replication and nucleoprotein nuclear export. Emerg Microbes Infect 2024; 13:2387910. [PMID: 39087696 PMCID: PMC11321118 DOI: 10.1080/22221751.2024.2387910] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2024] [Revised: 07/21/2024] [Accepted: 07/30/2024] [Indexed: 08/02/2024]
Abstract
Nuclear export of the viral ribonucleoprotein (vRNP) is a critical step in the influenza A virus (IAV) life cycle and may be an effective target for the development of anti-IAV drugs. The host factor ras-related nuclear protein (RAN) is known to participate in the life cycle of several viruses, but its role in influenza virus replication remains unknown. In the present study, we aimed to determine the function of RAN in influenza virus replication using different cell lines and subtype strains. We found that RAN is essential for the nuclear export of vRNP, as it enhances the binding affinity of XPO1 toward the viral nuclear export protein NS2. Depletion of RAN constrained the vRNP complex in the nucleus and attenuated the replication of various subtypes of influenza virus. Using in silico compound screening, we identified that bepotastine could dissociate the RAN-XPO1-vRNP trimeric complex and exhibit potent antiviral activity against influenza virus both in vitro and in vivo. This study demonstrates the important role of RAN in IAV replication and suggests its potential use as an antiviral target.
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Affiliation(s)
- Lei Cao
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Ziwei She
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Ya Zhao
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Chuxing Cheng
- Wuhan Keqian Biological Co. Ltd., Wuhan, People’s Republic of China
| | - Yaqin Li
- Wuhan Keqian Biological Co. Ltd., Wuhan, People’s Republic of China
| | - Ting Xu
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Haiying Mao
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Yumei Zhang
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Xianfeng Hui
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Xian Lin
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Ting Wang
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Xiaomei Sun
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Kun Huang
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
| | - Lianzhong Zhao
- College of Life Sciences and Oceanography, Shenzhen University, Shenzhen, People’s Republic of China
| | - Meilin Jin
- State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, People’s Republic of China
- College of Animal Medicine, Huazhong Agricultural University, Wuhan, People’s Republic of China
- The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, People’s Republic of China
- Hubei Jiangxia Laboratory, Wuhan, People’s Republic of China
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Zhang H, Li C, Sun R, Zhang X, Li Z, Hua D, Yin B, Yang L, Zhang L, Huang J. NEIL1 block IFN-β production and enhance vRNP function to facilitate influenza A virus proliferation. NPJ VIRUSES 2024; 2:57. [PMID: 40295715 PMCID: PMC11721407 DOI: 10.1038/s44298-024-00065-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/25/2024] [Accepted: 10/14/2024] [Indexed: 04/30/2025]
Abstract
Influenza A virus (IAV) has developed multiple tactics to hinder the innate immune response including the epigenetic regulation during IAV infection, but the novel epigenetic factors and their mechanism in innate immunity remain well studied. Here, through a non-biased high-throughput sgRNA screening of 1041 known epigenetic modifiers in a cellular model of IAV-induced interferon-beta (IFN-β) production, we identified nei endonuclease VIII-like 1 (NEIL1) as a critical regulator of IFN-β in response to viral infection. Further studies showed that NEIL1 promoted the replication of the influenza virus by regulating the methylation of cytonuclear IFN-β promoter (mainly CpG-345), inhibiting the expression of IFN-β and IFN-stimulating genes. The structural domains of NEIL1, especially the catalytic domain, were critical for the suppression of IFN-β production, but the enzymatic activity of NEIL1 was dispensable. Furthermore, our results revealed that NEIL1 relied on interactions with the N- and C-terminus of the nucleoprotein (NP) of IAV, and NEIL1 expression facilitated the entry of the NP into the nucleus, which further enhanced the stability of the viral ribonucleoprotein (vRNP) complex and thus contributed to IAV replication and transcription. These findings reveal an enzyme-independent mechanism of host NEIL1 that negatively regulates IFN-β expression, thereby facilitating IAV propagation. Our study provides new insights into the roles of NEIL1, both in directly promoting virus replication and in evading innate immunity in IAV infection.
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Affiliation(s)
- Huixia Zhang
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Changyan Li
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Ruiqi Sun
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Xinyi Zhang
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Zexing Li
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Deping Hua
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Boxuan Yin
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Liu Yang
- School of Life Sciences, Tianjin University, Tianjin, China
| | - Lilin Zhang
- School of Life Sciences, Tianjin University, Tianjin, China.
| | - Jinhai Huang
- School of Life Sciences, Tianjin University, Tianjin, China.
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5
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Shalamova L, Barth P, Pickin MJ, Kouti K, Ott B, Humpert K, Janssen S, Lorenzo G, Brun A, Goesmann A, Hain T, Hartmann RK, Rossbach O, Weber F. Nucleocapsids of the Rift Valley fever virus ambisense S segment contain an exposed RNA element in the center that overlaps with the intergenic region. Nat Commun 2024; 15:7602. [PMID: 39217162 PMCID: PMC11365940 DOI: 10.1038/s41467-024-52058-2] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/16/2023] [Accepted: 08/21/2024] [Indexed: 09/04/2024] Open
Abstract
Rift Valley fever virus (RVFV) is a mosquito-borne zoonotic pathogen. Its RNA genome consists of two negative-sense segments (L and M) with one gene each, and one ambisense segment (S) with two opposing genes separated by the noncoding "intergenic region" (IGR). These vRNAs and the complementary cRNAs are encapsidated by nucleoprotein (N). Using iCLIP2 (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to map all N-vRNA and N-cRNA interactions, we detect N coverage along the L and M segments. However, the S segment vRNA and cRNA each contain approximately 100 non-encapsidated nucleotides stretching from the IGR into the 5'-adjacent reading frame. These exposed regions are RNase-sensitive and predicted to form stem-loop structures with the mRNA transcription termination motif positioned near the top. Moreover, optimal S segment transcription and replication requires the entire exposed region rather than only the IGR. Thus, the RVFV S segment contains a central, non-encapsidated RNA region with a functional role.
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Affiliation(s)
- Lyudmila Shalamova
- Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, Giessen, Germany
| | - Patrick Barth
- Bioinformatics & Systems Biology, Justus-Liebig University, Giessen, Germany
- Cell Biology and Plant Biochemistry, University of Regensburg, Regensburg, Germany
| | - Matthew J Pickin
- Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, Giessen, Germany
| | - Kiriaki Kouti
- Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, Giessen, Germany
| | - Benjamin Ott
- Institute for Medical Microbiology, FB11-Medicine, Justus-Liebig University, Giessen, Germany
| | - Katharina Humpert
- Institute for Medical Microbiology, FB11-Medicine, Justus-Liebig University, Giessen, Germany
- Institute of Molecular Oncology, Genomics Core Facility, Philipps-University, Marburg, Germany
| | - Stefan Janssen
- Algorithmic Bioinformatics, Justus-Liebig University, Giessen, Germany
| | - Gema Lorenzo
- Centro de Investigación en Sanidad Animal (CISA-INIA/CSIC), Valdeolmos, Madrid, Spain
| | - Alejandro Brun
- Centro de Investigación en Sanidad Animal (CISA-INIA/CSIC), Valdeolmos, Madrid, Spain
| | - Alexander Goesmann
- Bioinformatics & Systems Biology, Justus-Liebig University, Giessen, Germany
| | - Torsten Hain
- Institute for Medical Microbiology, FB11-Medicine, Justus-Liebig University, Giessen, Germany
| | - Roland K Hartmann
- Institute of Pharmaceutical Chemistry, Philipps-University Marburg, Marburg, Germany
| | - Oliver Rossbach
- Institute for Biochemistry, FB 08-Biology and Chemistry, Justus-Liebig University, Giessen, Germany
| | - Friedemann Weber
- Institute for Virology, FB10-Veterinary Medicine, Justus-Liebig University, Giessen, Germany.
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6
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He Z, Li W, Zhang M, Huang M, Chen Z, Zhao X, Ding Y, Zhang J, Zhao L, Jiao P. RNF216 Inhibits the Replication of H5N1 Avian Influenza Virus and Regulates the RIG-I Signaling Pathway in Ducks. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2024; 213:187-203. [PMID: 38829131 DOI: 10.4049/jimmunol.2300540] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/09/2023] [Accepted: 04/20/2024] [Indexed: 06/05/2024]
Abstract
The RING finger (RNF) family, a group of E3 ubiquitin ligases, plays multiple essential roles in the regulation of innate immunity and resistance to viral infection in mammals. However, it is still unclear whether RNF proteins affect the production of IFN-I and the replication of avian influenza virus (AIV) in ducks. In this article, we found that duck RNF216 (duRNF216) inhibited the duRIG-I signaling pathway. Conversely, duRNF216 deficiency enhanced innate immune responses in duck embryonic fibroblasts. duRNF216 did not interacted with duRIG-I, duMDA5, duMAVS, duSTING, duTBK1, or duIRF7 in the duck RIG-I pathway. However, duRNF216 targeted duTRAF3 and inhibited duMAVS in the recruitment of duTRAF3 in a dose-dependent manner. duRNF216 catalyzed K48-linked polyubiquitination of duck TRAF3, which was degraded by the proteasome pathway. Additionally, AIV PB1 protein competed with duTRAF3 for binding to duRNF216 to reduce degradation of TRAF3 by proteasomes in the cytoplasm, thereby slightly weakening duRNF216-mediated downregulation of IFN-I. Moreover, although duRNF216 downregulated the IFN-β expression during virus infection, the expression level of IFN-β in AIV-infected duck embryonic fibroblasts overexpressing duRNF216 was still higher than that in uninfected cells, which would hinder the viral replication. During AIV infection, duRNF216 protein targeted the core protein PB1 of viral polymerase to hinder viral polymerase activity and viral RNA synthesis in the nucleus, ultimately strongly restricting viral replication. Thus, our study reveals a new mechanism by which duRNF216 downregulates innate immunity and inhibits AIV replication in ducks. These findings broaden our understanding of the mechanisms by which the duRNF216 protein affects AIV replication in ducks.
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Affiliation(s)
- Zhuoliang He
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, Guangzhou, China
| | - Weiqiang Li
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Meng Zhang
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Minfan Huang
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Zuxian Chen
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Xiya Zhao
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Yangbao Ding
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Junsheng Zhang
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Luxiang Zhao
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
| | - Peirong Jiao
- College of Veterinary Medicine, Guangdong Laboratory for Lingnan Modern Agriculture, South China Agricultural University, Guangzhou, China
- Guangdong Provincial Key Laboratory of Zoonosis Prevention and Control, Guangzhou, China
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7
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Quignon E, Ferhadian D, Hache A, Vivet-Boudou V, Isel C, Printz-Schweigert A, Donchet A, Crépin T, Marquet R. Structural Impact of the Interaction of the Influenza A Virus Nucleoprotein with Genomic RNA Segments. Viruses 2024; 16:421. [PMID: 38543786 PMCID: PMC10974462 DOI: 10.3390/v16030421] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2024] [Revised: 03/01/2024] [Accepted: 03/06/2024] [Indexed: 05/23/2024] Open
Abstract
Influenza A viruses (IAVs) possess a segmented genome consisting of eight viral RNAs (vRNAs) associated with multiple copies of viral nucleoprotein (NP) and a viral polymerase complex. Despite the crucial role of RNA structure in IAV replication, the impact of NP binding on vRNA structure is not well understood. In this study, we employed SHAPE chemical probing to compare the structure of NS and M vRNAs of WSN IAV in various states: before the addition of NP, in complex with NP, and after the removal of NP. Comparison of the RNA structures before the addition of NP and after its removal reveals that NP, while introducing limited changes, remodels local structures in both vRNAs and long-range interactions in the NS vRNA, suggesting a potentially biologically relevant RNA chaperone activity. In contrast, NP significantly alters the structure of vRNAs in vRNA/NP complexes, though incorporating experimental data into RNA secondary structure prediction proved challenging. Finally, our results suggest that NP not only binds single-stranded RNA but also helices with interruptions, such as bulges or small internal loops, with a preference for G-poor and C/U-rich regions.
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Affiliation(s)
- Erwan Quignon
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
| | - Damien Ferhadian
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
| | - Antoine Hache
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
| | - Valérie Vivet-Boudou
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
| | - Catherine Isel
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
| | - Anne Printz-Schweigert
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
| | - Amélie Donchet
- Université Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38000 Grenoble, France (T.C.)
| | - Thibaut Crépin
- Université Grenoble Alpes, CNRS, CEA, Institut de Biologie Structurale, 38000 Grenoble, France (T.C.)
| | - Roland Marquet
- Université de Strasbourg, CNRS, Architecture et Réactivité de l’ARN, UPR9002, 67000 Strasbourg, France; (E.Q.); (A.H.); (V.V.-B.); (C.I.)
