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1
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Hanson WA, Romero Agosto GA, Rouskin S. Viral RNA Interactome: The Ultimate Researcher's Guide to RNA-Protein Interactions. Viruses 2024; 16:1702. [PMID: 39599817 PMCID: PMC11599142 DOI: 10.3390/v16111702] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2024] [Revised: 10/18/2024] [Accepted: 10/25/2024] [Indexed: 11/29/2024] Open
Abstract
RNA molecules in the cell are bound by a multitude of RNA-binding proteins (RBPs) with a variety of regulatory consequences. Often, interactions with these RNA-binding proteins are facilitated by the complex secondary and tertiary structures of RNA molecules. Viral RNAs especially are known to be heavily structured and interact with many RBPs, with roles including genome packaging, immune evasion, enhancing replication and transcription, and increasing translation efficiency. As such, the RNA-protein interactome represents a critical facet of the viral replication cycle. Characterization of these interactions is necessary for the development of novel therapeutics targeted at the disruption of essential replication cycle events. In this review, we aim to summarize the various roles of RNA structures in shaping the RNA-protein interactome, the regulatory roles of these interactions, as well as up-to-date methods developed for the characterization of the interactome and directions for novel, RNA-directed therapeutics.
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Affiliation(s)
| | | | - Silvi Rouskin
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA 02115, USA; (W.A.H.); (G.A.R.A.)
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2
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Tapescu I, Cherry S. DDX RNA helicases: key players in cellular homeostasis and innate antiviral immunity. J Virol 2024; 98:e0004024. [PMID: 39212449 PMCID: PMC11494928 DOI: 10.1128/jvi.00040-24] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 09/04/2024] Open
Abstract
RNA helicases are integral in RNA metabolism, performing important roles in cellular homeostasis and stress responses. In particular, the DExD/H-box (DDX) helicase family possesses a conserved catalytic core that binds structural features rather than specific sequences in RNA targets. DDXs have critical roles in all aspects of RNA metabolism including ribosome biogenesis, translation, RNA export, and RNA stability. Importantly, functional specialization within this family arises from divergent N and C termini and is driven at least in part by gene duplications with 18 of the 42 human helicases having paralogs. In addition to their key roles in the homeostatic control of cellular RNA, these factors have critical roles in RNA virus infection. The canonical RIG-I-like receptors (RLRs) play pivotal roles in cytoplasmic sensing of viral RNA structures, inducing antiviral gene expression. Additional RNA helicases function as viral sensors or regulators, further diversifying the innate immune defense arsenal. Moreover, some of these helicases have been coopted by viruses to facilitate their replication. Altogether, DDX helicases exhibit functional specificity, playing intricate roles in RNA metabolism and host defense. This review will discuss the mechanisms by which these RNA helicases recognize diverse RNA structures in cellular and viral RNAs, and how this impacts RNA processing and innate immune responses.
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Affiliation(s)
- Iulia Tapescu
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
- Biochemistry and Biophysics Graduate Group, University of Pennsylvania, Philadelphia, Pennsylvania, USA
| | - Sara Cherry
- Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania, USA
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3
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Cousineau SE, Camargo C, Sagan SM. Poly(rC)-Binding Protein 2 Does Not Directly Participate in HCV Translation or Replication, but Rather Modulates Genome Packaging. Viruses 2024; 16:1220. [PMID: 39205194 PMCID: PMC11359930 DOI: 10.3390/v16081220] [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: 07/05/2024] [Revised: 07/22/2024] [Accepted: 07/26/2024] [Indexed: 09/04/2024] Open
Abstract
The hepatitis C virus (HCV) co-opts many cellular factors-including proteins and microRNAs-to complete its life cycle. A cellular RNA-binding protein, poly(rC)-binding protein 2 (PCBP2), was previously shown to bind to the hepatitis C virus (HCV) genome; however, its precise role in the viral life cycle remained unclear. Herein, using the HCV cell culture (HCVcc) system and assays that isolate each step of the viral life cycle, we found that PCBP2 does not have a direct role in viral entry, translation, genome stability, or HCV RNA replication. Rather, our data suggest that PCBP2 depletion only impacts viral RNAs that can undergo genome packaging. Taken together, our data suggest that endogenous PCBP2 modulates the early steps of genome packaging, and therefore only has an indirect effect on viral translation and RNA replication, likely by increasing the translating/replicating pool of viral RNAs to the detriment of virion assembly.
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Affiliation(s)
- Sophie E. Cousineau
- Department of Microbiology & Immunology, McGill University, Montreal, QC H3A 2B4, Canada
| | - Carolina Camargo
- Department of Microbiology & Immunology, University of British Columbia, 2350 Health Science Mall, Room 4.520, Vancouver, BC V6T 1Z3, Canada
| | - Selena M. Sagan
- Department of Microbiology & Immunology, McGill University, Montreal, QC H3A 2B4, Canada
- Department of Microbiology & Immunology, University of British Columbia, 2350 Health Science Mall, Room 4.520, Vancouver, BC V6T 1Z3, Canada
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4
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Wang H, Li W, Zheng SJ. Advances on Innate Immune Evasion by Avian Immunosuppressive Viruses. Front Immunol 2022; 13:901913. [PMID: 35634318 PMCID: PMC9133627 DOI: 10.3389/fimmu.2022.901913] [Citation(s) in RCA: 12] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/22/2022] [Accepted: 04/19/2022] [Indexed: 01/12/2023] Open
Abstract
Innate immunity is not only the first line of host defense against pathogenic infection, but also the cornerstone of adaptive immune response. Upon pathogenic infection, pattern recognition receptors (PRRs) of host engage pathogen-associated molecular patterns (PAMPs) of pathogens, which initiates IFN production by activating interferon regulatory transcription factors (IRFs), nuclear factor-kappa B (NF-κB), and/or activating protein-1 (AP-1) signal transduction pathways in host cells. In order to replicate and survive, pathogens have evolved multiple strategies to evade host innate immune responses, including IFN-I signal transduction, autophagy, apoptosis, necrosis, inflammasome and/or metabolic pathways. Some avian viruses may not be highly pathogenic but they have evolved varied strategies to evade or suppress host immune response for survival, causing huge impacts on the poultry industry worldwide. In this review, we focus on the advances on innate immune evasion by several important avian immunosuppressive viruses (infectious bursal disease virus (IBDV), Marek’s disease virus (MDV), avian leukosis virus (ALV), etc.), especially their evasion of PRRs-mediated signal transduction pathways (IFN-I signal transduction pathway) and IFNAR-JAK-STAT signal pathways. A comprehensive understanding of the mechanism by which avian viruses evade or suppress host immune responses will be of help to the development of novel vaccines and therapeutic reagents for the prevention and control of infectious diseases in chickens.
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Affiliation(s)
- Hongnuan Wang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Wei Li
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Shijun J. Zheng
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture, College of Veterinary Medicine, China Agricultural University, Beijing, China
- Department of Preventive Veterinary Medicine, College of Veterinary Medicine, China Agricultural University, Beijing, China
- *Correspondence: Shijun J. Zheng,
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5
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DEAD/H-box helicases:Anti-viral and pro-viral roles during infections. Virus Res 2021; 309:198658. [PMID: 34929216 DOI: 10.1016/j.virusres.2021.198658] [Citation(s) in RCA: 33] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2021] [Revised: 11/26/2021] [Accepted: 12/14/2021] [Indexed: 02/08/2023]
Abstract
DEAD/H-box RNA helicases make the prominent family of helicases super family-2 which take part in almost all RNA-related processes, from initiation of transcription to RNA decay pathways. In addition to these RNA-related activities, in recent years a certain number of these helicases are reported to play important roles in anti-viral immunity through various ways. Along with RLHs, endosomal TLRs, and cytosolic DNA receptors, many RNA helicases including DDX3, DHX9, DDX6, DDX41, DHX33, DDX60, DHX36 and DDX1-DDX21-DHX36 complex act as viral nucleic acid sensors or co-sensors. These helicases mostly follow RLHs-MAVS and STING mediated signaling cascades to trigger induction of type-I interferons and pro-inflammatory cytokines. Many of them also function as downstream adaptor molecules (DDX3), segments of stress and processing bodies (DDX3 and DDX6) or negative regulators (DDX19, DDX24, DDX25, DDX39A and DDX46). On the contrary, many studies indicated that several DEAD/H-box helicases such as DDX1, DDX3, DDX6, DDX24, and DHX9 could be exploited by viruses to evade innate immune responses, suggesting that these helicases seem to have a dual function as anti-viral innate immune mediators and viral replication cofactors. In this review, we summarized the current knowledge on several representative DEAD/H-box helicases, with an emphasis on their functions in innate immunity responses, involved in their anti-viral and pro-viral roles.
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Abstract
RNA viruses cause many routine illnesses, such as the common cold and the flu. Recently, more deadly diseases have emerged from this family of viruses. The hepatitis C virus has had a devastating impact worldwide. Despite the cures developed in the U.S. and Europe, economically disadvantaged countries remain afflicted by HCV infection due to the high cost of these medications. More recently, COVID-19 has swept across the world, killing millions and disrupting economies and lifestyles; the virus responsible for this pandemic is a coronavirus. Our understanding of HCV and SARS CoV-2 replication is still in its infancy. Helicases play a critical role in the replication, transcription and translation of viruses. These key enzymes need extensive study not only as an essential player in the viral lifecycle, but also as targets for antiviral therapeutics. In this review, we highlight the current knowledge for RNA helicases of high importance to human health.
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Affiliation(s)
- John C Marecki
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Binyam Belachew
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Jun Gao
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States
| | - Kevin D Raney
- Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR, United States.