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8
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Sabsay KR, te Velthuis AJW. Negative and ambisense RNA virus ribonucleocapsids: more than protective armor. Microbiol Mol Biol Rev 2023; 87:e0008223. [PMID: 37750733 PMCID: PMC10732063 DOI: 10.1128/mmbr.00082-23] [Citation(s) in RCA: 4] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/27/2023] Open
Abstract
SUMMARYNegative and ambisense RNA viruses are the causative agents of important human diseases such as influenza, measles, Lassa fever, and Ebola hemorrhagic fever. The viral genome of these RNA viruses consists of one or more single-stranded RNA molecules that are encapsidated by viral nucleocapsid proteins to form a ribonucleoprotein complex (RNP). This RNP acts as protection, as a scaffold for RNA folding, and as the context for viral replication and transcription by a viral RNA polymerase. However, the roles of the viral nucleoproteins extend beyond these functions during the viral infection cycle. Recent advances in structural biology techniques and analysis methods have provided new insights into the formation, function, dynamics, and evolution of negative sense virus nucleocapsid proteins, as well as the role that they play in host innate immune responses against viral infection. In this review, we discuss the various roles of nucleocapsid proteins, both in the context of RNPs and in RNA-free states, as well as the open questions that remain.
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Affiliation(s)
- Kimberly R. Sabsay
- Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA
- Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, USA
| | - Aartjan J. W. te Velthuis
- Lewis Thomas Laboratory, Department of Molecular Biology, Princeton University, Princeton, New Jersey, USA
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9
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Chenavier F, Estrozi LF, Teulon JM, Zarkadas E, Freslon LL, Pellequer JL, Ruigrok RW, Schoehn G, Ballandras-Colas A, Crépin T. Cryo-EM structure of influenza helical nucleocapsid reveals NP-NP and NP-RNA interactions as a model for the genome encapsidation. SCIENCE ADVANCES 2023; 9:eadj9974. [PMID: 38100595 PMCID: PMC10848707 DOI: 10.1126/sciadv.adj9974] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Accepted: 11/16/2023] [Indexed: 12/17/2023]
Abstract
Influenza virus genome encapsidation is essential for the formation of a helical viral ribonucleoprotein (vRNP) complex composed of nucleoproteins (NP), the trimeric polymerase, and the viral genome. Although low-resolution vRNP structures are available, it remains unclear how the viral RNA is encapsidated and how NPs assemble into the helical filament specific of influenza vRNPs. In this study, we established a biological tool, the RNP-like particles assembled from recombinant influenza A virus NP and synthetic RNA, and we present the first subnanometric cryo-electron microscopy structure of the helical NP-RNA complex (8.7 to 5.3 Å). The helical RNP-like structure reveals a parallel double-stranded conformation, allowing the visualization of NP-NP and NP-RNA interactions. The RNA, located at the interface of neighboring NP protomers, interacts with conserved residues previously described as essential for the NP-RNA interaction. The NP undergoes conformational changes to enable RNA binding and helix formation. Together, our findings provide relevant insights for understanding the mechanism for influenza genome encapsidation.
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Affiliation(s)
| | | | | | | | | | | | | | - Guy Schoehn
- Univ. Grenoble Alpes, CNRS, CEA, IBS, F-38000, Grenoble, France
| | | | - Thibaut Crépin
- Univ. Grenoble Alpes, CNRS, CEA, IBS, F-38000, Grenoble, France
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10
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Rashid F, Xie Z, Li M, Xie Z, Luo S, Xie L. Roles and functions of IAV proteins in host immune evasion. Front Immunol 2023; 14:1323560. [PMID: 38152399 PMCID: PMC10751371 DOI: 10.3389/fimmu.2023.1323560] [Citation(s) in RCA: 10] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2023] [Accepted: 11/30/2023] [Indexed: 12/29/2023] Open
Abstract
Influenza A viruses (IAVs) evade the immune system of the host by several regulatory mechanisms. Their genomes consist of eight single-stranded segments, including nonstructural proteins (NS), basic polymerase 1 (PB1), basic polymerase 2 (PB2), hemagglutinin (HA), acidic polymerase (PA), matrix (M), neuraminidase (NA), and nucleoprotein (NP). Some of these proteins are known to suppress host immune responses. In this review, we discuss the roles, functions and underlying strategies adopted by IAV proteins to escape the host immune system by targeting different proteins in the interferon (IFN) signaling pathway, such as tripartite motif containing 25 (TRIM25), inhibitor of nuclear factor κB kinase (IKK), mitochondrial antiviral signaling protein (MAVS), Janus kinase 1 (JAK1), type I interferon receptor (IFNAR1), interferon regulatory factor 3 (IRF3), IRF7, and nuclear factor-κB (NF-κB). To date, the IAV proteins NS1, NS2, PB1, PB1-F2, PB2, HA, and PA have been well studied in terms of their roles in evading the host immune system. However, the detailed mechanisms of NS3, PB1-N40, PA-N155, PA-N182, PA-X, M42, NA, and NP have not been well studied with respect to their roles in immune evasion. Moreover, we also highlight the future perspectives of research on IAV proteins.
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Affiliation(s)
- Farooq Rashid
- Department of Biotechnology, Guangxi Veterinary Research Institute, Nanning, China
- Guangxi Key Laboratory of Veterinary Biotechnology, Nanning, China
- Key Laboratory of China (Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China
| | - Zhixun Xie
- Department of Biotechnology, Guangxi Veterinary Research Institute, Nanning, China
- Guangxi Key Laboratory of Veterinary Biotechnology, Nanning, China
- Key Laboratory of China (Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China
| | - Meng Li
- Department of Biotechnology, Guangxi Veterinary Research Institute, Nanning, China
- Guangxi Key Laboratory of Veterinary Biotechnology, Nanning, China
- Key Laboratory of China (Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China
| | - Zhiqin Xie
- Department of Biotechnology, Guangxi Veterinary Research Institute, Nanning, China
- Guangxi Key Laboratory of Veterinary Biotechnology, Nanning, China
- Key Laboratory of China (Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China
| | - Sisi Luo
- Department of Biotechnology, Guangxi Veterinary Research Institute, Nanning, China
- Guangxi Key Laboratory of Veterinary Biotechnology, Nanning, China
- Key Laboratory of China (Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China
| | - Liji Xie
- Department of Biotechnology, Guangxi Veterinary Research Institute, Nanning, China
- Guangxi Key Laboratory of Veterinary Biotechnology, Nanning, China
- Key Laboratory of China (Guangxi)-ASEAN Cross-border Animal Disease Prevention and Control, Ministry of Agriculture and Rural Affairs of China, Nanning, China
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Fu Y, Wang T, Zhou S, Zhou J, Zhao Y, Chen D, Zheng L. A novel narnavirus isolated from Colletotrichum curcumae strain 780-2T. Arch Virol 2023; 168:226. [PMID: 37561160 DOI: 10.1007/s00705-023-05847-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Accepted: 07/09/2023] [Indexed: 08/11/2023]
Abstract
The complete genome of a novel mycovirus, Colletotrichum curcumae narnavirus 1 (CcNV1), derived from the phytopathogenic fungus Colletotrichum curcumae strain 780-2T, was sequenced and analyzed. The full sequence of CcNV1 is 3,374 nucleotides in length and contains a single large open reading frame (ORF) encoding an RNA-dependent RNA polymerase (RdRp) of 1,087 amino acids with a molecular mass of 124.2 kDa that shares the closest similarity with that of Monilinia narnavirus H (53.02% identity). RdRp phylogeny analysis showed that CcNV1 is a new member of the proposed genus "Betanarnavirus" within the family Narnaviridae. This is the first report of a novel narnavirus infecting the phytopathogenic fungus C. curcumae, the causal agent of leaf blight of Curcuma wenyujin.
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Affiliation(s)
- Yujia Fu
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China
| | - Tian Wang
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China
| | - Siyu Zhou
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China
| | - Jingyi Zhou
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China
| | - Yang Zhao
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China
| | - Daipeng Chen
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China
| | - Li Zheng
- Hainan Yazhou Bay Seed Laboratory, Sanya Nanfan Research Institute of Hainan University, 572025, Sanya, China.
- Key Laboratory of Green Prevention and Control of Tropical Plant Diseases and Pests, School of Plant Protection, Ministry of Education, Hainan University, 570228, Haikou, Hainan, China.
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12
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Liu K, Guo Y, Zheng H, Ji Z, Cai M, Gao R, Zhang P, Liu X, Xu X, Wang X, Liu X. Enhanced pathogenicity and transmissibility of H9N2 avian influenza virus in mammals by hemagglutinin mutations combined with PB2-627K. Virol Sin 2023; 38:47-55. [PMID: 36103978 PMCID: PMC10006187 DOI: 10.1016/j.virs.2022.09.006] [Citation(s) in RCA: 11] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Accepted: 09/07/2022] [Indexed: 10/14/2022] Open
Abstract
H9N2 avian influenza viruses (AIVs) circulate globally in poultry and have become the dominant AIV subtype in China in recent years. Previously, we demonstrated that the H9N2 virus (A/chicken/Eastern China/SDKD1/2015) naturally harbors a mammalian-adaptive molecular factor (627K) in the PB2 protein and is weakly pathogenic in mice. Here, we focused on new markers for virulence in mammals. A mouse-adapted H9N2 virus was serially passaged in mice by infecting their lungs. As expected, infected mice showed clinical symptoms and died at passage six. A comparison between the wild-type and mouse-adapted virus sequences identified amino acid substitutions in the hemagglutinin (HA) protein. H9N2 viruses with the T187P + M227L double mutation exhibited an increased affinity to human-type (SAα2,6Gal) receptors and significantly enhanced viral attachment to mouse lung tissues, which contributed to enhancing viral replication and virulence in mice. Additionally, HA with the T187P + M227L mutation enabled H9N2 viral transmission in guinea pigs via direct contact. AIV pathogenicity in mice is a polygenic trait. Our results demonstrated that these HA mutations might be combined with PB2-627K to significantly increase H9N2 virulence in mice, and this enhanced virulence was achieved in other H9N2 AIVs by generating the same combination of mutations. In summary, our study identified novel key elements in the HA protein that are required for H9N2 pathogenicity in mice and provided valuable insights into pandemic preparedness against emerging H9N2 strains.
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Affiliation(s)
- Kaituo Liu
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China; Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009, China
| | - Yaqian Guo
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China
| | - Huafen Zheng
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China
| | - Zhuxing Ji
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China
| | - Miao Cai
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China
| | - Ruyi Gao
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009, China
| | - Pinghu Zhang
- Institute of Translational Medicine, Key Laboratory of Geriatric Disease Prevention and Control of Jiangsu Province, Medical College, Yangzhou University, Yangzhou, 225009, China
| | - Xiaowen Liu
- Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009, China
| | - Xiulong Xu
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China; Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009, China
| | - Xiaoquan Wang
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China; Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009, China.
| | - Xiufan Liu
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, 225009, China; Animal Infectious Disease Laboratory, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, China; Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonosis, Yangzhou University, Yangzhou, 225009, China; Jiangsu Key Laboratory of Zoonosis, Yangzhou, 225009, China.
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13
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Wang X, Jiang L, Wang G, Shi W, Hu Y, Wang B, Zeng X, Tian G, Deng G, Shi J, Liu L, Li C, Chen H. Influenza A virus use of BinCARD1 to facilitate the binding of viral NP to importin α7 is counteracted by TBK1-p62 axis-mediated autophagy. Cell Mol Immunol 2022; 19:1168-1184. [PMID: 36056146 PMCID: PMC9508095 DOI: 10.1038/s41423-022-00906-w] [Citation(s) in RCA: 27] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/30/2022] [Accepted: 07/11/2022] [Indexed: 11/09/2022] Open
Abstract
As a major component of the viral ribonucleoprotein (vRNP) complex in influenza A virus (IAV), nucleoprotein (NP) interacts with isoforms of importin α family members, leading to the import of itself and vRNP complex into the nucleus, a process pivotal in the replication cycle of IAV. In this study, we found that BinCARD1, an isoform of Bcl10-interacting protein with CARD (BinCARD), was leveraged by IAV for efficient viral replication. BinCARD1 promoted the nuclear import of the vRNP complex and newly synthesized NP and thus enhanced vRNP complex activity. Moreover, we found that BinCARD1 interacted with NP to promote NP binding to importin α7, an adaptor in the host nuclear import pathway. However, we also found that BinCARD1 promoted RIG-I-mediated innate immune signaling by mediating Lys63-linked polyubiquitination of TRAF3, and that TBK1 appeared to degrade BinCARD1. We showed that BinCARD1 was polyubiquitinated at residue K103 through a Lys63 linkage, which was recognized by the TBK1-p62 axis for autophagic degradation. Overall, our data demonstrate that IAV leverages BinCARD1 as an important host factor that promotes viral replication, and two mechanisms in the host defense system are triggered-innate immune signaling and autophagic degradation-to mitigate the promoting effect of BinCARD1 on the life cycle of IAV.
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Affiliation(s)
- Xuyuan Wang
- College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China
- Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Li Jiang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Guangwen Wang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Wenjun Shi
- College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Yuzhen Hu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Bo Wang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Xianying Zeng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Guobin Tian
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Guohua Deng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Jianzhong Shi
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Liling Liu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China
| | - Chengjun Li
- Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China.
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
| | - Hualan Chen
- College of Veterinary Medicine, Gansu Agricultural University, Lanzhou, 730070, China.
- Guangdong Laboratory for Lingnan Modern Agriculture, Guangzhou, 510642, China.
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, China.
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14
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Jakob C, Paul-Stansilaus R, Schwemmle M, Marquet R, Bolte H. The influenza A virus genome packaging network - complex, flexible and yet unsolved. Nucleic Acids Res 2022; 50:9023-9038. [PMID: 35993811 PMCID: PMC9458418 DOI: 10.1093/nar/gkac688] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 07/19/2022] [Accepted: 08/18/2022] [Indexed: 12/24/2022] Open
Abstract
The genome of influenza A virus (IAV) consists of eight unique viral RNA segments. This genome organization allows genetic reassortment between co-infecting IAV strains, whereby new IAVs with altered genome segment compositions emerge. While it is known that reassortment events can create pandemic IAVs, it remains impossible to anticipate reassortment outcomes with pandemic prospects. Recent research indicates that reassortment is promoted by a viral genome packaging mechanism that delivers the eight genome segments as a supramolecular complex into the virus particle. This finding holds promise of predicting pandemic IAVs by understanding the intermolecular interactions governing this genome packaging mechanism. Here, we critically review the prevailing mechanistic model postulating that IAV genome packaging is orchestrated by a network of intersegmental RNA-RNA interactions. Although we find supporting evidence, including segment-specific packaging signals and experimentally proposed RNA-RNA interaction networks, this mechanistic model remains debatable due to a current shortage of functionally validated intersegmental RNA-RNA interactions. We speculate that identifying such functional intersegmental RNA-RNA contacts might be hampered by limitations of the utilized probing techniques and the inherent complexity of the genome packaging mechanism. Nevertheless, we anticipate that improved probing strategies combined with a mutagenesis-based validation could facilitate their discovery.
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Affiliation(s)
| | | | - Martin Schwemmle
- To whom correspondence should be addressed. Tel: +49 761 203 6526; Fax: +49 761 203 6626;
| | - Roland Marquet
- Correspondence may also be addressed to Roland Marquet. Tel: +33 3 88 41 70 54; Fax: +33 3 88 60 22 18;
| | - Hardin Bolte
- Institute of Virology, Medical Center – University of Freiburg, 79104 Freiburg, Germany,Faculty of Medicine, University of Freiburg, 79110 Freiburg, Germany
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15
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Wang G, Zhao Y, Zhou Y, Jiang L, Liang L, Kong F, Yan Y, Wang X, Wang Y, Wen X, Zeng X, Tian G, Deng G, Shi J, Liu L, Chen H, Li C. PIAS1-mediated SUMOylation of influenza A virus PB2 restricts viral replication and virulence. PLoS Pathog 2022; 18:e1010446. [PMID: 35377920 PMCID: PMC9009768 DOI: 10.1371/journal.ppat.1010446] [Citation(s) in RCA: 29] [Impact Index Per Article: 9.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/25/2021] [Revised: 04/14/2022] [Accepted: 03/14/2022] [Indexed: 11/28/2022] Open
Abstract
Host defense systems employ posttranslational modifications to protect against invading pathogens. Here, we found that protein inhibitor of activated STAT 1 (PIAS1) interacts with the nucleoprotein (NP), polymerase basic protein 1 (PB1), and polymerase basic protein 2 (PB2) of influenza A virus (IAV). Lentiviral-mediated stable overexpression of PIAS1 dramatically suppressed the replication of IAV, whereas siRNA knockdown or CRISPR/Cas9 knockout of PIAS1 expression significantly increased virus growth. The expression of PIAS1 was significantly induced upon IAV infection in both cell culture and mice, and PIAS1 was involved in the overall increase in cellular SUMOylation induced by IAV infection. We found that PIAS1 inhibited the activity of the viral RNP complex, whereas the C351S or W372A mutant of PIAS1, which lacks the SUMO E3 ligase activity, lost the ability to suppress the activity of the viral RNP complex. Notably, the SUMO E3 ligase activity of PIAS1 catalyzed robust SUMOylation of PB2, but had no role in PB1 SUMOylation and a minimal role in NP SUMOylation. Moreover, PIAS1-mediated SUMOylation remarkably reduced the stability of IAV PB2. When tested in vivo, we found that the downregulation of Pias1 expression in mice enhanced the growth and virulence of IAV. Together, our findings define PIAS1 as a restriction factor for the replication and pathogenesis of IAV.
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Affiliation(s)
- Guangwen Wang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Yuhui Zhao
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Yuan Zhou
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Li Jiang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Libin Liang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Fandi Kong
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Ya Yan
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Xuyuan Wang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Yihan Wang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Xia Wen
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Xianying Zeng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Guobin Tian
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Guohua Deng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Jianzhong Shi
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Liling Liu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Hualan Chen
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
| | - Chengjun Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, The People’s Republic of China
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16
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p21 restricts influenza A virus by perturbing the viral polymerase complex and upregulating type I interferon signaling. PLoS Pathog 2022; 18:e1010295. [PMID: 35180274 PMCID: PMC8920271 DOI: 10.1371/journal.ppat.1010295] [Citation(s) in RCA: 14] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 03/14/2022] [Accepted: 01/20/2022] [Indexed: 11/19/2022] Open
Abstract
Many cellular genes and networks induced in human lung epithelial cells infected with the influenza virus remain uncharacterized. Here, we find that p21 levels are elevated in response to influenza A virus (IAV) infection, which is independent of p53. Silencing, pharmacological inhibition or deletion of p21 promotes virus replication in vitro and in vivo, indicating that p21 is an influenza restriction factor. Mechanistically, p21 binds to the C-terminus of IAV polymerase subunit PA and competes with PB1 to limit IAV polymerase activity. Besides, p21 promotes IRF3 activation by blocking K48-linked ubiquitination degradation of HO-1 to enhance type I interferons expression. Furthermore, a synthetic p21 peptide (amino acids 36 to 43) significantly inhibits IAV replication in vitro and in vivo. Collectively, our findings reveal that p21 restricts IAV by perturbing the viral polymerase complex and activating the host innate immune response, which may aid the design of desperately needed new antiviral therapeutics. Influenza A virus (IAV) poses a continuous threat to public health and economic stability. The ribonucleoprotein (RNP) of IAV is responsible for the transcription and replication of the viral RNA. These processes require interplay between host factors and RNP components. Here, we report that p21 can be activated by IAV infection and is controlled by a p53-independent pathway. We demonstrate that p21 directly binds to the viral polymerase acidic protein and limits IAV polymerase activity through disrupting the formation of the ribonucleoprotein complex. Additionally, p21 activation promotes IRF3 activation by blocking K48-linked polyubiquitination degradation of HO-1, thereby activating the type I interferon pathway. We further identify an 8-amino-acid peptide of p21 as the minimum motif that effectively inhibits IAV replication and presents therapeutic efficacy both in vitro and in vivo. Thus, our studies not only identify p21 as an antiviral protein, but also provide mechanistic insight to facilitate drug development.
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17
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Waters K, Wan HJ, Han L, Xue J, Ykema M, Tao YJ, Wan XF. Variations outside the conserved motifs of PB1 catalytic active site may affect replication efficiency of the RNP complex of influenza A virus. Virology 2021; 559:145-155. [PMID: 33887645 PMCID: PMC8579824 DOI: 10.1016/j.virol.2021.04.001] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/30/2021] [Revised: 03/31/2021] [Accepted: 04/05/2021] [Indexed: 12/24/2022]
Abstract
PB1 functions as the catalytic subunit of influenza virus RNA polymerase complex and plays an essential role in viral RNA transcription and replication. To determine plasticity in the PB1 enzymatic site and map catalytically important residues, 658 mutants were constructed, each with one to seven mutations in the enzymatic site of PB1. The polymerase activities of these mutants were quantified using a minigenome assay, and polymerase activity-associated residues were identified using sparse learning. Results showed that polymerase activities are affected by the residues not only within the conserved motifs, but also across the inter-motif regions of PB1, and the latter are primarily located at the base of the palm domain, a region that is conserved in avian PB1 but with high sequence diversity in swine PB1. Our results suggest that mutations outside the PB1 conserved motifs may affect RNA replication and could be associated with influenza virus host adaptation.
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Affiliation(s)
- Kaitlyn Waters
- Center for Influenza and Emerging Infectious Diseases, University of Missouri, Columbia, MO, USA; Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, 65212, USA; Bond Life Sciences Center, University of Missouri, Columbia, MO, USA; Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762, USA
| | - Hamilton J Wan
- Center for Influenza and Emerging Infectious Diseases, University of Missouri, Columbia, MO, USA; Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, 65212, USA; Bond Life Sciences Center, University of Missouri, Columbia, MO, USA
| | - Lei Han
- Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762, USA
| | - Jianli Xue
- Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762, USA
| | - Matthew Ykema
- Department of BioSciences, Rice University, Houston, TX, 77251, USA
| | - Yizhi J Tao
- Department of BioSciences, Rice University, Houston, TX, 77251, USA
| | - Xiu-Feng Wan
- Center for Influenza and Emerging Infectious Diseases, University of Missouri, Columbia, MO, USA; Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, 65212, USA; Bond Life Sciences Center, University of Missouri, Columbia, MO, USA; Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Mississippi State, MS, 39762, USA; Department of Electrical Engineering & Computer Science, College of Engineering, University of Missouri, Columbia, MO, USA.
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18
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Weis S, te Velthuis AJW. Influenza Virus RNA Synthesis and the Innate Immune Response. Viruses 2021; 13:v13050780. [PMID: 33924859 PMCID: PMC8146608 DOI: 10.3390/v13050780] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/10/2021] [Revised: 04/25/2021] [Accepted: 04/25/2021] [Indexed: 12/25/2022] Open
Abstract
Infection with influenza A and B viruses results in a mild to severe respiratory tract infection. It is widely accepted that many factors affect the severity of influenza disease, including viral replication, host adaptation, innate immune signalling, pre-existing immunity, and secondary infections. In this review, we will focus on the interplay between influenza virus RNA synthesis and the detection of influenza virus RNA by our innate immune system. Specifically, we will discuss the generation of various RNA species, host pathogen receptors, and host shut-off. In addition, we will also address outstanding questions that currently limit our knowledge of influenza virus replication and host adaption. Understanding the molecular mechanisms underlying these factors is essential for assessing the pandemic potential of future influenza virus outbreaks.
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19
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Interaction of NEP with G Protein Pathway Suppressor 2 Facilitates Influenza A Virus Replication by Weakening the Inhibition of GPS2 to RNA Synthesis and Ribonucleoprotein Assembly. J Virol 2021; 95:JVI.00008-21. [PMID: 33658351 PMCID: PMC8139649 DOI: 10.1128/jvi.00008-21] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/17/2022] Open
Abstract
The nuclear export protein (NEP) serves multiple functions in the life cycle of influenza A virus (IAV). Identifying novel host proteins that interact with NEP and understanding their functions in IAV replication are of great interest. In this study, we screened and confirmed the direct interaction of G protein pathway suppressor 2 (GPS2) with NEP through a yeast two-hybrid screening assay and glutathione S-transferase-pulldown and co-immunoprecipitation assays. Knockdown or knockout of GPS2 enhanced IAV titers, whereas overexpression of GPS2 impaired IAV replication, demonstrating that GPS2 acted as a negative host factor in IAV replication. Meanwhile, GPS2 inhibited viral RNA synthesis by reducing the assembly of IAV polymerase. Interestingly, IAV NEP interacted with GPS2 and mediated its nuclear export, thereby activated the degradation of GPS2. Thus, NEP-GPS2 interaction weakened the inhibition of GPS2 to viral polymerase activity and benefited virus replication. Overall, this study identified the novel NEP-binding host partner GPS2 as a critical host factor to participate in IAV replication. These findings provided novel insights into the interactions between IAV and host cells, revealing a new function for GPS2 during IAV replication.Importance: NEP is proposed to play multiple biologically important roles in the life cycle of IAV, which largely relies on host factors by interaction. Our study demonstrated that GPS2 could reduce the interaction between PB1 and PB2 and interfere with vRNP assembly. Thus, GPS2 inhibited the RNA synthesis of IAV and negatively regulated its replication. Importantly, IAV NEP interacted with GPS2 and mediated the nuclear export of GPS2, thereby activated the degradation of GPS2. Thus, NEP-GPS2 interaction weakened the inhibition of GPS2 to viral polymerase activity and benefited virus replication.