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7
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Function of Host Protein Staufen1 in Rabies Virus Replication. Viruses 2021; 13:v13081426. [PMID: 34452292 PMCID: PMC8402631 DOI: 10.3390/v13081426] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/12/2021] [Revised: 07/15/2021] [Accepted: 07/20/2021] [Indexed: 12/14/2022] Open
Abstract
Rabies virus is a highly neurophilic negative-strand RNA virus with high lethality and remains a huge public health problem in developing countries to date. The double-stranded RNA-binding protein Staufen1 (STAU1) has multiple functions in RNA virus replication, transcription, and translation. However, its function in RABV infection and its mechanism of action are not clear. In this study, we investigated the role of host factor STAU1 in RABV infection of SH-SY-5Y cells. Immunofluorescence, TCID50 titers, confocal microscopy, quantitative real-time PCR and Western blotting were carried out to determine the molecular function and subcellular distribution of STAU1 in these cell lines. Expression of STAU1 in SH-SY-5Y cells was down-regulated by RNA interference or up-regulated by transfection of eukaryotic expression vectors. The results showed that N proficiently colocalized with STAU1 in SH-SY-5Y at 36 h post-infection, and the expression level of STAU1 was also proportional to the time of infection. Down-regulation of STAU1 expression increased the number of Negri body-like structures, enhanced viral replication, and a caused 10-fold increase in viral titers. Meanwhile, N protein and G protein mRNA levels also accumulated gradually with increasing infection time, which implied that STAU1 inhibited rabies virus infection of SH-SY-5Y cells in vitro. In conclusion, our results provide important clues for the detailed replication mechanism of rabies virus and the discovery of therapeutic targets.
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8
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Ali MAM. DEAD-box RNA helicases: The driving forces behind RNA metabolism at the crossroad of viral replication and antiviral innate immunity. Virus Res 2021; 296:198352. [PMID: 33640359 DOI: 10.1016/j.virusres.2021.198352] [Citation(s) in RCA: 26] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2020] [Revised: 02/22/2021] [Accepted: 02/22/2021] [Indexed: 02/07/2023]
Abstract
DEAD-box RNA helicases, the largest family of superfamily 2 helicases, are a profoundly conserved family of RNA-binding proteins, containing a distinctive Asp-Glu-Ala-Asp (D-E-A-D) sequence motif, which is the origin of their name. Aside from the ATP-dependent unwinding of RNA duplexes, which set up these proteins as RNA helicases, DEAD-box proteins have been found to additionally stimulate RNA duplex fashioning and to uproot proteins from RNA, aiding the reformation of RNA and RNA-protein complexes. There is accumulating evidence that DEAD-box helicases play functions in the recognition of foreign nucleic acids and the modification of viral infection. As intracellular parasites, viruses must avoid identification by innate immune sensing mechanisms and disintegration by cellular machinery, whilst additionally exploiting host cell activities to assist replication. The capability of DEAD-box helicases to sense RNA in a sequence-independent way, as well as the broadness of cellular roles performed by members of this family, drive them to affect innate sensing and viral infections in numerous manners. Undoubtedly, DEAD-box helicases have been demonstrated to contribute to intracellular immune recognition, function as antiviral effectors, and even to be exploited by viruses to support their replication. Relying on the virus or the viral cycle phase, a DEAD-box helicase can function either in a proviral manner or as an antiviral factor. This review gives a comprehensive perspective on the various biochemical characteristics of DEAD-box helicases and their links to structural data. It additionally outlines the multiple functions that members of the DEAD-box helicase family play during viral infections.
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Affiliation(s)
- Mohamed A M Ali
- Department of Biochemistry, Faculty of Science, Ain Shams University, Abbassia, 11566, Cairo, Egypt.
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9
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Zhang H, Song X, Li T, Wang J, Xing B, Zhai X, Luo J, Hu X, Hou X, Wei L. DDX1 from Cherry valley duck mediates signaling pathways and anti-NDRV activity. Vet Res 2021; 52:9. [PMID: 33472667 PMCID: PMC7816157 DOI: 10.1186/s13567-020-00889-4] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/14/2020] [Accepted: 12/29/2020] [Indexed: 11/23/2022] Open
Abstract
Novel duck reovirus (NDRV) causes severe economic losses to the duck industry, which is characterized by hemorrhagic spots and necrotic foci of the livers and spleens. DEAD-box helicase 1 (DDX1) plays a critical role in the innate immune system against viral infection. However, the role of duck DDX1 (duDDX1) in anti-RNA virus infection, especially in the anti-NDRV infection, has yet to be elucidated. In the present study, the full-length cDNA of duDDX1 (2223 bp encode 740 amino acids) was firstly cloned from the spleen of healthy Cherry valley ducks, and the phylogenetic tree indicated that the duDDX1 has the closest relationship with Anas platyrhynchos in the bird branch. The duDDX1 mRNA was widely distributed in all tested tissues, especially in the duodenum, liver, and spleen. Overexpression of duDDX1 in primary duck embryo fibroblast (DEF) cells triggered the activation of transcription factors IRF-7 and NF-κB, as well as IFN-β expression, and the expression of the Toll-like receptors (TLR2, TLR3, and TLR4) was significantly increased. Importantly, after overexpressing or knocking down duDDX1 and infecting NDRV in DEF cells, duDDX1 inhibits the replication of NDRV virus and also regulates the expression of pattern recognition receptors and cytokines. This indicates that duDDX1 may play an important role in the innate immune response of ducks to NDRV. Collectively, we first cloned DDX1 from ducks and analyzed its biological functions. Secondly, we proved that duck DDX1 participates in anti-NDRV infection, and innovated new ideas for the prevention and control of duck virus infection.
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Affiliation(s)
- Huihui Zhang
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Xingdong Song
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Tianxu Li
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Jinchao Wang
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Bin Xing
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Xinyu Zhai
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Jinjian Luo
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Xiaofang Hu
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Xiaolan Hou
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China
| | - Liangmeng Wei
- Sino-German Cooperative Research Centre for Zoonosis of Animal Origin of Shandong Province, Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, College of Animal Science and Veterinary Medicine, Shandong Agricultural University, 61 Daizong Road, Tai'an, 271018, Shandong Province, China. .,Collaborative Innovation Center for the Origin and Control of Emerging Infectious Diseases, College of Basic Medical Sciences, Shandong First Medical University, Tai'an, 271000, Shandong Province, China.
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10
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Human DDX3X Unwinds Japanese Encephalitis and Zika Viral 5' Terminal Regions. Int J Mol Sci 2021; 22:ijms22010413. [PMID: 33401776 PMCID: PMC7795613 DOI: 10.3390/ijms22010413] [Citation(s) in RCA: 12] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/30/2020] [Revised: 12/26/2020] [Accepted: 12/29/2020] [Indexed: 12/27/2022] Open
Abstract
Flavivirus genus includes many deadly viruses such as the Japanese encephalitis virus (JEV) and Zika virus (ZIKV). The 5' terminal regions (TR) of flaviviruses interact with human proteins and such interactions are critical for viral replication. One of the human proteins identified to interact with the 5' TR of JEV is the DEAD-box helicase, DDX3X. In this study, we in vitro transcribed the 5' TR of JEV and demonstrated its direct interaction with recombinant DDX3X (Kd of 1.66 ± 0.21 µM) using microscale thermophoresis (MST). Due to the proposed structural similarities of 5' and 3' TRs of flaviviruses, we investigated if the ZIKV 5' TR could also interact with human DDX3X. Our MST studies suggested that DDX3X recognizes ZIKV 5' TR with a Kd of 7.05 ± 0.75 µM. Next, we performed helicase assays that suggested that the binding of DDX3X leads to the unwinding of JEV and ZIKV 5' TRs. Overall, our data indicate, for the first time, that DDX3X can directly bind and unwind in vitro transcribed flaviviral TRs. In summary, our work indicates that DDX3X could be further explored as a therapeutic target to inhibit Flaviviral replication.
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11
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Analytical ultracentrifuge: an ideal tool for characterization of non-coding RNAs. EUROPEAN BIOPHYSICS JOURNAL: EBJ 2020; 49:809-818. [PMID: 33067686 DOI: 10.1007/s00249-020-01470-9] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/13/2020] [Revised: 09/26/2020] [Accepted: 10/05/2020] [Indexed: 12/25/2022]
Abstract
Analytical ultracentrifugation (AUC) has emerged as a robust and reliable technique for biomolecular characterization with extraordinary sensitivity. AUC is widely used to study purity, conformational changes, biomolecular interactions, and stoichiometry. Furthermore, AUC is used to determine the molecular weight of biomolecules such as proteins, carbohydrates, and DNA and RNA. Due to the multifaceted role(s) of non-coding RNAs from viruses, prokaryotes, and eukaryotes, research aimed at understanding the structure-function relationships of non-coding RNAs is rapidly increasing. However, due to their large size, flexibility, complicated secondary structures, and conformations, structural studies of non-coding RNAs are challenging. In this review, we are summarizing the application of AUC to evaluate the homogeneity, interactions, and conformational changes of non-coding RNAs from adenovirus as well as from Murray Valley, Powassan, and West Nile viruses. We also discuss the application of AUC to characterize eukaryotic long non-coding RNAs, Xist, and HOTAIR. These examples highlight the significant role AUC can play in facilitating the structural determination of non-coding RNAs and their complexes.
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12
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Liu Y, Zhang Y, Wang M, Cheng A, Yang Q, Wu Y, Jia R, Liu M, Zhu D, Chen S, Zhang S, Zhao X, Huang J, Mao S, Ou X, Gao Q, Wang Y, Xu Z, Chen Z, Zhu L, Luo Q, Liu Y, Yu Y, Zhang L, Tian B, Pan L, Chen X. Structures and Functions of the 3' Untranslated Regions of Positive-Sense Single-Stranded RNA Viruses Infecting Humans and Animals. Front Cell Infect Microbiol 2020; 10:453. [PMID: 32974223 PMCID: PMC7481400 DOI: 10.3389/fcimb.2020.00453] [Citation(s) in RCA: 23] [Impact Index Per Article: 4.6] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/20/2020] [Accepted: 07/23/2020] [Indexed: 12/20/2022] Open
Abstract
The 3′ untranslated region (3′ UTR) of positive-sense single-stranded RNA [ssRNA(+)] viruses is highly structured. Multiple elements in the region interact with other nucleotides and proteins of viral and cellular origin to regulate various aspects of the virus life cycle such as replication, translation, and the host-cell response. This review attempts to summarize the primary and higher order structures identified in the 3′UTR of ssRNA(+) viruses and their functional roles.