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20
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Zeng Y, Xu S, Wei Y, Zhang X, Wang Q, Jia Y, Wang W, Han L, Chen Z, Wang Z, Zhang B, Chen H, Lei CQ, Zhu Q. The PB1 protein of influenza A virus inhibits the innate immune response by targeting MAVS for NBR1-mediated selective autophagic degradation. PLoS Pathog 2021; 17:e1009300. [PMID: 33577621 PMCID: PMC7880438 DOI: 10.1371/journal.ppat.1009300] [Citation(s) in RCA: 97] [Impact Index Per Article: 24.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2020] [Accepted: 01/08/2021] [Indexed: 12/28/2022] Open
Abstract
Influenza A virus (IAV) has evolved various strategies to counteract the innate immune response using different viral proteins. However, the mechanism is not fully elucidated. In this study, we identified the PB1 protein of H7N9 virus as a new negative regulator of virus- or poly(I:C)-stimulated IFN induction and specifically interacted with and destabilized MAVS. A subsequent study revealed that PB1 promoted E3 ligase RNF5 to catalyze K27-linked polyubiquitination of MAVS at Lys362 and Lys461. Moreover, we found that PB1 preferentially associated with a selective autophagic receptor neighbor of BRCA1 (NBR1) that recognizes ubiquitinated MAVS and delivers it to autophagosomes for degradation. The degradation cascade mediated by PB1 facilitates H7N9 virus infection by blocking the RIG-I-MAVS-mediated innate signaling pathway. Taken together, these data uncover a negative regulatory mechanism involving the PB1-RNF5-MAVS-NBR1 axis and provide insights into an evasion strategy employed by influenza virus that involves selective autophagy and innate signaling pathways. In 2013, H7N9 influenza viruses appeared in China and other countries resulting in 1, 567 human infections and 615 deaths. Understanding the cross-talk between virus and host is vital for the development of effective vaccines and therapeutics. Here, we identified the PB1 protein of H7N9 virus as a novel negative regulator that enhances the degradation of MAVS, an essential adaptor protein in the innate signaling pathway. Mechanistically, PB1 promoted the E3 ligase RNF5-mediated ubiquitination of MAVS and recruited the selective autophagic receptor NBR1 to associate with and deliver the ubiquitinated MAVS to the autophagosomes for degradation. Thus, the PB1-RNF5-MAVS-NBR1 axis inhibited innate immune antiviral response and facilitated virus replication by mediating MAVS degradation in an autophagosome-dependent manner. Our findings reveal a novel mechanism by which influenza virus negatively regulates the innate immune response.
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Affiliation(s)
- Yan Zeng
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Shuai Xu
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Yanli Wei
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Xuegang Zhang
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Qian Wang
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Yane Jia
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Wanbing Wang
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Lu Han
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Zhaoshan Chen
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Zhengxiang Wang
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
| | - Bo Zhang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Hualan Chen
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Cao-Qi Lei
- Hubei Key Laboratory of Cell Homeostasis, Frontier Science Center for Immunology and Metabolism, State Key Laboratory of Virology, College of Life Sciences, Wuhan University, Wuhan, China
- * E-mail: (C-QL); (QZ)
| | - Qiyun Zhu
- State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, China
- * E-mail: (C-QL); (QZ)
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21
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Wang X, Lin L, Zhong Y, Feng M, Yu T, Yan Y, Zhou J, Liao M. Cellular hnRNPAB binding to viral nucleoprotein inhibits flu virus replication by blocking nuclear export of viral mRNA. iScience 2021; 24:102160. [PMID: 33681726 PMCID: PMC7918295 DOI: 10.1016/j.isci.2021.102160] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/01/2020] [Revised: 11/27/2020] [Accepted: 02/02/2021] [Indexed: 12/21/2022] Open
Abstract
Heterogeneous nuclear ribonucleoproteins (hnRNPs) play critical roles in the nuclear export, splicing, and sensing of RNA. However, the role of heterogeneous nuclear ribonucleoprotein A/B (hnRNPAB) is poorly understood. In this study, we report that hnRNPAB cooperates with nucleoprotein (NP) to restrict viral mRNA nuclear export via inhibiting viral mRNA binding to ALY and NXF1. HnRNPAB restricts mRNA transfer from ALY to NXF1, inhibiting the mRNA nuclear export. Moreover, when cells are invaded by influenza A virus, NP interacts with hnRNPAB and interrupts the ALY-UAP56 interaction, leading to repression of ALY-viral mRNA binding, and then inhibits the viral mRNA binding to NXF1, leading to nuclear stimulation of viral mRNA. Collectively, these observations provide a new role of hnRNPAB to act as an mRNA nuclear retention factor, which is also effective for viral mRNA of influenza A virus, and NP cooperates with hnRNPAB to further restrict the viral mRNA nuclear export.
HnRNPAB inhibits influenza A virus replication by repressing viral mRNA nuclear export HnRNPAB interrupts viral mRNA transferring from ALY to NXF1 NP cooperates with hnRNPAB to further restrict viral mRNA nuclear export The ALY-viral mRNA binding is restricted by NP-hnRNPAB complex
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Affiliation(s)
- Xingbo Wang
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
| | - Lulu Lin
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
| | - Yiye Zhong
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
| | - Mingfang Feng
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
| | - Tianqi Yu
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
| | - Yan Yan
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
| | - Jiyong Zhou
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
- State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, First Affiliated Hospital, Zhejiang University, Hangzhou 310003, P.R. China
- Corresponding author
| | - Min Liao
- MOA Key Laboratory of Animal Virology, Zhejiang University Center for Veterinary Sciences, 866 Yuhangtang Road, Hangzhou, Zhejiang 310058, PR China
- Corresponding author
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22
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Zhang X, Lin X, Qin C, Huang K, Sun X, Zhao L, Jin M. Avian Chaperonin Containing TCP1 Subunit 5 Supports Influenza A Virus Replication by Interacting With Viral Nucleoprotein, PB1, and PB2 Proteins. Front Microbiol 2020; 11:538355. [PMID: 33178142 PMCID: PMC7593399 DOI: 10.3389/fmicb.2020.538355] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2020] [Accepted: 08/26/2020] [Indexed: 11/30/2022] Open
Abstract
Humans and avian species are prone to influenza viral infection, which may cause serious clinical consequences. Many studies have documented the critical role of host factors in the influenza virus life cycle based on human models, but knowledge about their roles in birds is very limited. In this study, using immunoprecipitation coupled with mass spectrometry, a total of 72 potential interacting proteins of influenza nucleoprotein (NP) were identified in DF-1 cells. Among these proteins, avian chaperonin containing TCP1 subunit 5 (CCT5) was demonstrated to interact with influenza A virus (IAV) NP directly, as well as polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2) but not with polymerase acidic protein (PA). Further investigation showed that viral infection profoundly elevated the expression level of cellular CCT5, whose expression, in turn, promoted the nuclear export of NP, as well as viral polymerase activity, thereby facilitating the replication of IAV. The obtained results suggested an important role of avian CCT5 in supporting influenza virus replication, which may serve as an anti-influenza target.
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Affiliation(s)
- Xiaohan Zhang
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China
| | - Xian Lin
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China
| | - Chenghuang Qin
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China
| | - Kun Huang
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China
| | - Xiaomei Sun
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.,Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People's Republic of China, Wuhan, China
| | - Lianzhong Zhao
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China
| | - Meilin Jin
- State Key Laboratory of Agricultural Microbiology, College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.,Key Laboratory of Preventive Veterinary Medicine in Hubei Province, The Cooperative Innovation Center for Sustainable Pig Production, Wuhan, China.,Key Laboratory of Development of Veterinary Diagnostic Products, Ministry of Agriculture of the People's Republic of China, Wuhan, China
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23
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Dawson AR, Wilson GM, Freiberger EC, Mondal A, Coon JJ, Mehle A. Phosphorylation controls RNA binding and transcription by the influenza virus polymerase. PLoS Pathog 2020; 16:e1008841. [PMID: 32881973 PMCID: PMC7494117 DOI: 10.1371/journal.ppat.1008841] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/14/2020] [Revised: 09/16/2020] [Accepted: 07/25/2020] [Indexed: 12/03/2022] Open
Abstract
The influenza virus polymerase transcribes and replicates the viral genome. The proper timing and balance of polymerase activity is important for successful replication. Genome replication is controlled in part by phosphorylation of NP that regulates assembly of the replication machinery. However, it remains unclear whether phosphorylation directly regulated polymerase activity. Here we identified polymerase phosphosites that control its function. Mutating phosphosites in the catalytic subunit PB1 altered polymerase activity and virus replication. Biochemical analyses revealed phosphorylation events that disrupted global polymerase function by blocking the NTP entry channel or preventing RNA binding. We also identified a regulatory site that split polymerase function by specifically suppressing transcription. These experiments show that host kinases phospho-regulate viral RNA synthesis directly by modulating polymerase activity and indirectly by controlling assembly of replication machinery. Further, they suggest polymerase phosphorylation may bias replication versus transcription at discrete times or locations during the infectious cycle. The influenza virus polymerase is a multifunctional enzyme directing viral gene expression and genome replication. Immediately following infection, the polymerase primarily performs transcription to make the viral mRNAs that program the replication cycle. The polymerase then shifts output to produce more copies of the viral genome at later stages of infection. The balance between transcription and replication is critical for successful infection. Here we identify phosphorylation sites within the viral polymerase and describe how these post-translational modifications control polymerase activity. Cellular kinases modify the viral polymerase. We identified a phosphorylation site in the catalytic subunit PB1 that selectively disables transcription, but not replication. We also describe a phosphorylation site in PB1 that disrupts binding to viral RNAs, disabling all activities of the polymerase. These modifications may establish polymerases with specialized function, and help regulate the balance between transcription and replication throughout the viral life cycle.
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Affiliation(s)
- Anthony R. Dawson
- Department of Medical Microbiology & Immunology, University of Wisconsin–Madison, Madison, WI, United States of America
| | - Gary M. Wilson
- Department of Chemistry, University of Wisconsin–Madison, Madison, WI, United States of America
| | - Elyse C. Freiberger
- Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI, United States of America
| | - Arindam Mondal
- Department of Medical Microbiology & Immunology, University of Wisconsin–Madison, Madison, WI, United States of America
| | - Joshua J. Coon
- Department of Chemistry, University of Wisconsin–Madison, Madison, WI, United States of America
- Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI, United States of America
| | - Andrew Mehle
- Department of Medical Microbiology & Immunology, University of Wisconsin–Madison, Madison, WI, United States of America
- * E-mail:
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24
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Differential Behaviours and Preferential Bindings of Influenza Nucleoproteins on Importins-α. Viruses 2020; 12:v12080834. [PMID: 32751671 PMCID: PMC7472415 DOI: 10.3390/v12080834] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2020] [Revised: 07/23/2020] [Accepted: 07/27/2020] [Indexed: 12/20/2022] Open
Abstract
Influenza viruses are negative single-stranded RNA viruses with nuclear transcription and replication. They enter the nucleus by using the cellular importin-α/-β nuclear import machinery. Influenza nucleoproteins from influenza A, B, C and D viruses possess a nuclear localization signal (NLS) localized on an intrinsically disordered extremity (NPTAIL). In this paper, using size exclusion chromatography (SEC), SEC-multi-angle laser light scattering (SEC-MALLS) analysis, surface plasmon resonance (SPR) and fluorescence anisotropy, we provide the first comparative study designed to dissect the interaction between the four NPTAILs and four importins-α identified as partners. All interactions between NPTAILs and importins-α have high association and dissociation rates and present a distinct and specific behaviour. D/NPTAIL interacts strongly with all importins-α while B/NPTAIL shows weak affinity for importins-α. A/NPTAIL and C/NPTAIL present preferential importin-α partners. Mutations in B/NPTAIL and D/NPTAIL show a loss of importin-α binding, confirming key NLS residues. Taken together, our results provide essential highlights of this complex translocation mechanism.