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Affiliation(s)
- Yuanzhi Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Yu Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Mingshu Wang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Anchun Cheng
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Qiao Yang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Ying Wu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Renyong Jia
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Mafeng Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Dekang Zhu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Shun Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Shaqiu Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - XinXin Zhao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Juan Huang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Sai Mao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Xumin Ou
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Qun Gao
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Yin Wang
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Zhiwen Xu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Zhengli Chen
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Ling Zhu
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Qihui Luo
- Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China
| | - Yunya Liu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Yanling Yu
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Ling Zhang
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Bin Tian
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Leichang Pan
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
| | - Xiaoyue Chen
- Institute of Preventive Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, China.,Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China
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13
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Mrozowich T, Henrickson A, Demeler B, Patel TR. Nanoscale Structure Determination of Murray Valley Encephalitis and Powassan Virus Non-Coding RNAs. Viruses 2020; 12:E190. [PMID: 32046304 PMCID: PMC7077200 DOI: 10.3390/v12020190] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2020] [Revised: 02/05/2020] [Accepted: 02/06/2020] [Indexed: 01/02/2023] Open
Abstract
Viral infections are responsible for numerous deaths worldwide. Flaviviruses, which contain RNA as their genetic material, are one of the most pathogenic families of viruses. There is an increasing amount of evidence suggesting that their 5' and 3' non-coding terminal regions are critical for their survival. Information on their structural features is essential to gain detailed insights into their functions and interactions with host proteins. In this study, the 5' and 3' terminal regions of Murray Valley encephalitis virus and Powassan virus were examined using biophysical and computational modeling methods. First, we used size exclusion chromatography and analytical ultracentrifuge methods to investigate the purity of in-vitro transcribed RNAs. Next, we employed small-angle X-ray scattering techniques to study solution conformation and low-resolution structures of these RNAs, which suggest that the 3' terminal regions are highly extended as compared to the 5' terminal regions for both viruses. Using computational modeling tools, we reconstructed 3-dimensional structures of each RNA fragment and compared them with derived small-angle X-ray scattering low-resolution structures. This approach allowed us to reinforce that the 5' terminal regions adopt more dynamic structures compared to the mainly double-stranded structures of the 3' terminal regions.
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Affiliation(s)
- Tyler Mrozowich
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; (T.M.); (A.H.); (B.D.)
| | - Amy Henrickson
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; (T.M.); (A.H.); (B.D.)
| | - Borries Demeler
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; (T.M.); (A.H.); (B.D.)
- Department of Chemistry And Biochemistry, University of Montana, Missoula, MT 59812, USA
- NorthWest Biophysics Consortium, University of Lethbridge, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada
| | - Trushar R Patel
- Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada; (T.M.); (A.H.); (B.D.)
- NorthWest Biophysics Consortium, University of Lethbridge, University of Lethbridge, 4401 University Drive, Lethbridge, AB T1K 3M4, Canada
- Department of Microbiology, Immunology and Infectious Disease, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
- Li Ka Shing Institute of Virology and Discovery Lab, University of Alberta, Edmonton, AB T6G 2E1, Canada
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14
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Kaur R, Lal SK. The multifarious roles of heterogeneous ribonucleoprotein A1 in viral infections. Rev Med Virol 2020; 30:e2097. [PMID: 31989716 PMCID: PMC7169068 DOI: 10.1002/rmv.2097] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/11/2019] [Revised: 12/19/2019] [Accepted: 12/30/2019] [Indexed: 12/13/2022]
Abstract
Viruses are obligate parasites known to interact with a wide variety of host proteins at different stages of infection. Current antiviral treatments target viral proteins and may be compromised due to the emergence of drug resistant viral strains. Targeting viral-host interactions is now gaining recognition as an alternative approach against viral infections. Recent research has revealed that heterogeneous ribonucleoprotein A1, an RNA-binding protein, plays an essential functional and regulatory role in the life cycle of many viruses. In this review, we summarize the interactions between heterogeneous ribonucleoprotein A1 (hnRNPA1) and multiple viral proteins during the life cycle of RNA and DNA viruses. hnRNPA1 protein levels are modulated differently, in different viruses, which further dictates its stability, function, and intracellular localization. Multiple reports have emphasized that in Sindbis virus, enteroviruses, porcine endemic diarrhea virus, and rhinovirus infection, hnRNPA1 enhances viral replication and survival. However, in others like hepatitis C virus and human T-cell lymphotropic virus, it exerts a protective response. The involvement of hnRNPA1 in viral infections highlights its importance as a central regulator of host and viral gene expression. Understanding the nature of these interactions will increase our understanding of specific viral infections and pathogenesis and eventually aid in the development of novel and robust antiviral intervention strategies.
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Affiliation(s)
- Ramandeep Kaur
- Tropical Medicine and Biology Platform & School of Science, Monash University, 47500 Bandar Sunway, Selangor DE, Malaysia
| | - Sunil K Lal
- Tropical Medicine and Biology Platform & School of Science, Monash University, 47500 Bandar Sunway, Selangor DE, Malaysia
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15
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Staufen1 Protein Participates Positively in the Viral RNA Replication of Enterovirus 71. Viruses 2019; 11:v11020142. [PMID: 30744035 PMCID: PMC6409738 DOI: 10.3390/v11020142] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2018] [Revised: 01/29/2019] [Accepted: 02/06/2019] [Indexed: 12/28/2022] Open
Abstract
The double-stranded RNA-binding protein Staufen1 (Stau1) has multiple functions during RNA virus infection. In this study, we investigated the role of Stau1 in viral translation by using a combination of enterovirus 71 (EV-A71) infection, RNA reporter transfection, and in vitro functional and biochemical assays. We demonstrated that Stau1 specifically binds to the 5′-untranslated region of EV-A71 viral RNA. The RNA-binding domain 2-3 of Stau1 is responsible for this binding ability. Subsequently, we created a Stau1 knockout cell line using the CRISPR/Cas9 approach to further characterize the functional role of Stau1’s interaction with viral RNA in the EV-A71-infected cells. Both the viral RNA accumulation and viral protein expression were downregulated in the Stau1 knockout cells compared with the wild-type naïve cells. Moreover, dysregulation of viral RNA translation was observed in the Stau1 knockout cells using ribosome fractionation assay, and a reduced RNA stability of 5′-UTR of the EV-A71 was also identified using an RNA stability assay, which indicated that Stau1 has a role in facilitating viral translation during EV-A71 infection. In conclusion, we determined the functional relevance of Stau1 in the EV-A71 infection cycle and herein describe the mechanism of Stau1 participation in viral RNA translation through its interaction with viral RNA. Our results suggest that Stau1 is an important host factor involved in viral translation and influential early in the EV-A71 replication cycle.
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16
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Rasputin a decade on and more promiscuous than ever? A review of G3BPs. BIOCHIMICA ET BIOPHYSICA ACTA-MOLECULAR CELL RESEARCH 2018; 1866:360-370. [PMID: 30595162 PMCID: PMC7114234 DOI: 10.1016/j.bbamcr.2018.09.001] [Citation(s) in RCA: 48] [Impact Index Per Article: 6.9] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/24/2018] [Revised: 08/29/2018] [Accepted: 09/04/2018] [Indexed: 12/12/2022]
Abstract
Ras-GTPase-activating protein (SH3 domain)-binding proteins (G3BPs, also known as Rasputin) are a family of RNA binding proteins that regulate gene expression in response to environmental stresses by controlling mRNA stability and translation. G3BPs appear to facilitate this activity through their role in stress granules for which they are considered a core component, however, it should be noted that not all stress granules contain G3BPs and this appears to be contextual depending on the environmental stress and the cell type. Although the role of G3BPs in stress granules appears to be one of its major roles, data also strongly suggests that they interact with mRNAs outside of stress granules to regulate gene expression. G3BPs have been implicated in several diseases including cancer progression, invasion, and metastasis as well as virus survival. There is now a body of evidence that suggests targeting of G3BPs could be explored as a form of cancer therapeutic. This review discusses the important discoveries and advancements made in the field of G3BPs biology over the last two decades including their roles in RNA stability, translational control of cellular transcripts, stress granule formation, cancer progression and its interactions with viruses during infection. An emerging theme for G3BPs is their ability to regulate gene expression in response to environmental stimuli, disease progression and virus infection making it an intriguing target for disease therapies.
Triage of many cellular mRNA occurs via stress granules in a G3BP-dependant manner. G3BPs control intra cellular responses to viral infection. Transcript stability, degradation and translation are controlled by G3BPs. G3BPs can control cancer progression.
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17
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Meier-Stephenson V, Mrozowich T, Pham M, Patel TR. DEAD-box helicases: the Yin and Yang roles in viral infections. Biotechnol Genet Eng Rev 2018; 34:3-32. [PMID: 29742983 DOI: 10.1080/02648725.2018.1467146] [Citation(s) in RCA: 29] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022]
Abstract
Viruses hijack the host cell machinery and recruit host proteins to aid their replication. Several host proteins also play vital roles in inhibiting viral replication. Emerging class of host proteins central to both of these processes are the DEAD-box helicases: a highly conserved family of ATP-dependent RNA helicases, bearing a common D-E-A-D (Asp-Glu-Ala-Asp) motif. They play key roles in numerous cellular processes, including transcription, splicing, miRNA biogenesis and translation. Though their sequences are highly conserved, these helicases have quite diverse roles in the cell. Interestingly, often these helicases display contradictory actions in terms of the support and/or clearance of invading viruses. Increasing evidence highlights the importance of these enzymes, however, little is known about the structural basis of viral RNA recognition by the members of the DEAD-box family. This review summarizes the current knowledge in the field for selected DEAD-box helicases and highlights their diverse actions upon viral invasion of the host cell. We anticipate that through a better understanding of how these helicases are being utilized by viral RNAs and proteins to aid viral replication, it will be possible to address the urgent need to develop novel therapeutic approaches to combat viral infections.
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Affiliation(s)
- Vanessa Meier-Stephenson
- a Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute , University of Lethbridge , Lethbridge , Canada.,b Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine , University of Calgary , Calgary , Canada
| | - Tyler Mrozowich
- a Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute , University of Lethbridge , Lethbridge , Canada
| | - Mimi Pham
- a Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute , University of Lethbridge , Lethbridge , Canada
| | - Trushar R Patel
- a Department of Chemistry and Biochemistry, Alberta RNA Research and Training Institute , University of Lethbridge , Lethbridge , Canada.,b Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine , University of Calgary , Calgary , Canada.,c Faculty of Medicine & Dentistry, DiscoveryLab , University of Alberta , Edmonton , Canada
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18
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Niepmann M, Shalamova LA, Gerresheim GK, Rossbach O. Signals Involved in Regulation of Hepatitis C Virus RNA Genome Translation and Replication. Front Microbiol 2018; 9:395. [PMID: 29593672 PMCID: PMC5857606 DOI: 10.3389/fmicb.2018.00395] [Citation(s) in RCA: 31] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/09/2018] [Accepted: 02/21/2018] [Indexed: 12/15/2022] Open
Abstract
Hepatitis C virus (HCV) preferentially replicates in the human liver and frequently causes chronic infection, often leading to cirrhosis and liver cancer. HCV is an enveloped virus classified in the genus Hepacivirus in the family Flaviviridae and has a single-stranded RNA genome of positive orientation. The HCV RNA genome is translated and replicated in the cytoplasm. Translation is controlled by the Internal Ribosome Entry Site (IRES) in the 5' untranslated region (5' UTR), while also downstream elements like the cis-replication element (CRE) in the coding region and the 3' UTR are involved in translation regulation. The cis-elements controlling replication of the viral RNA genome are located mainly in the 5'- and 3'-UTRs at the genome ends but also in the protein coding region, and in part these signals overlap with the signals controlling RNA translation. Many long-range RNA-RNA interactions (LRIs) are predicted between different regions of the HCV RNA genome, and several such LRIs are actually involved in HCV translation and replication regulation. A number of RNA cis-elements recruit cellular RNA-binding proteins that are involved in the regulation of HCV translation and replication. In addition, the liver-specific microRNA-122 (miR-122) binds to two target sites at the 5' end of the viral RNA genome as well as to at least three additional target sites in the coding region and the 3' UTR. It is involved in the regulation of HCV RNA stability, translation and replication, thereby largely contributing to the hepatotropism of HCV. However, we are still far from completely understanding all interactions that regulate HCV RNA genome translation, stability, replication and encapsidation. In particular, many conclusions on the function of cis-elements in HCV replication have been obtained using full-length HCV genomes or near-full-length replicon systems. These include both genome ends, making it difficult to decide if a cis-element in question acts on HCV replication when physically present in the plus strand genome or in the minus strand antigenome. Therefore, it may be required to use reduced systems that selectively focus on the analysis of HCV minus strand initiation and/or plus strand initiation.
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Affiliation(s)
- Michael Niepmann
- Medical Faculty, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
| | - Lyudmila A Shalamova
- Medical Faculty, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany.,Faculty of Biology and Chemistry, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
| | - Gesche K Gerresheim
- Medical Faculty, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany.,Faculty of Biology and Chemistry, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
| | - Oliver Rossbach
- Faculty of Biology and Chemistry, Institute of Biochemistry, Justus Liebig University Giessen, Giessen, Germany
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19
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Shobahah J, Xue S, Hu D, Zhao C, Wei M, Quan Y, Yu W. Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus. Virol J 2017. [PMID: 28629377 PMCID: PMC5477107 DOI: 10.1186/s12985-017-0783-8] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
Background Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. Method Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. Results Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. Conclusion The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.
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Affiliation(s)
- Jauharotus Shobahah
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China
| | - Shengjie Xue
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China
| | - Dongbing Hu
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China
| | - Cui Zhao
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China
| | - Ming Wei
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China
| | - Yanping Quan
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China.,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China
| | - Wei Yu
- Institute of Biochemistry, College of Life Sciences, Zhejiang Sci-Tech University, Xiasha High-Tech Zone No.2 Road, Zhejiang Province, Hangzhou, 310018, People's Republic of China. .,Zhejiang Provincial Key Laboratory of Silkworm Bioreactor and Biomedicine, Zhejiang Province, Hangzhou, 310018, People's Republic of China.
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20
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Rösch K, Kwiatkowski M, Schlüter H, Herker E. Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis. J Vis Exp 2017. [PMID: 28448054 DOI: 10.3791/55585] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/24/2022] Open
Abstract
Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with "heavy" amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e. HCV-infected/"light" amino acids and uninfected control/"heavy" amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.
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Affiliation(s)
- Kathrin Rösch
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology
| | - Marcel Kwiatkowski
- Core Facility Mass Spectrometric Proteomics, University Medical Center Hamburg-Eppendorf
| | - Hartmut Schlüter
- Core Facility Mass Spectrometric Proteomics, University Medical Center Hamburg-Eppendorf
| | - Eva Herker
- Heinrich Pette Institute, Leibniz Institute for Experimental Virology;
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21
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Jiang Y, Zhu Y, Liu ZJ, Ouyang S. The emerging roles of the DDX41 protein in immunity and diseases. Protein Cell 2017; 8:83-89. [PMID: 27502187 PMCID: PMC5291771 DOI: 10.1007/s13238-016-0303-4] [Citation(s) in RCA: 75] [Impact Index Per Article: 9.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2016] [Accepted: 07/14/2016] [Indexed: 12/12/2022] Open
Abstract
RNA helicases are involved in almost every aspect of RNA, from transcription to RNA decay. DExD/H-box helicases comprise the largest SF2 helicase superfamily, which are characterized by two conserved RecA-like domains. In recent years, an increasing number of unexpected functions of these proteins have been discovered. They play important roles not only in innate immune response but also in diseases like cancers and chronic hepatitis C. In this review, we summarize the recent literatures on one member of the SF2 superfamily, the DEAD-box protein DDX41. After bacterial or viral infection, DNA or cyclic-di-GMP is released to cells. After phosphorylation of Tyr414 by BTK kinase, DDX41 will act as a sensor to recognize the invaders, followed by induction of type I interferons (IFN). After the immune response, DDX41 is degraded by the E3 ligase TRIM21, using Lys9 and Lys115 of DDX41 as the ubiquitination sites. Besides the roles in innate immunity, DDX41 is also related to diseases. An increasing number of both inherited and acquired mutations in DDX41 gene are identified from myelodysplastic syndrome and/or acute myeloid leukemia (MDS/AML) patients. The review focuses on DDX41, as well as its homolog Abstrakt in Drosophila, which is important for survival at all stages throughout the life cycle of the fly.
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Affiliation(s)
- Yan Jiang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Yanping Zhu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
| | - Zhi-Jie Liu
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China
- iHuman Institute, Shanghai Tech University, Shanghai, 201210, China
| | - Songying Ouyang
- National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, 100101, China.
- Department of Microbiology, Immunology and Molecular Genetics, University of California Los Angeles, Los Angeles, CA, 90095, USA.
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22
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Zhang X, Hua L, Yan D, Zhao F, Liu J, Zhou H, Liu J, Wu M, Zhang C, Chen Y, Chen B, Hu B. Overexpression of PCBP2 contributes to poor prognosis and enhanced cell growth in human hepatocellular carcinoma. Oncol Rep 2016; 36:3456-3464. [PMID: 27748915 DOI: 10.3892/or.2016.5167] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/25/2016] [Accepted: 06/06/2016] [Indexed: 11/06/2022] Open
Abstract
Poly(C)‑binding protein 2 (PCBP2) is a member of the PCBP family, and plays an important role in post‑transcriptional and translational regulation of various signaling molecules through direct binding to single‑stranded poly(C) motifs. PCBP2 has been reported to play a critical role in the development of multiple human tumors. However, whether PCBP2 participates in hepatocellular carcinoma (HCC) development remains largely elusive. Herein, we showed that PCBP2 was upregulated in human HCC tissues and cell lines. Overexpression of PCBP2 predicted significantly worsened prognosis in HCC patients, suggesting that PCBP2 may serve as a prognostic marker of HCC. In addition, we found that depletion of PCBP2 inhibited HCC cell proliferation, accompanying the increase in the cyclin‑dependent kinase inhibitor p27 level. Moreover, we found that high expression of PCBP2 may contribute to sorafenib resistance in HCC cells, involving dysregulated expression of Bax and Bcl‑2 proteins. In conclusion, our results suggest that PCBP2 may serve as a prognostic marker and potential therapeutic target of HCC.