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25
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Audi A, Soudani N, Dbaibo G, Zaraket H. Depletion of Host and Viral Sphingomyelin Impairs Influenza Virus Infection. Front Microbiol 2020; 11:612. [PMID: 32425895 PMCID: PMC7203554 DOI: 10.3389/fmicb.2020.00612] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2019] [Accepted: 03/19/2020] [Indexed: 12/19/2022] Open
Abstract
Influenza A virus (IAV) is a major human respiratory pathogen causing annual epidemics as well as periodic pandemics. A complete understanding of the virus pathogenesis and host factors involved in the viral lifecycle is crucial for developing novel therapeutic approaches. Sphingomyelin (SM) is the most abundant membrane sphingolipid. It preferentially associates with cholesterol to form distinct domains named lipid rafts. Sphingomyelinases, including acid sphingomyelinase (ASMase), catalyzes the hydrolysis of membrane SM and consequently transform lipid rafts into ceramide-enriched membrane platforms. In this study, we investigated the effect of SM hydrolysis on IAV propagation. Depleting plasma membrane SM by exogenous bacterial SMase (bSMase) impaired virus infection and reduced virus entry, whereas exogenous SM enhanced infection. Moreover, the depletion of virus envelope SM also reduced virus infectivity and impaired its attachment and internalization. Nonetheless, inhibition of ASMase by desipramine did not affect IAV infection. Similarly, virus replication was not impaired in Niemann-Pick disease type A (NPA) cells, which lack functional ASMase. IAV infection in A549 cells was associated with suppression of ASMase activity starting at 6 h post-infection. Our data reveals that intact cellular and viral envelope SM is required for efficient IAV infection. Therefore, SM metabolism can be a potential target for therapeutic intervention against influenza virus infection.
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Affiliation(s)
- Amani Audi
- Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.,Center for Infectious Diseases Research, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Nadia Soudani
- Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.,Center for Infectious Diseases Research, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.,Doctoral School of Science and Technology, Research Platform for Environmental Science (PRASE), Faculty of Sciences, Lebanese University, Beirut, Lebanon
| | - Ghassan Dbaibo
- Center for Infectious Diseases Research, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.,Department of Pediatrics and Adolescent Medicine, Faculty of Medicine, American University of Beirut Medical Center, Beirut, Lebanon.,Department of Biochemistry and Molecular Genetics, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
| | - Hassan Zaraket
- Department of Experimental Pathology, Immunology and Microbiology, Faculty of Medicine, American University of Beirut, Beirut, Lebanon.,Center for Infectious Diseases Research, Faculty of Medicine, American University of Beirut, Beirut, Lebanon
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26
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Boukharta M, El Amri H, Zakham F, Ennaji MM. Role of Genetic and Molecular Dynamics in the Emergence, Reemergence, and Interspecies Transmission of Equine Influenza Viruses. EMERGING AND REEMERGING VIRAL PATHOGENS 2020:745-780. [DOI: 10.1016/b978-0-12-819400-3.00034-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/05/2025]
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27
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[Structural studies on negative-strand RNA virus]. Uirusu 2020; 70:91-100. [PMID: 33967118 DOI: 10.2222/jsv.70.91] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022]
Abstract
Negative-strand RNA viruses do not possess a rigid viral shell, and their structures are flexible and fragile. We have applied various electron microscopies to analyze the morphologies of influenza and Ebola virus. Our studies have revealed the native interior and exterior ultrastructures of influenza virus as well as the assembly of Ebola virus core in atomic detail.
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28
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Shafiuddin M, Boon ACM. RNA Sequence Features Are at the Core of Influenza A Virus Genome Packaging. J Mol Biol 2019; 431:4217-4228. [PMID: 30914291 PMCID: PMC6756997 DOI: 10.1016/j.jmb.2019.03.018] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/04/2019] [Revised: 02/18/2019] [Accepted: 03/11/2019] [Indexed: 11/23/2022]
Abstract
The influenza A virus (IAV), a respiratory pathogen for humans, poses serious medical and economic challenges to global healthcare systems. The IAV genome, consisting of eight single-stranded viral RNA segments, is incorporated into virions by a complex process known as genome packaging. Specific RNA sequences within the viral RNA segments serve as signals that are necessary for genome packaging. Although efficient packaging is a prerequisite for viral infectivity, many of the mechanistic details about this process are still missing. In this review, we discuss the recent advances toward the understanding of IAV genome packaging and focus on the RNA features that play a role in this process.
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Affiliation(s)
- Md Shafiuddin
- Department of Internal Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA
| | - Adrianus C M Boon
- Department of Internal Medicine, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA; Department of Molecular Microbiology and Microbial Pathogenesis, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA; Department of Pathology and Immunology, Washington University in Saint Louis School of Medicine, St. Louis, MO 63110, USA.
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29
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Smertina E, Urakova N, Strive T, Frese M. Calicivirus RNA-Dependent RNA Polymerases: Evolution, Structure, Protein Dynamics, and Function. Front Microbiol 2019; 10:1280. [PMID: 31244803 PMCID: PMC6563846 DOI: 10.3389/fmicb.2019.01280] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2019] [Accepted: 05/22/2019] [Indexed: 12/11/2022] Open
Abstract
The Caliciviridae are viruses with a positive-sense, single-stranded RNA genome that is packaged into an icosahedral, environmentally stable protein capsid. The family contains five genera (Norovirus, Nebovirus, Sapovirus, Lagovirus, and Vesivirus) that infect vertebrates including amphibians, reptiles, birds, and mammals. The RNA-dependent RNA polymerase (RdRp) replicates the genome of RNA viruses and can speed up evolution due to its error-prone nature. Studying calicivirus RdRps in the context of genuine virus replication is often hampered by a lack of suitable model systems. Enteric caliciviruses and RHDV in particular are notoriously difficult to propagate in cell culture; therefore, molecular studies of replication mechanisms are challenging. Nevertheless, research on recombinant proteins has revealed several unexpected characteristics of calicivirus RdRps. For example, the RdRps of RHDV and related lagoviruses possess the ability to expose a hydrophobic motif, to rearrange Golgi membranes, and to copy RNA at unusually high temperatures. This review is focused on the structural dynamics, biochemical properties, kinetics, and putative interaction partners of these RdRps. In addition, we discuss the possible existence of a conserved but as yet undescribed structural element that is shared amongst the RdRps of all caliciviruses.
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Affiliation(s)
- Elena Smertina
- Commonwealth Scientific and Industrial Research Organisation, Health and Biosecurity, Canberra, ACT, Australia
- Faculty of Science and Technology, University of Canberra, Canberra, ACT, Australia
| | - Nadya Urakova
- Department of Entomology, Pennsylvania State University, University Park, PA, United States
| | - Tanja Strive
- Commonwealth Scientific and Industrial Research Organisation, Health and Biosecurity, Canberra, ACT, Australia
- Invasive Animals Cooperative Research Centre, University of Canberra, Canberra, ACT, Australia
| | - Michael Frese
- Faculty of Science and Technology, University of Canberra, Canberra, ACT, Australia
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30
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Wu X, Wang J, Wang S, Wu F, Chen Z, Li C, Cheng G, Qin FXF. Inhibition of Influenza A Virus Replication by TRIM14 via Its Multifaceted Protein-Protein Interaction With NP. Front Microbiol 2019; 10:344. [PMID: 30873142 PMCID: PMC6401474 DOI: 10.3389/fmicb.2019.00344] [Citation(s) in RCA: 52] [Impact Index Per Article: 8.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/13/2018] [Accepted: 02/08/2019] [Indexed: 01/12/2023] Open
Abstract
Influenza A virus (IAV) is a worldwide ongoing health threat causing diseases in both humans and animals. The interaction between IAV and host is a dynamic and evolving process that influences the pathogenicity and host specificity of the virus. TRIM14, a member of tripartite motif (TRIM) family, has been demonstrated to possess a strong capability of regulating type I interferon and NF-κB induction in host defense against viral infection. In this study, we found that TRIM14 could restrict the replication of IAV in a type I interferon and NF-κB independent manner. Mechanistically, different domains of TRIM14 could selectively interact with the viral nucleoprotein (NP), resulting in disparate influences on the RNP formation and viral replication. In particular, the PRYSPRY domain of TRIM14 exhibited a potent inhibitory activity on NP protein stability and IAV replication. On the contrary, the ΔS2 domain could rather antagonize the function of PRYSPRY domain and promote the IAV RNP formation by stabilizing NP. At the biochemical level, TRIM14-NP interaction could induce the K48-linked ubiquitination and proteasomal degradation of NP. Moreover, due to the rapid degradation of newly synthesized NP, TRIM14 could effectively block the translocation of NP from cytoplasm to nucleus thus further restrain the propagation of IAV in host cells. Taken together, our study has unraveled a previously unknown mechanism of TRIM14 mediated inhibition on RNP formation and influenza virus replication, and provides a new paradigm of complex and multifaceted host-pathogen interaction between ISG and viral protein.
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Affiliation(s)
- Xiangwei Wu
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Jingfeng Wang
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Shanshan Wang
- Department of Pathology, Institute of Precision Medicine, Jining Medical University, Jining, China
| | - Fei Wu
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Zhigao Chen
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Chunfeng Li
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Genhong Cheng
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - F. Xiao-Feng Qin
- Institute of Systems Medicine, Chinese Academy of Medical Sciences and Peking Union Medical College, Suzhou, China
- Center for Systems Medicine, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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Autophagy Promotes Replication of Influenza A Virus In Vitro. J Virol 2019; 93:JVI.01984-18. [PMID: 30541828 DOI: 10.1128/jvi.01984-18] [Citation(s) in RCA: 76] [Impact Index Per Article: 12.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Accepted: 11/27/2018] [Indexed: 12/20/2022] Open
Abstract
Influenza A virus (IAV) infection could induce autophagosome accumulation. However, the impact of the autophagy machinery on IAV infection remains controversial. Here, we showed that induction of cellular autophagy by starvation or rapamycin treatment increases progeny virus production, while disruption of autophagy using a small interfering RNA (siRNA) and pharmacological inhibitor reduces progeny virus production. Further studies revealed that alteration of autophagy significantly affects the early stages of the virus life cycle or viral RNA synthesis. Importantly, we demonstrated that overexpression of both the IAV M2 and NP proteins alone leads to the lipidation of LC3 to LC3-II and a redistribution of LC3 from the cytosol to punctate vesicles indicative of authentic autophagosomes. Intriguingly, both M2 and NP colocalize and interact with LC3 puncta during M2 or NP transfection alone and IAV infection, leading to an increase in viral ribonucleoprotein (vRNP) export and infectious viral particle formation, which indicates that the IAV-host autophagy interaction plays a critical role in regulating IAV replication. We showed that NP and M2 induce the AKT-mTOR-dependent autophagy pathway and an increase in HSP90AA1 expression. Finally, our studies provided evidence that IAV replication needs an autophagy pathway to enhance viral RNA synthesis via the interaction of PB2 and HSP90AA1 by modulating HSP90AA1 expression and the AKT-mTOR signaling pathway in host cells. Collectively, our studies uncover a new mechanism that NP- and M2-mediated autophagy functions in different stages of virus replication in the pathogenicity of influenza A virus.IMPORTANCE Autophagy impacts the replication cycle of many viruses. However, the role of the autophagy machinery in IAV replication remains unclear. Therefore, we explored the detailed mechanisms utilized by IAV to promote its replication. We demonstrated that IAV NP- and M2-mediated autophagy promotes IAV replication by regulating the AKT-mTOR signaling pathway and HSP90AA1 expression. The interaction of PB2 and HSP90AA1 results in the increase of viral RNA synthesis first; subsequently the binding of NP to LC3 favors vRNP export, and later the interaction of M2 and LC3 leads to an increase in the production of infectious viral particles, thus accelerating viral progeny production. These findings improve our understanding of IAV pathogenicity in host cells.
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The structure of the nucleoprotein of Influenza D shows that all Orthomyxoviridae nucleoproteins have a similar NP CORE, with or without a NP TAIL for nuclear transport. Sci Rep 2019; 9:600. [PMID: 30679709 PMCID: PMC6346101 DOI: 10.1038/s41598-018-37306-y] [Citation(s) in RCA: 13] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2018] [Accepted: 11/30/2018] [Indexed: 02/07/2023] Open
Abstract
This paper focuses on the nucleoprotein (NP) of the newly identified member of the Orthomyxoviridae family, Influenza D virus. To date several X-ray structures of NP of Influenza A (A/NP) and B (B/NP) viruses and of infectious salmon anemia (ISA/NP) virus have been solved. Here we purified, characterized and solved the X-ray structure of the tetrameric D/NP at 2.4 Å resolution. The crystal structure of its core is similar to NP of other Influenza viruses. However, unlike A/NP and B/NP which possess a flexible amino-terminal tail containing nuclear localization signals (NLS) for their nuclear import, D/NP possesses a carboxy-terminal tail (D/NPTAIL). We show that D/NPTAIL harbors a bipartite NLS and designed C-terminal truncated mutants to demonstrate the role of D/NPTAIL for nuclear transport.