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Affiliation(s)
- Xiubing Zhang
- Department of Oncology, Nantong Second People's Hospital, Nantong, Jiangsu 226001, P.R. China
| | - Lu Hua
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Daliang Yan
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Fengbo Zhao
- Basic Medical Research Centre, Medical College, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Jinxia Liu
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Huiling Zhou
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Jie Liu
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Miaomiao Wu
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Chengliang Zhang
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Yingying Chen
- Jiangsu Province Key Laboratory for Inflammation and Molecular Drug Target, Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Buyou Chen
- Department of Radiotherapy, Affiliated Hospital of Nantong University, Nantong, Jiangsu 226001, P.R. China
| | - Baoying Hu
- Basic Medical Research Centre, Medical College, Nantong University, Nantong, Jiangsu 226001, P.R. China
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Sorenson A, Owens L, Caltabiano M, Cadet-James Y, Hall R, Govan B, Clancy P. The Impact of Prior Flavivirus Infections on the Development of Type 2 Diabetes Among the Indigenous Australians. Am J Trop Med Hyg 2016; 95:265-8. [PMID: 27001762 PMCID: PMC4973169 DOI: 10.4269/ajtmh.15-0727] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/07/2015] [Accepted: 02/22/2016] [Indexed: 12/21/2022] Open
Abstract
It is estimated that 5% of Australians over the age of 18 have diabetes, with the number of new cases increasing every year. Type 2 diabetes (T2D) also represents a significant disease burden in the Australian indigenous population, where prevalence is three times greater than that of non-indigenous Australians. Prevalence of T2D has been found to be higher in rural and remote indigenous Australian populations compared with urban indigenous Australian populations. Several studies have also found that body mass index and waist circumference are not appropriate for the prediction of T2D risk in indigenous Australians. Regional and remote areas of Australia are endemic for a variety of mosquito-borne flaviviruses. Studies that have investigated seroprevalence of flaviviruses in remote aboriginal communities have found high proportions of seroconversion. The family Flaviviridae comprises several genera of viruses with non-segmented single-stranded positive sense RNA genomes, and includes the flaviviruses and hepaciviruses. Hepatitis C virus (HCV) has been shown to be associated with insulin resistance and subsequent development of T2D. Flaviviruses and HCV possess conserved proteins and subgenomic RNA structures that may play similar roles in the development of insulin resistance. Although dietary and lifestyle factors are associated with increased risk of developing T2D, the impact of infectious diseases such as arboviruses has not been assessed. Flaviviruses circulating in indigenous Australian communities may play a significant role in inducing glucose intolerance and exacerbating T2D.
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Affiliation(s)
- Alanna Sorenson
- College of Public Health, Medical and Veterinary Sciences, James Cook University, Queensland, Australia.
| | - Leigh Owens
- College of Public Health, Medical and Veterinary Sciences, James Cook University, Queensland, Australia
| | - Marie Caltabiano
- College of Healthcare Sciences, James Cook University, Queensland, Australia
| | - Yvonne Cadet-James
- Anton Breinl Research Centre for Health Systems Strengthening, James Cook University, Queensland, Australia
| | - Roy Hall
- School of Chemistry and Molecular Biosciences, University of Queensland, Queensland, Australia
| | - Brenda Govan
- College of Public Health, Medical and Veterinary Sciences, James Cook University, Queensland, Australia
| | - Paula Clancy
- College of Public Health, Medical and Veterinary Sciences, James Cook University, Queensland, Australia
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24
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Ye J, Zhou G, Zhang Z, Sun L, He X, Zhou J. Poly (C)-binding protein 2 (PCBP2) promotes the progression of esophageal squamous cell carcinoma (ESCC) through regulating cellular proliferation and apoptosis. Pathol Res Pract 2016; 212:717-25. [DOI: 10.1016/j.prp.2016.05.008] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/28/2015] [Revised: 04/21/2016] [Accepted: 05/25/2016] [Indexed: 12/14/2022]
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25
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PCBP2 Modulates Neural Apoptosis and Astrocyte Proliferation After Spinal Cord Injury. Neurochem Res 2016; 41:2401-14. [DOI: 10.1007/s11064-016-1953-6] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/16/2015] [Revised: 04/05/2016] [Accepted: 05/09/2016] [Indexed: 12/22/2022]
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26
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Ríos-Marco P, Romero-López C, Berzal-Herranz A. The cis-acting replication element of the Hepatitis C virus genome recruits host factors that influence viral replication and translation. Sci Rep 2016; 6:25729. [PMID: 27165399 PMCID: PMC4863150 DOI: 10.1038/srep25729] [Citation(s) in RCA: 28] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2015] [Accepted: 04/21/2016] [Indexed: 02/08/2023] Open
Abstract
The cis-acting replication element (CRE) of the hepatitis C virus (HCV) RNA genome is a region of conserved sequence and structure at the 3' end of the open reading frame. It participates in a complex and dynamic RNA-RNA interaction network involving, among others, essential functional domains of the 3' untranslated region and the internal ribosome entry site located at the 5' terminus of the viral genome. A proper balance between all these contacts is critical for the control of viral replication and translation, and is likely dependent on host factors. Proteomic analyses identified a collection of proteins from a hepatoma cell line as CRE-interacting candidates. A large fraction of these were RNA-binding proteins sharing highly conserved RNA recognition motifs. The vast majority of these proteins were validated by bioinformatics tools that consider RNA-protein secondary structure. Further characterization of representative proteins indicated that hnRNPA1 and HMGB1 exerted negative effects on viral replication in a subgenomic HCV replication system. Furthermore DDX5 and PARP1 knockdown reduced the HCV IRES activity, suggesting an involvement of these proteins in HCV translation. The identification of all these host factors provides new clues regarding the function of the CRE during viral cycle progression.
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Affiliation(s)
- Pablo Ríos-Marco
- Instituto de Parasitología y Biomedicina López-Neyra, (IPBLN-CSIC). PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain
| | - Cristina Romero-López
- Instituto de Parasitología y Biomedicina López-Neyra, (IPBLN-CSIC). PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain
| | - Alfredo Berzal-Herranz
- Instituto de Parasitología y Biomedicina López-Neyra, (IPBLN-CSIC). PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain
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27
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Venezuelan equine encephalitis virus non-structural protein 3 (nsP3) interacts with RNA helicases DDX1 and DDX3 in infected cells. Antiviral Res 2016; 131:49-60. [PMID: 27105836 PMCID: PMC7113772 DOI: 10.1016/j.antiviral.2016.04.008] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/08/2016] [Revised: 04/12/2016] [Accepted: 04/13/2016] [Indexed: 12/17/2022]
Abstract
The mosquito-borne New World alphavirus, Venezuelan equine encephalitis virus (VEEV) is a Category B select agent with no approved vaccines or therapies to treat infected humans. Therefore it is imperative to identify novel targets that can be targeted for effective therapeutic intervention. We aimed to identify and validate interactions of VEEV nonstructural protein 3 (nsP3) with host proteins and determine the consequences of these interactions to viral multiplication. We used a HA tagged nsP3 infectious clone (rTC-83-nsP3-HA) to identify and validate two RNA helicases: DDX1 and DDX3 that interacted with VEEV-nsP3. In addition, DDX1 and DDX3 knockdown resulted in a decrease in infectious viral titers. Furthermore, we propose a functional model where the nsP3:DDX3 complex interacts with the host translational machinery and is essential in the viral life cycle. This study will lead to future investigations in understanding the importance of VEEV-nsP3 to viral multiplication and apply the information for the discovery of novel host targets as therapeutic options.
VEEV nsP3 interacted with the host helicases DDX1 and DDX3 in infected cells. Depletion of DDX1 or DDX3 negatively impacted viral multiplication and decreased infectious viral titers. nsP3 may interact with the host translational machinery through DDX3. The small molecule DDX3 inhibitor RK33 negatively impacted VEEV multiplication.
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28
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Ye F, Xin Z, Han W, Fan J, Yin B, Wu S, Yang W, Yuan J, Qiang B, Sun W, Peng X. Quantitative Proteomics Analysis of the Hepatitis C Virus Replicon High-Permissive and Low-Permissive Cell Lines. PLoS One 2015; 10:e0142082. [PMID: 26544179 PMCID: PMC4636247 DOI: 10.1371/journal.pone.0142082] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/29/2015] [Accepted: 10/16/2015] [Indexed: 01/16/2023] Open
Abstract
Chronic hepatitis C virus (HCV) infection is one of the leading causes of severe hepatitis. The molecular mechanisms underlying HCV replication and pathogenesis remain unclear. The development of the subgenome replicon model system significantly enhanced study of HCV. However, the permissiveness of the HCV subgenome replicon greatly differs among different hepatoma cell lines. Proteomic analysis of different permissive cell lines might provide new clues in understanding HCV replication. In this study, to detect potential candidates that might account for the differences in HCV replication. Label-free and iTRAQ labeling were used to analyze the differentially expressed protein profiles between Huh7.5.1 wt and HepG2 cells. A total of 4919 proteins were quantified in which 114 proteins were commonly identified as differentially expressed by both quantitative methods. A total of 37 differential proteins were validated by qRT-PCR. The differential expression of Glutathione S-transferase P (GSTP1), Ubiquitin carboxyl-terminal hydrolase isozyme L1 (UCHL1), carboxylesterase 1 (CES1), vimentin, Proteasome activator complex subunit1 (PSME1), and Cathepsin B (CTSB) were verified by western blot. And over-expression of CTSB or knock-down of vimentin induced significant changes to HCV RNA levels. Additionally, we demonstrated that CTSB was able to inhibit HCV replication and viral protein translation. These results highlight the potential role of CTSB and vimentin in virus replication.
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Affiliation(s)
- Fei Ye
- The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Zhongshuai Xin
- Division of Hormone, National Institute for Food and Drug Control, Beijing, China
| | - Wei Han
- The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Jingjing Fan
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Bin Yin
- The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Shuzhen Wu
- Core facility of instrument, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Wei Yang
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Jiangang Yuan
- The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Boqin Qiang
- The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
| | - Wei Sun
- Core facility of instrument, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
- * E-mail: (XP); (WS)
| | - Xiaozhong Peng
- The State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
- * E-mail: (XP); (WS)
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29
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Loss of the Drosophila melanogaster DEAD box protein Ddx1 leads to reduced size and aberrant gametogenesis. Dev Biol 2015; 407:232-45. [PMID: 26433063 PMCID: PMC7094483 DOI: 10.1016/j.ydbio.2015.09.012] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/28/2015] [Revised: 07/31/2015] [Accepted: 09/24/2015] [Indexed: 02/07/2023]
Abstract
Mammalian DDX1 has been implicated in RNA trafficking, DNA double-strand break repair and RNA processing; however, little is known about its role during animal development. Here, we report phenotypes associated with a null Ddx1 (Ddx1AX) mutation generated in Drosophila melanogaster. Ddx1 null flies are viable but significantly smaller than control and Ddx1 heterozygous flies. Female Ddx1 null flies have reduced fertility with egg chambers undergoing autophagy, whereas males are sterile due to disrupted spermatogenesis. Comparative RNA sequencing of control and Ddx1 null third instars identified several transcripts affected by Ddx1 inactivation. One of these, Sirup mRNA, was previously shown to be overexpressed under starvation conditions and implicated in mitochondrial function. We demonstrate that Sirup is a direct binding target of Ddx1 and that Sirup mRNA is differentially spliced in the presence or absence of Ddx1. Combining Ddx1 null mutation with Sirup dsRNA-mediated knock-down causes epistatic lethality not observed in either single mutant. Our data suggest a role for Drosophila Ddx1 in stress-induced regulation of splicing.
We describe a new Ddx1 null Drosophila line. Ddx1 null flies are smaller in size and display aberrant gametogenesis. Sirup splicing is altered in Ddx1 null flies. We show both a physical and a genetic interaction between Ddx1 and Sirup.
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30
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Zhang Y, Si Y, Ma N, Mei J. The RNA-binding protein PCBP2 inhibits Ang II-induced hypertrophy of cardiomyocytes though promoting GPR56 mRNA degeneration. Biochem Biophys Res Commun 2015; 464:679-84. [PMID: 26116532 DOI: 10.1016/j.bbrc.2015.06.139] [Citation(s) in RCA: 33] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/18/2015] [Accepted: 06/21/2015] [Indexed: 12/25/2022]
Abstract
Poly(C)-binding proteins (PCBPs) are known as RNA-binding proteins that interact in a sequence-specific fashion with single-stranded poly(C). This family can be divided into two groups: hnRNP K and PCBP1-4. PCBPs are expressed broadly in human and mouse tissues and all members of the PCBP family are related evolutionarily. However, their physiological or pathological functions in the hearts remain unknown. Here we reported that PCBP2 is an anti-hypertrophic factor by inhibiting GPR56 mRNA stability. We found the downregulation of PCBP2 in human failing hearts and mouse hypertrophic hearts. PCBP2 knockdown promoted angiotensin II (Ang II)-induced hypertrophy (increase in cell size, protein synthesis and activation of fetal genes) of neonatal cardiomyocytes and H9C2 cells, while PCBP2 overexpression obtained oppose effects. Furthermore, PCBP2 was shown to inhibit GPR56 expression by promoting its mRNA degeneration in cardiomyocytes. Finally, we knocked down GPR56 in cardiomyocytes and found that GPR56 promoted Ang II-induced cardiomyocyte hypertrophy and it contributed to PCBP2 effects on cardiomyocyte hypertrophy.
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Affiliation(s)
- Yunjiao Zhang
- Department of Cardiothoracic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
| | - Yi Si
- Department of Cardiothoracic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
| | - Nan Ma
- Department of Cardiothoracic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China
| | - Ju Mei
- Department of Cardiothoracic Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200092, China.
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31
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Douam F, Ploss A. Proteomic approaches to analyzing hepatitis C virus biology. Proteomics 2015; 15:2051-65. [PMID: 25809442 PMCID: PMC4559851 DOI: 10.1002/pmic.201500009] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Revised: 02/25/2015] [Accepted: 03/19/2015] [Indexed: 12/15/2022]
Abstract
Hepatitis C virus (HCV) is a major cause of liver disease worldwide. Acute infection often progresses to chronicity resulting frequently in fibrosis, cirrhosis, and in rare cases, in the development of hepatocellular carcinoma. Although HCV has proven to be an arduous object of research and has raised important technical challenges, several experimental models have been developed all over the last two decades in order to improve our understanding of the virus life cycle, pathogenesis and virus-host interactions. The recent development of direct acting-agents, leading to considerable progress in treatment of patients, represents the direct outcomes of these achievements. Proteomic approaches have been of critical help to shed light on several aspect of the HCV biology such as virion composition, viral replication, and virus assembly and to unveil diagnostic or prognostic markers of HCV-induced liver disease. Here, we review how proteomic approaches have led to improve our understanding of HCV life cycle and liver disease, thus highlighting the relevance of these approaches for studying the complex interactions between other challenging human viral pathogens and their host.
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Affiliation(s)
- Florian Douam
- Department of Molecular Biology, Princeton University, 110 Lewis Thomas Laboratory, Washington Road, Princeton, NJ 08544
| | - Alexander Ploss
- Department of Molecular Biology, Princeton University, 110 Lewis Thomas Laboratory, Washington Road, Princeton, NJ 08544
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32
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Schwerk J, Jarret AP, Joslyn RC, Savan R. Landscape of post-transcriptional gene regulation during hepatitis C virus infection. Curr Opin Virol 2015; 12:75-84. [PMID: 25890065 DOI: 10.1016/j.coviro.2015.02.006] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2015] [Accepted: 02/11/2015] [Indexed: 12/11/2022]
Abstract
Post-transcriptional regulation of gene expression plays a pivotal role in various gene regulatory networks including, but not limited to metabolism, embryogenesis and immune responses. Different mechanisms of post-transcriptional regulation, which can act individually, synergistically, or even in an antagonistic manner have been described. Hepatitis C virus (HCV) is notorious for subverting host immune responses and indeed exploits several components of the host's post-transcriptional regulatory machinery for its own benefit. At the same time, HCV replication is post-transcriptionally targeted by host cell components to blunt viral propagation. This review discusses the interplay of post-transcriptional mechanisms that affect host immune responses in the setting of HCV infection and highlights the sophisticated mechanisms both host and virus have evolved in the race for superiority.
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Affiliation(s)
- Johannes Schwerk
- Department of Immunology, University of Washington, Seattle, WA 98109, USA
| | - Abigail P Jarret
- Department of Immunology, University of Washington, Seattle, WA 98109, USA
| | - Rochelle C Joslyn
- Department of Immunology, University of Washington, Seattle, WA 98109, USA
| | - Ram Savan
- Department of Immunology, University of Washington, Seattle, WA 98109, USA.
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33
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Kellner JN, Reinstein J, Meinhart A. Synergistic effects of ATP and RNA binding to human DEAD-box protein DDX1. Nucleic Acids Res 2015; 43:2813-28. [PMID: 25690890 PMCID: PMC4357711 DOI: 10.1093/nar/gkv106] [Citation(s) in RCA: 17] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
RNA helicases of the DEAD-box protein family form the largest group of helicases. The human DEAD-box protein 1 (DDX1) plays an important role in tRNA and mRNA processing, is involved in tumor progression and is also hijacked by several virus families such as HIV-1 for replication and nuclear export. Although important in many cellular processes, the mechanism of DDX1′s enzymatic function is unknown. We have performed equilibrium titrations and transient kinetics to determine affinities for nucleotides and RNA. We find an exceptional tight binding of DDX1 to adenosine diphosphate (ADP), one of the strongest affinities observed for DEAD-box helicases. ADP binds tighter by three orders of magnitude when compared to adenosine triphosphate (ATP), arresting the enzyme in a potential dead-end ADP conformation under physiological conditions. We thus suggest that a nucleotide exchange factor leads to DDX1 recycling. Furthermore, we find a strong cooperativity in binding of RNA and ATP to DDX1 that is also reflected in ATP hydrolysis. We present a model in which either ATP or RNA binding alone can partially shift the equilibrium from an ‘open’ to a ‘closed’-state; this shift appears to be not further pronounced substantially even in the presence of both RNA and ATP as the low rate of ATP hydrolysis does not change.
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Affiliation(s)
- Julian N Kellner
- Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany
| | - Jochen Reinstein
- Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany
| | - Anton Meinhart
- Department of Biomolecular Mechanisms, Max-Planck-Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany
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34
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Ariumi Y. Multiple functions of DDX3 RNA helicase in gene regulation, tumorigenesis, and viral infection. Front Genet 2014; 5:423. [PMID: 25538732 PMCID: PMC4257086 DOI: 10.3389/fgene.2014.00423] [Citation(s) in RCA: 99] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2014] [Accepted: 11/19/2014] [Indexed: 12/11/2022] Open
Abstract
The DEAD-box RNA helicase DDX3 is a multifunctional protein involved in all aspects of RNA metabolism, including transcription, splicing, mRNA nuclear export, translation, RNA decay and ribosome biogenesis. In addition, DDX3 is also implicated in cell cycle regulation, apoptosis, Wnt-β-catenin signaling, tumorigenesis, and viral infection. Notably, recent studies suggest that DDX3 is a component of anti-viral innate immune signaling pathways. Indeed, DDX3 contributes to enhance the induction of anti-viral mediators, interferon (IFN) regulatory factor 3 and type I IFN. However, DDX3 seems to be an important target for several viruses, such as human immunodeficiency virus type 1 (HIV-1), hepatitis C virus (HCV), hepatitis B virus (HBV), and poxvirus. DDX3 interacts with HIV-1 Rev or HCV Core protein and modulates its function. At least, DDX3 is required for both HIV-1 and HCV replication. Therefore, DDX3 could be a novel therapeutic target for the development of drug against HIV-1 and HCV.