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CypA Regulates AIP4-Mediated M1 Ubiquitination of Influenza A Virus. Virol Sin 2018; 33:440-448. [PMID: 30328013 DOI: 10.1007/s12250-018-0058-6] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2018] [Accepted: 09/13/2018] [Indexed: 10/28/2022] Open
Abstract
Cyclophilin A (CypA) is a peptidyl-prolyl cis/trans isomerase that interacts with the matrix protein (M1) of influenza A virus (IAV) and restricts virus replication by regulating the ubiquitin-proteasome-mediated degradation of M1. However, the mechanism by which CypA regulates M1 ubiquitination remains unknown. In this study, we reported that E3 ubiquitin ligase AIP4 promoted K48-linked ubiquitination of M1 at K102 and K104, and accelerated ubiquitin-proteasome-mediated degradation of M1. The recombinant IAV with mutant M1 (K102R/K104R) could not be rescued, suggesting that the ubiquitination of M1 at K102/K104 was essential for IAV replication. Furthermore, CypA inhibited AIP4-mediated M1 ubiquitination by impairing the interaction between AIP4 and M1. More importantly, both the mutations of M1 (K102R/K104R) and CypA inhibited the nuclear export of M1, indicating that CypA regulates the cellular localization of M1 via inhibition of AIP4-mediated M1 ubiquitination at K102 and K104, which results in the reduced replication of IAV. Collectively, our findings reveal a novel ubiquitination-based mechanism by which CypA regulates the replication of IAV.
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Nncube NB, Ramharack P, Soliman MES. Using bioinformatics tools for the discovery of Dengue RNA-dependent RNA polymerase inhibitors. PeerJ 2018; 6:e5068. [PMID: 30280009 PMCID: PMC6161702 DOI: 10.7717/peerj.5068] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2018] [Accepted: 06/04/2018] [Indexed: 12/29/2022] Open
Abstract
Background Dengue fever has rapidly manifested into a serious global health concern. The emergence of various viral serotypes has prompted the urgent need for innovative drug design techniques. Of the viral non-structural enzymes, the NS5 RNA-dependent RNA polymerase has been established as a promising target due to its lack of an enzymatic counterpart in mammalian cells and its conserved structure amongst all serotypes. The onus is now on scientists to probe further into understanding this enzyme and its mechanism of action. The field of bioinformatics has evolved greatly over recent decades, with updated drug design tools now being publically available. Methods In this study, bioinformatics tools were used to provide a comprehensive sequence and structural analysis of the two most prominent serotypes of Dengue RNA-dependent RNA polymerase. A list of popular flavivirus inhibitors were also chosen to dock to the active site of the enzyme. The best docked compound was then used as a template to generate a pharmacophore model that may assist in the design of target-specific Dengue virus inhibitors. Results Comparative sequence alignment exhibited similarity between all three domains of serotype 2 and 3.Sequence analysis revealed highly conserved regions at residues Meth530, Thr543 Asp597, Glu616, Arg659 and Pro671. Mapping of the active site demonstrated two highly conserved residues: Ser710 and Arg729. Of the active site interacting residues, Ser796 was common amongst all ten docked compounds, indicating its importance in the drug design process. Of the ten docked flavivirus inhibitors, NITD-203 showed the best binding affinity to the active site. Further pharmacophore modeling of NITD-203 depicted significant pharmacophoric elements that are necessary for stable binding to the active site. Discussion This study utilized publically available bioinformatics tools to provide a comprehensive framework on Dengue RNA-dependent RNA polymerase. Based on docking studies, a pharmacophore model was also designed to unveil the crucial pharmacophoric elements that are required when constructing an efficacious DENV inhibitor. We believe that this study will be a cornerstone in paving the road toward the design of target-specific inhibitors against DENV RdRp.
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Affiliation(s)
- Nomagugu B Nncube
- Molecular Bio-computation and Drug Design laboratory, School of Health Sciences, University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa
| | - Pritika Ramharack
- Molecular Bio-computation and Drug Design laboratory, School of Health Sciences, University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa
| | - Mahmoud E S Soliman
- Molecular Bio-computation and Drug Design laboratory, School of Health Sciences, University of KwaZulu-Natal, Durban, KwaZulu-Natal, South Africa
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Li Y, Sun L, Zheng W, Madina Mahesutihan, Li J, Bi Y, Wang H, Liu W, Luo TR. Phosphorylation and dephosphorylation of threonine 188 in nucleoprotein is crucial for the replication of influenza A virus. Virology 2018; 520:30-38. [PMID: 29775781 DOI: 10.1016/j.virol.2018.05.002] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/19/2018] [Revised: 05/03/2018] [Accepted: 05/04/2018] [Indexed: 10/16/2022]
Abstract
Nucleoprotein (NP) is a major component of the viral ribonucleoprotein (vRNP) complex that is responsible for viral replication, transcription and packaging of influenza A virus. Phosphorylation of NP plays an important role during viral infection. In the present study, we identified threonine 188 (T188) as a novel phosphorylated residue in the NP of influenza A virus by using mass spectrometry. T188 is located within nuclear export signal 2 (NES2) which is chromosome region maintenance 1 (CRM1)-independent. We observed that the phosphorylation and dephosphorylation of residue T188 regulated viral replication by controlling NES2-dependent NP nuclear export and the polymerase activity of the vRNP complex. Our findings provide further insights for understanding the replication of influenza A virus.
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Affiliation(s)
- Yun Li
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses & Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China; CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Lei Sun
- CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Weinan Zheng
- CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Madina Mahesutihan
- CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Jing Li
- CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China
| | - Yuhai Bi
- CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
| | - Heran Wang
- International Department, Beijing National Day School, Beijing 100039, China
| | - Wenjun Liu
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses & Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China; CAS Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China; University of Chinese Academy of Sciences, Beijing 100049, China.
| | - Ting Rong Luo
- State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresourses & Laboratory of Animal Infectious Diseases, College of Animal Sciences and Veterinary Medicine, Guangxi University, Nanning 530004, Guangxi, China.
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González C, Tabernero D, Cortese MF, Gregori J, Casillas R, Riveiro-Barciela M, Godoy C, Sopena S, Rando A, Yll M, Lopez-Martinez R, Quer J, Esteban R, Buti M, Rodríguez-Frías F. Detection of hyper-conserved regions in hepatitis B virus X gene potentially useful for gene therapy. World J Gastroenterol 2018; 24:2095-2107. [PMID: 29785078 PMCID: PMC5960815 DOI: 10.3748/wjg.v24.i19.2095] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/16/2018] [Revised: 03/26/2018] [Accepted: 05/06/2018] [Indexed: 02/06/2023] Open
Abstract
AIM To detect hyper-conserved regions in the hepatitis B virus (HBV) X gene (HBX) 5' region that could be candidates for gene therapy. METHODS The study included 27 chronic hepatitis B treatment-naive patients in various clinical stages (from chronic infection to cirrhosis and hepatocellular carcinoma, both HBeAg-negative and HBeAg-positive), and infected with HBV genotypes A-F and H. In a serum sample from each patient with viremia > 3.5 log IU/mL, the HBX 5' end region [nucleotide (nt) 1255-1611] was PCR-amplified and submitted to next-generation sequencing (NGS). We assessed genotype variants by phylogenetic analysis, and evaluated conservation of this region by calculating the information content of each nucleotide position in a multiple alignment of all unique sequences (haplotypes) obtained by NGS. Conservation at the HBx protein amino acid (aa) level was also analyzed. RESULTS NGS yielded 1333069 sequences from the 27 samples, with a median of 4578 sequences/sample (2487-9279, IQR 2817). In 14/27 patients (51.8%), phylogenetic analysis of viral nucleotide haplotypes showed a complex mixture of genotypic variants. Analysis of the information content in the haplotype multiple alignments detected 2 hyper-conserved nucleotide regions, one in the HBX upstream non-coding region (nt 1255-1286) and the other in the 5' end coding region (nt 1519-1603). This last region coded for a conserved amino acid region (aa 63-76) that partially overlaps a Kunitz-like domain. CONCLUSION Two hyper-conserved regions detected in the HBX 5' end may be of value for targeted gene therapy, regardless of the patients' clinical stage or HBV genotype.
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Affiliation(s)
- Carolina González
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - David Tabernero
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Maria Francesca Cortese
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Josep Gregori
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
- Roche Diagnostics SL, Sant Cugat del Vallès 08174, Spain
| | - Rosario Casillas
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Mar Riveiro-Barciela
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
- Rafael Esteban and Maria Buti, Liver Unit, Department of Internal Medicine, Hospital Universitari Vall d'Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Cristina Godoy
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Marçall Yll, Josep Quer, Liver Unit, Liver Disease Laboratory-Viral Hepatitis, Vall d’Hebron Institut Recerca-Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Sara Sopena
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Ariadna Rando
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Marçal Yll
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Rosa Lopez-Martinez
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
| | - Josep Quer
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Rafael Esteban
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Maria Buti
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
| | - Francisco Rodríguez-Frías
- Liver Pathology Unit, Departments of Biochemistry and Microbiology, Hospital Universitari Vall d’Hebron, Universitat Autònoma de Barcelona, Barcelona 08035, Spain
- Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBERehd), Instituto de Salud Carlos III, Madrid 28029, Spain
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Williams GD, Townsend D, Wylie KM, Kim PJ, Amarasinghe GK, Kutluay SB, Boon ACM. Nucleotide resolution mapping of influenza A virus nucleoprotein-RNA interactions reveals RNA features required for replication. Nat Commun 2018; 9:465. [PMID: 29386621 PMCID: PMC5792457 DOI: 10.1038/s41467-018-02886-w] [Citation(s) in RCA: 65] [Impact Index Per Article: 9.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/22/2017] [Accepted: 01/04/2018] [Indexed: 02/03/2023] Open
Abstract
Influenza A virus nucleoprotein (NP) association with viral RNA (vRNA) is essential for packaging, but the pattern of NP binding to vRNA is unclear. Here we applied photoactivatable ribonucleoside enhanced cross-linking and immunoprecipitation (PAR-CLIP) to assess the native-state of NP-vRNA interactions in infected human cells. NP binds short fragments of RNA (~12 nucleotides) non-uniformly and without apparent sequence specificity. Moreover, NP binding is reduced at specific locations within the viral genome, including regions previously identified as required for viral genome segment packaging. Synonymous mutations designed to alter the predicted RNA structures in these low-NP-binding regions impact genome packaging and result in virus attenuation, whereas control mutations or mutagenesis of NP-bound regions have no effect. Finally, we demonstrate that the sequence conservation of low-NP-binding regions is required in multiple genome segments for propagation of diverse mammalian and avian IAV in host cells.
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Affiliation(s)
- Graham D Williams
- Department of Medicine at Washington University School of Medicine, St Louis, MO, 63110, USA
- Department of Molecular Microbiology at Washington University School of Medicine, St Louis, MO, 63110, USA
| | - Dana Townsend
- Department of Molecular Microbiology at Washington University School of Medicine, St Louis, MO, 63110, USA
| | - Kristine M Wylie
- Department of Pediatrics at Washington University School of Medicine, St Louis, MO, 63110, USA
- The McDonnell Genome Institute at Washington University School of Medicine, St Louis, MO, 63110, USA
| | - Preston J Kim
- Department of Pathology and Immunology at Washington University School of Medicine, St Louis, MO, 63110, USA
| | - Gaya K Amarasinghe
- Department of Pathology and Immunology at Washington University School of Medicine, St Louis, MO, 63110, USA
- Department of Biochemistry and Biophysics at Washington University School of Medicine, St Louis, MO, 63110, USA
| | - Sebla B Kutluay
- Department of Molecular Microbiology at Washington University School of Medicine, St Louis, MO, 63110, USA
| | - Adrianus C M Boon
- Department of Medicine at Washington University School of Medicine, St Louis, MO, 63110, USA.
- Department of Molecular Microbiology at Washington University School of Medicine, St Louis, MO, 63110, USA.
- Department of Pathology and Immunology at Washington University School of Medicine, St Louis, MO, 63110, USA.
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Luo W, Zhang J, Liang L, Wang G, Li Q, Zhu P, Zhou Y, Li J, Zhao Y, Sun N, Huang S, Zhou C, Chang Y, Cui P, Chen P, Jiang Y, Deng G, Bu Z, Li C, Jiang L, Chen H. Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication. PLoS Pathog 2018; 14:e1006851. [PMID: 29352288 PMCID: PMC5792031 DOI: 10.1371/journal.ppat.1006851] [Citation(s) in RCA: 74] [Impact Index Per Article: 10.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2017] [Revised: 01/31/2018] [Accepted: 01/03/2018] [Indexed: 12/21/2022] Open
Abstract
Transcription and replication of the influenza A virus (IAV) genome occur in the nucleus of infected cells and are carried out by the viral ribonucleoprotein complex (vRNP). As a major component of the vRNP complex, the viral nucleoprotein (NP) mediates the nuclear import of the vRNP complex via its nuclear localization signals (NLSs). Clearly, an effective way for the host to antagonize IAV infection would be by targeting vRNP nuclear import. Here, we identified phospholipid scramblase 1 (PLSCR1) as a binding partner of NP by using a yeast two-hybrid (Y2H) screen. The interaction between NP and PLSCR1 in mammalian cells was demonstrated by using co-immunoprecipitation and pull-down assays. We found that the stable overexpression of PLSCR1 suppressed the nuclear import of NP, hindered the virus life cycle, and significantly inhibited the replication of various influenza subtypes. In contrast, siRNA knockdown or CRISPR/Cas9 knockout of PLSCR1 increased virus propagation. Further analysis indicated that the inhibitory effect of PLSCR1 on the nuclear import of NP was not caused by affecting the phosphorylation status of NP or by stimulating the interferon (IFN) pathways. Instead, PLSCR1 was found to form a trimeric complex with NP and members of the importin α family, which inhibited the incorporation of importin β, a key mediator of the classical nuclear import pathway, into the complex, thus impairing the nuclear import of NP and suppressing virus replication. Our results demonstrate that PLSCR1 negatively regulates virus replication by interacting with NP in the cytoplasm and preventing its nuclear import.