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Affiliation(s)
- Yasuo Ariumi
- Ariumi Project Laboratory, Center for AIDS Research - International Research Center for Medical Sciences, Kumamoto University Kumamoto, Japan
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35
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Greco TM, Diner BA, Cristea IM. The Impact of Mass Spectrometry-Based Proteomics on Fundamental Discoveries in Virology. Annu Rev Virol 2014; 1:581-604. [PMID: 26958735 DOI: 10.1146/annurev-virology-031413-085527] [Citation(s) in RCA: 46] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
In recent years, mass spectrometry has emerged as a core component of fundamental discoveries in virology. As a consequence of their coevolution, viruses and host cells have established complex, dynamic interactions that function either in promoting virus replication and dissemination or in host defense against invading pathogens. Thus, viral infection triggers an impressive range of proteome changes. Alterations in protein abundances, interactions, posttranslational modifications, subcellular localizations, and secretion are temporally regulated during the progression of an infection. Consequently, understanding viral infection at the molecular level requires versatile approaches that afford both breadth and depth of analysis. Mass spectrometry is uniquely positioned to bridge this experimental dichotomy. Its application to both unbiased systems analyses and targeted, hypothesis-driven studies has accelerated discoveries in viral pathogenesis and host defense. Here, we review the contributions of mass spectrometry-based proteomic approaches to understanding viral morphogenesis, replication, and assembly and to characterizing host responses to infection.
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Affiliation(s)
- Todd M Greco
- Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544;
| | - Benjamin A Diner
- Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544;
| | - Ileana M Cristea
- Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544;
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36
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Romero-López C, Berzal-Herranz A. Structure-function relationship in viral RNA genomes: The case of hepatitis C virus. World J Med Genet 2014; 4:6-18. [DOI: 10.5496/wjmg.v4.i2.6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/10/2013] [Revised: 01/23/2014] [Accepted: 04/03/2014] [Indexed: 02/06/2023] Open
Abstract
The acquisition of a storage information system beyond the nucleotide sequence has been a crucial issue for the propagation and dispersion of RNA viruses. This system is composed by highly conserved, complex structural units in the genomic RNA, termed functional RNA domains. These elements interact with other regions of the viral genome and/or proteins to direct viral translation, replication and encapsidation. The genomic RNA of the hepatitis C virus (HCV) is a good model for investigating about conserved structural units. It contains functional domains, defined by highly conserved structural RNA motifs, mostly located in the 5’-untranslatable regions (5’UTRs) and 3’UTR, but also occupying long stretches of the coding sequence. Viral translation initiation is mediated by an internal ribosome entry site located at the 5’ terminus of the viral genome and regulated by distal functional RNA domains placed at the 3’ end. Subsequent RNA replication strongly depends on the 3’UTR folding and is also influenced by the 5’ end of the HCV RNA. Further increase in the genome copy number unleashes the formation of homodimers by direct interaction of two genomic RNA molecules, which are finally packed and released to the extracellular medium. All these processes, as well as transitions between them, are controlled by structural RNA elements that establish a complex, direct and long-distance RNA-RNA interaction network. This review summarizes current knowledge about functional RNA domains within the HCV RNA genome and provides an overview of the control exerted by direct, long-range RNA-RNA contacts for the execution of the viral cycle.
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37
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Hu CE, Liu YC, Zhang HD, Huang GJ. The RNA-binding protein PCBP2 facilitates gastric carcinoma growth by targeting miR-34a. Biochem Biophys Res Commun 2014; 448:437-42. [PMID: 24796666 DOI: 10.1016/j.bbrc.2014.04.124] [Citation(s) in RCA: 31] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2014] [Accepted: 04/24/2014] [Indexed: 12/31/2022]
Abstract
Gastric carcinoma is the fourth most common cancer worldwide, with a high rate of death and low 5-year survival rate. However, the mechanism underling gastric cancer is still not fully understood. Here in the present study, we identify the RNA-binding protein PCBP2 as an oncogenic protein in human gastric carcinoma. Our results show that PCBP2 is up-regulated in human gastric cancer tissues compared to adjacent normal tissues, and that high level of PCBP2 predicts poor overall and disease-free survival. Knockdown of PCBP2 in gastric cancer cells inhibits cell proliferation and colony formation in vitro, whereas opposing results are obtained when PCBP2 is overexpressed. Our in vivo subcutaneous xenograft results also show that PCBP2 can critically regulate gastric cancer cell growth. In addition, we find that PCBP2-depletion induces apoptosis in gastric cancer cells via up-regulating expression of pro-apoptotic proteins and down-regulating anti-apoptotic proteins. Mechanically, we identify that miR-34a as a target of PCBP2, and that miR-34a is critically essential for the function of PCBP2. In summary, PCBP2 promotes gastric carcinoma development by regulating the level of miR-34a.
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Affiliation(s)
- Cheng-En Hu
- Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China
| | - Yong-Chao Liu
- Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China
| | - Hui-Dong Zhang
- Department of General Surgery, Shanghai Children's Medical Center, Shanghai, China
| | - Guang-Jian Huang
- Department of General Surgery, Huashan Hospital, Fudan University, Shanghai, China.
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38
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Upadya MH, Aweya JJ, Tan YJ. Understanding the interaction of hepatitis C virus with host DEAD-box RNA helicases. World J Gastroenterol 2014; 20:2913-2926. [PMID: 24659882 PMCID: PMC3961968 DOI: 10.3748/wjg.v20.i11.2913] [Citation(s) in RCA: 28] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/27/2013] [Revised: 12/06/2013] [Accepted: 01/20/2014] [Indexed: 02/06/2023] Open
Abstract
The current therapeutic regimen to combat chronic hepatitis C is not optimal due to substantial side effects and the failure of a significant proportion of patients to achieve a sustained virological response. Recently developed direct-acting antivirals targeting hepatitis C virus (HCV) enzymes reportedly increase the virologic response to therapy but may lead to a selection of drug-resistant variants. Besides direct-acting antivirals, another promising class of HCV drugs in development include host targeting agents that are responsible for interfering with the host factors crucial for the viral life cycle. A family of host proteins known as DEAD-box RNA helicases, characterized by nine conserved motifs, is known to play an important role in RNA metabolism. Several members of this family such as DDX3, DDX5 and DDX6 have been shown to play a role in HCV replication and this review will summarize our current knowledge on their interaction with HCV. As chronic hepatitis C is one of the leading causes of hepatocellular carcinoma, the involvement of DEAD-box RNA helicases in the development of HCC will also be highlighted. Continuing research on the interaction of host DEAD-box proteins with HCV and the contribution to viral replication and pathogenesis could be the panacea for the development of novel therapeutics against HCV.
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Romero-López C, Barroso-delJesus A, García-Sacristán A, Briones C, Berzal-Herranz A. End-to-end crosstalk within the hepatitis C virus genome mediates the conformational switch of the 3'X-tail region. Nucleic Acids Res 2014; 42:567-582. [PMID: 24049069 PMCID: PMC3874160 DOI: 10.1093/nar/gkt841] [Citation(s) in RCA: 52] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/28/2013] [Revised: 08/17/2013] [Accepted: 08/27/2013] [Indexed: 02/06/2023] Open
Abstract
The hepatitis C virus (HCV) RNA genome contains multiple structurally conserved domains that make long-distance RNA-RNA contacts important in the establishment of viral infection. Microarray antisense oligonucleotide assays, improved dimethyl sulfate probing methods and 2' acylation chemistry (selective 2'-hydroxyl acylation and primer extension, SHAPE) showed the folding of the genomic RNA 3' end to be regulated by the internal ribosome entry site (IRES) element via direct RNA-RNA interactions. The essential cis-acting replicating element (CRE) and the 3'X-tail region adopted different 3D conformations in the presence and absence of the genomic RNA 5' terminus. Further, the structural transition in the 3'X-tail from the replication-competent conformer (consisting of three stem-loops) to the dimerizable form (with two stem-loops), was found to depend on the presence of both the IRES and the CRE elements. Complex interplay between the IRES, the CRE and the 3'X-tail region would therefore appear to occur. The preservation of this RNA-RNA interacting network, and the maintenance of the proper balance between different contacts, may play a crucial role in the switch between different steps of the HCV cycle.
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Affiliation(s)
- Cristina Romero-López
- Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Unidad de Genómica, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Laboratorio de Evolución Molecular, Centro de Astrobiología (CSIC-INTA), Carretera de Ajalvir km 4, 28850 Torrejón de Ardoz, Madrid, Spain and Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain
| | - Alicia Barroso-delJesus
- Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Unidad de Genómica, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Laboratorio de Evolución Molecular, Centro de Astrobiología (CSIC-INTA), Carretera de Ajalvir km 4, 28850 Torrejón de Ardoz, Madrid, Spain and Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain
| | - Ana García-Sacristán
- Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Unidad de Genómica, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Laboratorio de Evolución Molecular, Centro de Astrobiología (CSIC-INTA), Carretera de Ajalvir km 4, 28850 Torrejón de Ardoz, Madrid, Spain and Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain
| | - Carlos Briones
- Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Unidad de Genómica, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Laboratorio de Evolución Molecular, Centro de Astrobiología (CSIC-INTA), Carretera de Ajalvir km 4, 28850 Torrejón de Ardoz, Madrid, Spain and Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain
| | - Alfredo Berzal-Herranz
- Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Unidad de Genómica, Instituto de Parasitología y Biomedicina López-Neyra, IPBLN-CSIC, PTS Granada, Avda. del Conocimiento s/n, Armilla, 18016 Granada, Spain, Laboratorio de Evolución Molecular, Centro de Astrobiología (CSIC-INTA), Carretera de Ajalvir km 4, 28850 Torrejón de Ardoz, Madrid, Spain and Centro de Investigación Biomédica en Red de enfermedades hepáticas y digestivas (CIBERehd), Spain
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Blackham SL, McGarvey MJ. A host cell RNA-binding protein, Staufen1, has a role in hepatitis C virus replication before virus assembly. J Gen Virol 2013; 94:2429-2436. [DOI: 10.1099/vir.0.051383-0] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 08/30/2023] Open
Abstract
Staufen1 is a dsRNA-binding protein involved in the regulation of translation and the trafficking and degradation of cellular RNAs. Staufen1 has also been shown to stimulate translation of human immunodeficiency virus type 1 (HIV-1) RNA, regulate HIV-1 and influenza A virus assembly, and there is also indication that it can interact with hepatitis C virus (HCV) RNA. To investigate the role of Staufen1 in the HCV replication cycle, the effects of small interfering RNA knockout of Staufen1 on HCV strain JFH-1 replication and the intracellular distribution of the Staufen1 protein during HCV infection were examined. Silencing Staufen1 in HCV-infected Huh7 cells reduced virus secretion by around 70 %, intracellular HCV RNA levels by around 40 %, and core and NS3 proteins by around 95 and 45 %, respectively. Staufen1 appeared to be predominantly localized in the endoplasmic reticulum at the nuclear periphery in both uninfected and HCV-infected Huh7 cells. However, Staufen1 showed significant co-localization with NS3 and dsRNA, indicating that it may bind to replicating HCV RNA that is associated with the non-structural proteins. Staufen1 and HCV core protein localized very closely to one another during infection, but did not appear to overlap, indicating that Staufen1 may not bind to core protein or localize to the core-coated lipid droplets, suggesting that it may not be directly involved in HCV virus assembly. These findings indicate that Staufen1 is an important factor in HCV replication and that it might play a role early in the HCV replication cycle, e.g. in translation, replication or trafficking of the HCV genome, rather than in virion morphogenesis.