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Affiliation(s)
- Weiyu Luo
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Jie Zhang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Libin Liang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Guangwen Wang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Qibing Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Pengyang Zhu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Yuan Zhou
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Junping Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Yuhui Zhao
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Nan Sun
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Shanyu Huang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Chenchen Zhou
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Yu Chang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Pengfei Cui
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Pucheng Chen
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Yongping Jiang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Guohua Deng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Zhigao Bu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Chengjun Li
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Li Jiang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
| | - Hualan Chen
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, China
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39
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Mondal A, Dawson AR, Potts GK, Freiberger EC, Baker SF, Moser LA, Bernard KA, Coon JJ, Mehle A. Influenza virus recruits host protein kinase C to control assembly and activity of its replication machinery. eLife 2017; 6:26910. [PMID: 28758638 PMCID: PMC5791932 DOI: 10.7554/elife.26910] [Citation(s) in RCA: 50] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/17/2017] [Accepted: 07/29/2017] [Indexed: 12/24/2022] Open
Abstract
Influenza virus expresses transcripts early in infection and transitions towards genome replication at later time points. This process requires de novo assembly of the viral replication machinery, large ribonucleoprotein complexes (RNPs) composed of the viral polymerase, genomic RNA and oligomeric nucleoprotein (NP). Despite the central role of RNPs during infection, the factors dictating where and when they assemble are poorly understood. Here we demonstrate that human protein kinase C (PKC) family members regulate RNP assembly. Activated PKCδ interacts with the polymerase subunit PB2 and phospho-regulates NP oligomerization and RNP assembly during infection. Consistent with its role in regulating RNP assembly, knockout of PKCδ impairs virus infection by selectively disrupting genome replication. However, primary transcription from pre-formed RNPs deposited by infecting particles is unaffected. Thus, influenza virus exploits host PKCs to regulate RNP assembly, a step required for the transition from primary transcription to genome replication during the infectious cycle.
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Affiliation(s)
- Arindam Mondal
- Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States
| | - Anthony R Dawson
- Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States.,Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, United States
| | - Gregory K Potts
- Department of Chemistry, University of Wisconsin-Madison, Madison, United States
| | - Elyse C Freiberger
- Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, United States
| | - Steven F Baker
- Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States
| | - Lindsey A Moser
- Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, United States
| | - Kristen A Bernard
- Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, United States
| | - Joshua J Coon
- Department of Chemistry, University of Wisconsin-Madison, Madison, United States.,Department of Biomolecular Chemistry, University of Wisconsin-Madison, Madison, United States
| | - Andrew Mehle
- Medical Microbiology and Immunology, University of Wisconsin-Madison, Madison, United States
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40
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Model-based structural and functional characterization of the Rice stripe tenuivirus nucleocapsid protein interacting with viral genomic RNA. Virology 2017; 506:73-83. [PMID: 28359901 DOI: 10.1016/j.virol.2017.03.010] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Revised: 03/19/2017] [Accepted: 03/21/2017] [Indexed: 11/21/2022]
Abstract
Rice stripe tenuivirus (RSV) is a filamentous, negative-strand RNA virus causing severe diseases on rice in Asian countries. The viral particle is composed predominantly of a nucleocapsid protein (NP) and genomic RNA. However, the molecular details of how the RSV NP interacts with genomic RNA during particle assembly remain largely unknown. Here, we modeled the NP-RNA complex and show that polar amino acids within a predicted groove of NP are critical for RNA binding and protecting the RNA from RNase digestion. RSV NP formed pentamers, hexamers, heptamers, and octamers. By modeling the higher-order structures, we found that oligomer formation was driven by the N-terminal amino arm of the NP. Deletion of this arm abolished oligomerization; the N-terminally truncated NP was less able to interact with RNA and protect RNA than was the wild type. These findings afford valuable new insights into molecular mechanism of RSV NPs interacting with genomic RNA.
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Pflug A, Lukarska M, Resa-Infante P, Reich S, Cusack S. Structural insights into RNA synthesis by the influenza virus transcription-replication machine. Virus Res 2017; 234:103-117. [PMID: 28115197 DOI: 10.1016/j.virusres.2017.01.013] [Citation(s) in RCA: 126] [Impact Index Per Article: 15.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/28/2016] [Revised: 12/31/2016] [Accepted: 01/13/2017] [Indexed: 12/22/2022]
Abstract
Influenza virus is a segmented, negative strand RNA virus with each genome segment being packaged in a distinct ribonucleoprotein particle (RNP). The RNP consists of the heterotrimeric viral RNA-dependent RNA polymerase bound to the conserved 5' and 3' ends of the genome segment (the viral promoter) with the rest of the viral RNA (vRNA) being covered by multiple copies of nucleoprotein. This review focusses on the new insights that recent crystal structures have given into the detailed molecular mechanisms by which the polymerase performs both transcription and replication of the vRNA genome. Promoter binding, in particular that of 5' end, is essential to allosterically activate all polymerase functions. Transcription is initiated by the hijacking of nascent, capped host transcripts by the process of 'cap-snatching', for which the viral polymerase makes an essential interaction with the C-terminal domain (CTD) of cellular RNA polymerase II. The structures allow a coherent mechanistic model of the subsequent cap-snatching, cap-dependent priming, elongation and self-polyadenylation steps of viral mRNA synthesis. During replication, the vRNA is copied without modification into complementary RNA (cRNA) which is packaged into cRNPs. A priming loop located in the polymerase active site is required for the unprimed synthesis of cRNA from vRNA, but is not required for cRNA to vRNA replication due to differences in the mode of initiation of RNA synthesis. Overall a picture emerges of influenza polymerase being a highly complex, flexible and dynamic machine. The challenge remains to understand in more detail how it functions within the RNP and how interacting host factors modulate its activity in the cellular context. Finally, these detailed insights have opened up new opportunities for structure-based antiviral drug design targeting multiple aspects of polymerase function.
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Affiliation(s)
- Alexander Pflug
- European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France
| | - Maria Lukarska
- European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France
| | - Patricia Resa-Infante
- European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France
| | - Stefan Reich
- European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France
| | - Stephen Cusack
- European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
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42
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Gallagher JR, Torian U, McCraw DM, Harris AK. Structural studies of influenza virus RNPs by electron microscopy indicate molecular contortions within NP supra-structures. J Struct Biol 2016; 197:294-307. [PMID: 28007449 DOI: 10.1016/j.jsb.2016.12.007] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/13/2016] [Revised: 11/19/2016] [Accepted: 12/16/2016] [Indexed: 12/29/2022]
Abstract
Ribonucleoprotein (RNP) complexes of influenza viruses are composed of multiple copies of the viral nucleoprotein (NP) that can form filamentous supra-structures. RNPs package distinct viral genomic RNA segments of different lengths into pleomorphic influenza virions. RNPs also function in viral RNA transcription and replication. Different RNP segments have varying lengths, but all must be incorporated into virions during assembly and then released during viral entry for productive infection cycles. RNP structures serve varied functions in the viral replication cycle, therefore understanding their molecular organization and flexibility is essential to understanding these functions. Here, we show using electron tomography and image analyses that isolated RNP filaments are not rigid helical structures, but instead display variations in lengths, curvatures, and even tolerated kinks and local unwinding. Additionally, we observed NP rings within RNP preparations, which were commonly composed of 5, 6, or 7 NP molecules and were of similar widths to filaments, suggesting plasticity in NP-NP interactions mediate RNP structural polymorphism. To demonstrate that NP alone could generate rings of variable oligomeric state, we performed 2D single particle image analysis on recombinant NP and found that rings of 4 and 5 protomers dominated, but rings of all compositions up to 7 were directly observed with variable frequency. This structural flexibility may be needed as RNPs carry out the interactions and conformational changes required for RNP assembly and genome packaging as well as virus uncoating.
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Affiliation(s)
- John R Gallagher
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6351, Bethesda, MD 20892, USA
| | - Udana Torian
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6351, Bethesda, MD 20892, USA
| | - Dustin M McCraw
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6351, Bethesda, MD 20892, USA
| | - Audray K Harris
- Laboratory of Infectious Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 50 South Drive, Room 6351, Bethesda, MD 20892, USA.
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43
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Li J, Zheng W, Hou L, Chen C, Fan W, Qu H, Jiang J, Liu J, Gao GF, Zhou J, Sun L, Liu W. Differential nucleocytoplasmic shuttling of the nucleoprotein of influenza a viruses and association with host tropism. Cell Microbiol 2016; 19. [DOI: 10.1111/cmi.12692] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/04/2016] [Revised: 10/13/2016] [Accepted: 11/02/2016] [Indexed: 01/01/2023]
Affiliation(s)
- Jing Li
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
| | - Weinan Zheng
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
| | - Lidan Hou
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
- China Institute of Veterinary Drug Control; Beijing China
| | - Can Chen
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
- University of Chinese Academy of Sciences; Beijing China
| | - Wenhui Fan
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
| | - Hongren Qu
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
| | - Jingwen Jiang
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
- School of Life Sciences; University of Science and Technology of China; Hefei China
| | - Jinhua Liu
- Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine; China Agricultural University; Beijing China
| | - George F. Gao
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
- University of Chinese Academy of Sciences; Beijing China
- Beijing Institutes of Life Science; Chinese Academy of Sciences; Beijing China
- Office of Director-General; Chinese Center for Disease Control and Prevention; Beijing China
| | - Jiyong Zhou
- College of Veterinary Medicine; Nanjing Agricultural University; Nanjing China
| | - Lei Sun
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
- University of Chinese Academy of Sciences; Beijing China
| | - Wenjun Liu
- CAS Key Laboratory of Pathogenic Microbiology and Immunology; Institute of Microbiology, Chinese Academy of Sciences; Beijing China
- University of Chinese Academy of Sciences; Beijing China
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44
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Kobayashi Y, Dadonaite B, van Doremalen N, Suzuki Y, Barclay WS, Pybus OG. Computational and molecular analysis of conserved influenza A virus RNA secondary structures involved in infectious virion production. RNA Biol 2016; 13:883-94. [PMID: 27399914 DOI: 10.1080/15476286.2016.1208331] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/21/2022] Open
Abstract
As well as encoding viral proteins, genomes of RNA viruses harbor secondary and tertiary RNA structures that have been associated with functions essential for successful replication and propagation. Here, we identified stem-loop structures that are extremely conserved among 1,884 M segment sequences of influenza A virus (IAV) strains from various subtypes and host species using computational and evolutionary methods. These structures were predicted within the 3' and 5' ends of the coding regions of M1 and M2, respectively, where packaging signals have been previously proposed to exist. These signals are thought to be required for the incorporation of a single copy of 8 different negative-strand RNA segments (vRNAs) into an IAV particle. To directly test the functionality of conserved stem-loop structures, we undertook reverse genetic experiments to introduce synonymous mutations designed to disrupt secondary structures predicted at 3 locations and found them to attenuate infectivity of recombinant virus. In one mutant, predicted to disrupt stem loop structure at nucleotide positions 219-240, attenuation was more evident at increased temperature and was accompanied by an increase in the production of defective virus particles. Our results suggest that the conserved secondary structures predicted in the M segment are involved in the production of infectious viral particles during IAV replication.
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Affiliation(s)
- Yuki Kobayashi
- a Nihon University Veterinary Research Center , Fujisawa , Kanagawa , Japan.,b Department of Zoology , University of Oxford , Oxford , UK
| | - Bernadeta Dadonaite
- c Section of Virology, Department of Medicine, Imperial College London , London , UK
| | - Neeltje van Doremalen
- c Section of Virology, Department of Medicine, Imperial College London , London , UK.,d Laboratory of Virology, Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health , Hamilton , MT , USA
| | - Yoshiyuki Suzuki
- e Graduate School of Natural Sciences, Nagoya City University , Nagoya , Japan
| | - Wendy S Barclay
- c Section of Virology, Department of Medicine, Imperial College London , London , UK
| | - Oliver G Pybus
- b Department of Zoology , University of Oxford , Oxford , UK
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Lenartowicz E, Kesy J, Ruszkowska A, Soszynska-Jozwiak M, Michalak P, Moss WN, Turner DH, Kierzek R, Kierzek E. Self-Folding of Naked Segment 8 Genomic RNA of Influenza A Virus. PLoS One 2016; 11:e0148281. [PMID: 26848969 PMCID: PMC4743857 DOI: 10.1371/journal.pone.0148281] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/31/2015] [Accepted: 01/15/2016] [Indexed: 01/10/2023] Open
Abstract
Influenza A is a negative sense RNA virus that kills hundreds of thousands of humans each year. Base pairing in RNA is very favorable, but possibilities for RNA secondary structure of the influenza genomic RNA have not been investigated. This work presents the first experimentally-derived exploration of potential secondary structure in an influenza A naked (protein-free) genomic segment. Favorable folding regions are revealed by in vitro chemical structure mapping, thermodynamics, bioinformatics, and binding to isoenergetic microarrays of an entire natural sequence of the 875 nt segment 8 vRNA and of a smaller fragment. Segment 8 has thermodynamically stable and evolutionarily conserved RNA structure and encodes essential viral proteins NEP and NS1. This suggests that vRNA self-folding may generate helixes and loops that are important at one or more stages of the influenza life cycle.
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Affiliation(s)
- Elzbieta Lenartowicz
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Julita Kesy
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Agnieszka Ruszkowska
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Marta Soszynska-Jozwiak
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Paula Michalak
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Walter N. Moss
- Department of Chemistry, University of Rochester, Rochester, New York, 14627, United States of America
| | - Douglas H. Turner
- Department of Chemistry, University of Rochester, Rochester, New York, 14627, United States of America
| | - Ryszard Kierzek
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
| | - Elzbieta Kierzek
- Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61–704 Poznan, Poland
- * E-mail:
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46
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Jang YH, Jung EJ, Lee KH, Byun YH, Yang SW, Seong BL. Genetic analysis of attenuation markers of cold-adapted X-31 influenza live vaccine donor strain. Vaccine 2016; 34:1343-9. [PMID: 26851733 DOI: 10.1016/j.vaccine.2016.01.053] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/12/2015] [Revised: 01/05/2016] [Accepted: 01/26/2016] [Indexed: 02/05/2023]
Abstract
Cold-adapted live attenuated influenza vaccines (CAIVs) have been considered as a safe prophylactic measure to prevent influenza virus infections. The safety of a CAIV depends largely on genetic markers that confer specific attenuation phenotypes. Previous studies with other CAIVs reported that polymerase genes were primarily responsible for the attenuation. Here, we analyzed the genetic mutations and their phenotypic contribution in the X-31 ca strain, a recently developed alternative CAIV donor strain. During the cold-adaptation of its parental X-31 virus, various numbers of sequence changes were accumulated in all six internal genes. Phenotypic analysis with single-gene and multiple-gene reassortant viruses suggests that NP gene makes the largest contribution to the cold-adapted (ca) and temperature-sensitive (ts) characters, while the remaining other internal genes also impart attenuation characters with varying degrees. A balanced contribution of all internal genes to the attenuation suggests that X-31 ca could serve as an ideal master donor strain for CAIVs preventing influenza epidemics and pandemics.
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Affiliation(s)
- Yo Han Jang
- Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea
| | - Eun-Ju Jung
- Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea
| | - Kwang-Hee Lee
- Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea
| | - Young Ho Byun
- Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea
| | - Seung Won Yang
- Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea
| | - Baik Lin Seong
- Laboratory of Molecular Medicine, Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul, South Korea; Vaccine Translational Research Center, Yonsei University, Seoul, South Korea.
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Codon Deletions in the Influenza A Virus PA Gene Generate Temperature-Sensitive Viruses. J Virol 2016; 90:3684-93. [PMID: 26792748 DOI: 10.1128/jvi.03101-15] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2015] [Accepted: 01/14/2016] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED The influenza virus RNA-dependent RNA polymerase, which is composed of three subunits, PB1, PB2, and PA, catalyzes genome replication and transcription within the cell nucleus. The PA linker (residues 197 to 256) can be altered by nucleotide substitutions to engineer temperature-sensitive (ts), attenuated mutants that display a defect in the transport of the PA-PB1 complex to the nucleus at a restrictive temperature. In this study, we investigated the ability of the PA linker to tolerate deletion mutations for further in vitro and in vivo characterization. Four viable mutants with single-codon deletions were generated; all of them exhibited a ts phenotype that was associated with the reduced efficiency of replication/transcription of a pseudoviral reporter RNA in a minireplicon assay. Using fluorescently tagged PB1, we observed that the deletion mutants did not efficiently recruit PB1 to reach the nucleus at a restrictive temperature (39.5°C). Mouse infections showed that the four mutants were attenuated and induced antibodies that were able to protect mice from challenge with a lethal homologous wild-type virus. Serial in vitro passages of two deletion mutants at 39.5°C and 37°C did not allow the restoration of a wild-type phenotype among virus progeny. Thus, our results identify codons that can be deleted in the PA gene to engineer genetically stable ts mutants that could be used to design novel attenuated vaccines. IMPORTANCE In order to generate genetically stable live influenza A virus vaccines, we constructed viruses with single-codon deletions in a discrete domain of the RNA polymerase PA gene. The four rescued viruses exhibited a temperature-sensitive phenotype that we found was associated with a defect in the transport of the PA-PB1 dimer to the nucleus, where viral replication occurs. These ts deletion mutants were shown to be attenuated and to be able to produce antibodies in mice and to protect them from a lethal challenge. Assays to select revertants that were able to grow efficiently at a restrictive temperature failed, showing that these deletion mutants are genetically more stable than conventional substitution mutants. These results are of interest for the design of genetically stable live influenza virus vaccines.
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48
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Fragile X mental retardation protein stimulates ribonucleoprotein assembly of influenza A virus. Nat Commun 2015; 5:3259. [PMID: 24514761 DOI: 10.1038/ncomms4259] [Citation(s) in RCA: 35] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2013] [Accepted: 01/15/2014] [Indexed: 12/16/2022] Open
Abstract
The ribonucleoprotein (RNP) of the influenza A virus is responsible for the transcription and replication of viral RNA in the nucleus. These processes require interplay between host factors and RNP components. Here, we report that the Fragile X mental retardation protein (FMRP) targets influenza virus RNA synthesis machinery and facilitates virus replication both in cell culture and in mice. We demonstrate that FMRP transiently associates with viral RNP and stimulates viral RNP assembly through RNA-mediated interaction with the nucleoprotein. Furthermore, the KH2 domain of FMRP mediates its association with the nucleoprotein. A point mutation (I304N) in the KH2 domain, identified from a Fragile X syndrome patient, disrupts the FMRP-nucleoprotein association and abolishes the ability of FMRP to participate in viral RNP assembly. We conclude that FMRP is a critical host factor used by influenza viruses to facilitate viral RNP assembly. Our observation reveals a mechanism of influenza virus RNA synthesis and provides insights into FMRP functions.
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Kakisaka M, Sasaki Y, Yamada K, Kondoh Y, Hikono H, Osada H, Tomii K, Saito T, Aida Y. A Novel Antiviral Target Structure Involved in the RNA Binding, Dimerization, and Nuclear Export Functions of the Influenza A Virus Nucleoprotein. PLoS Pathog 2015. [PMID: 26222066 PMCID: PMC4519322 DOI: 10.1371/journal.ppat.1005062] [Citation(s) in RCA: 31] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/01/2023] Open
Abstract
Developing antiviral therapies for influenza A virus (IAV) infection is an ongoing process because of the rapid rate of antigenic mutation and the emergence of drug-resistant viruses. The ideal strategy is to develop drugs that target well-conserved, functionally restricted, and unique surface structures without affecting host cell function. We recently identified the antiviral compound, RK424, by screening a library of 50,000 compounds using cell-based infection assays. RK424 showed potent antiviral activity against many different subtypes of IAV in vitro and partially protected mice from a lethal dose of A/WSN/1933 (H1N1) virus in vivo. Here, we show that RK424 inhibits viral ribonucleoprotein complex (vRNP) activity, causing the viral nucleoprotein (NP) to accumulate in the cell nucleus. In silico docking analysis revealed that RK424 bound to a small pocket in the viral NP. This pocket was surrounded by three functionally important domains: the RNA binding groove, the NP dimer interface, and nuclear export signal (NES) 3, indicating that it may be involved in the RNA binding, oligomerization, and nuclear export functions of NP. The accuracy of this binding model was confirmed in a NP-RK424 binding assay incorporating photo-cross-linked RK424 affinity beads and in a plaque assay evaluating the structure-activity relationship of RK424. Surface plasmon resonance (SPR) and pull-down assays showed that RK424 inhibited both the NP-RNA and NP-NP interactions, whereas size exclusion chromatography showed that RK424 disrupted viral RNA-induced NP oligomerization. In addition, in vitro nuclear export assays confirmed that RK424 inhibited nuclear export of NP. The amino acid residues comprising the NP pocket play a crucial role in viral replication and are highly conserved in more than 7,000 NP sequences from avian, human, and swine influenza viruses. Furthermore, we found that the NP pocket has a surface structure different from that of the pocket in host molecules. Taken together, these results describe a promising new approach to developing influenza virus drugs that target a novel pocket structure within NP. Influenza A virus nucleoprotein (NP) is a highly conserved multifunctional protein that plays an essential role in infection by all subtypes of influenza A virus, making it an attractive target for new antiviral drugs. NP regulates viral polymerase activity and transport of the viral genome into/from the host cell nucleus by forming the viral ribonucleoprotein complex (vRNP). Because NP regulates replication and transcription of the viral genome in addition to its role in nuclear export (all of which are essential for the production of viral progeny), it is a promising drug target. Here, we used the antiviral compound RK424 to identify a novel pocket structure within NP. This structure encompassed three different functional domains that are involved in the above-mentioned replication steps. RK424 inhibits viral genome replication/transcription and nuclear export of NP by destabilizing the NP oligomer and inhibiting the binding of chromosome region maintenance 1 (CRM1) to NP via nuclear export signal (NES) 3, which is located in close proximity to the NP pocket. Taken together, these findings suggest that this small NP pocket is a novel antiviral target.
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Affiliation(s)
| | - Yutaka Sasaki
- Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan
| | - Kazunori Yamada
- Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan
- Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan
| | | | - Hirokazu Hikono
- Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
| | - Hiroyuki Osada
- Chemical Biology Group, RIKEN CSRS, Wako, Saitama, Japan
| | - Kentaro Tomii
- Computational Biology Research Center (CBRC), National Institute of Advanced Industrial Science and Technology (AIST), Koto-ku, Tokyo, Japan
| | - Takehiko Saito
- Influenza and Prion Disease Research Center, National Institute of Animal Health, National Agriculture and Food Research Organization (NARO), Tsukuba, Ibaraki, Japan
| | - Yoko Aida
- Viral Infectious Diseases Unit, RIKEN, Wako, Saitama, Japan
- * E-mail:
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50
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Narkpuk J, Jaru-Ampornpan P, Subali T, Bertulfo FCT, Wongthida P, Jongkaewwattana A. Mechanistic study of intertypic nucleoprotein complex formation and its inhibitory effect toward influenza A virus. Virology 2015. [PMID: 26218215 DOI: 10.1016/j.virol.2015.06.032] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Co-infection of influenza A and B viruses (IAV and IBV) results in marked decreases in IAV replication. Multiple mechanisms have been proposed for this phenomenon. Recently, we reported that IBV nucleoprotein (BNP) alone can suppress IAV replication and proposed an inhibition model in which BNP binds IAV nucleoprotein (ANP) and disrupts IAV polymerase complexes. Here, using mutagenesis and co-immunoprecipitation, we determined the protein motifs mediating the intertypic ANP-BNP complex and showed that it specifically interferes with ANP׳s interaction with the PB2 subunit of the IAV polymerase but not with the other subunit PB1. We further demonstrated that BNP only suppresses growth of IAVs but not other RNA viruses. However, different IAV strains display varied sensitivity toward the BNP׳s inhibitory effect. Together, our data provide mechanistic insights into intertypic nucleoprotein complex formation and highlight the role of BNP as a potential broad-spectrum anti-IAV agent.
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Affiliation(s)
- Jaraspim Narkpuk
- Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand
| | - Peera Jaru-Ampornpan
- Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand.
| | - Theressa Subali
- Faculty of Biotechnology, Atma Jaya Catholic University of Indonesia, Jakarta, Indonesia
| | - Fatima Carla T Bertulfo
- Department of Molecular Biology and Biotechnology, University of the Phillipines Los Banos, Laguna, Phillipines
| | - Phonphimon Wongthida
- Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand
| | - Anan Jongkaewwattana
- Virology and Cell Technology Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Pathumthani, Thailand.
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