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Rosner A, Moiseeva E, Rabinowitz C, Rinkevich B. Germ lineage properties in the urochordate Botryllus schlosseri - from markers to temporal niches. Dev Biol 2013; 384:356-74. [PMID: 24120376 DOI: 10.1016/j.ydbio.2013.10.002] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2012] [Revised: 08/25/2013] [Accepted: 10/03/2013] [Indexed: 01/28/2023]
Abstract
The primordial germ cells (PGCs) in the colonial urochordate Botryllus schlosseri are sequestered in late embryonic stage. PGC-like populations, located at any blastogenic stage in specific niches, inside modules with curtailed lifespan, survive throughout the life of the colony by repeated weekly migration to newly formed buds. This cyclical migration and the lack of specific markers for PGC-like populations are obstacles to the study on PGCs. For that purpose, we isolated the Botryllus DDX1 (BS-DDX1) and characterized it by normal expression patterns and by specific siRNA knockdown experiments. Expression of BS-DDX1 concurrent with BS-Vasa, γ-H2AX, BS-cadherin and phospho-Smad1/5/8, demarcate PGC cells from soma cells and from more differentiated germ cells lineages, which enabled the detection of additional putative transient niches in zooids. Employing BS-cadherin siRNA knockdown, retinoic acid (RA) administration or β-estradiol administration affirmed the BS-Vasa(+)BS-DDX1(+)BS-cadherin(+)γ-H2AX(+)phospho-Smad1/5/8(+) population as the B. schlosseri PGC-like cells. By striving to understand the PGC-like cells trafficking between transient niches along blastogenic cycles, CM-DiI-stained PGC-like enriched populations from late blastogenic stage D zooids were injected into genetically matched colonial ramets at blastogenic stages A or C and their fates were observed for 9 days. Based on the accumulated data, we conceived a novel network of several transient and short lived 'germ line niches' that preserve PGCs homeostasis, protecting these cells from the weekly astogenic senescence processes, thus enabling the survival of the PGCs throughout the organism's life.
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Affiliation(s)
- Amalia Rosner
- National Institute of Oceanography, Israel Oceanography & Limnological Research, Tel Shikmona, P.O. Box 8030, Haifa 31080, Israel.
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Han W, Xin Z, Zhao Z, Bao W, Lin X, Yin B, Zhao J, Yuan J, Qiang B, Peng X. RNA-binding protein PCBP2 modulates glioma growth by regulating FHL3. J Clin Invest 2013; 123:2103-18. [PMID: 23585479 DOI: 10.1172/jci61820] [Citation(s) in RCA: 85] [Impact Index Per Article: 7.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/21/2012] [Accepted: 02/07/2013] [Indexed: 02/01/2023] Open
Abstract
PCBP2 is a member of the poly(C)-binding protein (PCBP) family, which plays an important role in posttranscriptional and translational regulation by interacting with single-stranded poly(C) motifs in target mRNAs. Several PCBP family members have been reported to be involved in human malignancies. Here, we show that PCBP2 is upregulated in human glioma tissues and cell lines. Knockdown of PCBP2 inhibited glioma growth in vitro and in vivo through inhibition of cell-cycle progression and induction of caspase-3-mediated apoptosis. Thirty-five mRNAs were identified as putative PCBP2 targets/interactors using RIP-ChIP protein-RNA interaction arrays in a human glioma cell line, T98G. Four-and-a-half LIM domain 3 (FHL3) mRNA was downregulated in human gliomas and was identified as a PCBP2 target. Knockdown of PCBP2 enhanced the expression of FHL3 by stabilizing its mRNA. Overexpression of FHL3 attenuated cell growth and induced apoptosis. This study establishes a link between PCBP2 and FHL3 proteins and identifies a new pathway for regulating glioma progression.
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Affiliation(s)
- Wei Han
- State Key Laboratory of Medical Molecular Biology, Department of Molecular Biology and Biochemistry, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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Fullam A, Schröder M. DExD/H-box RNA helicases as mediators of anti-viral innate immunity and essential host factors for viral replication. BIOCHIMICA ET BIOPHYSICA ACTA-GENE REGULATORY MECHANISMS 2013; 1829:854-65. [PMID: 23567047 PMCID: PMC7157912 DOI: 10.1016/j.bbagrm.2013.03.012] [Citation(s) in RCA: 140] [Impact Index Per Article: 11.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 11/02/2012] [Revised: 03/27/2013] [Accepted: 03/29/2013] [Indexed: 12/17/2022]
Abstract
Traditional functions of DExD/H-box helicases are concerned with RNA metabolism; they have been shown to play a part in nearly every cellular process that involves RNA. On the other hand, it is accepted that DexD/H-box helicases also engage in activities that do not require helicase activity. A number of DExD/H-box helicases have been shown to be involved in anti-viral immunity. The RIG-like helicases, RIG-I, mda5 and lgp2, act as important cytosolic pattern recognition receptors for viral RNA. Detection of viral nucleic acids by the RIG-like helicases or other anti-viral pattern recognition receptors leads to the induction of type I interferons and pro-inflammatory cytokines. More recently, additional DExD/H-box helicases have also been implicated to act as cytosolic sensors of viral nucleic acids, including DDX3, DDX41, DHX9, DDX60, DDX1 and DHX36. However, there is evidence that at least some of these helicases might have more downstream functions in pattern recognition receptor signalling pathways, as signalling adaptors or transcriptional regulators. In an interesting twist, a lot of DExD/H-box helicases have also been identified as essential host factors for the replication of different viruses, suggesting that viruses 'hijack' their RNA helicase activities for their benefit. Interestingly, DDX3, DDX1 and DHX9 are among the helicases that are required for the replication of a diverse range of viruses. This might suggest that these helicases are highly contested targets in the ongoing 'arms race' between viruses and the host immune system. This article is part of a Special Issue entitled: The Biology of RNA helicases - Modulation for life.
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Affiliation(s)
- Anthony Fullam
- National University of Ireland, Maynooth, Kildare, Ireland.
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Pager CT, Schütz S, Abraham TM, Luo G, Sarnow P. Modulation of hepatitis C virus RNA abundance and virus release by dispersion of processing bodies and enrichment of stress granules. Virology 2012; 435:472-84. [PMID: 23141719 DOI: 10.1016/j.virol.2012.10.027] [Citation(s) in RCA: 67] [Impact Index Per Article: 5.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/10/2012] [Revised: 10/11/2012] [Accepted: 10/16/2012] [Indexed: 12/12/2022]
Abstract
Components of cytoplasmic processing bodies (P-bodies) and stress granules can be subverted during viral infections to modulate viral gene expression. Because hepatitis C virus (HCV) RNA abundance is regulated by P-body components such as microRNA miR-122, Argonaute 2 and RNA helicase RCK/p54, we examined whether HCV infection modulates P-bodies and stress granules during viral infection. It was discovered that HCV infection decreased the number of P-bodies, but induced the formation of stress granules. Immunofluorescence studies revealed that a number of P-body and stress granule proteins co-localized with viral core protein at lipid droplets, the sites for viral RNA packaging. Depletion of selected P-body proteins decreased overall HCV RNA and virion abundance. Depletion of stress granule proteins also decreased overall HCV RNA abundance, but surprisingly enhanced the accumulation of infectious, extracellular virus. These data argue that HCV subverts P-body and stress granule components to aid in viral gene expression at particular sites in the cytoplasm.
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Affiliation(s)
- Cara T Pager
- Department of Microbiology and Immunology, Stanford University, Stanford, CA 94305-5124, United States
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Abstract
RNA helicases unwind their RNA substrates in an ATP-dependent reaction, and are central to all cellular processes involving RNA. They have important roles in viral life cycles, where RNA helicases are either virus-encoded or recruited from the host. Vertebrate RNA helicases sense viral infections, and trigger the innate antiviral immune response. RNA helicases have been implicated in protozoic, bacterial and fungal infections. They are also linked to neurological disorders, cancer, and aging processes. Genome-wide studies continue to identify helicase genes that change their expression patterns after infection or disease outbreak, but the mechanism of RNA helicase action has been defined for only a few diseases. RNA helicases are prognostic and diagnostic markers and suitable drug targets, predominantly for antiviral and anti-cancer therapies. This review summarizes the current knowledge on RNA helicases in infection and disease, and their growing potential as drug targets.
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Affiliation(s)
- Lenz Steimer
- University of Muenster, Institute for Physical Chemistry, Muenster, Germany
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Edgcomb SP, Carmel AB, Naji S, Ambrus-Aikelin G, Reyes JR, Saphire ACS, Gerace L, Williamson JR. DDX1 is an RNA-dependent ATPase involved in HIV-1 Rev function and virus replication. J Mol Biol 2011; 415:61-74. [PMID: 22051512 PMCID: PMC3249508 DOI: 10.1016/j.jmb.2011.10.032] [Citation(s) in RCA: 61] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2011] [Revised: 10/18/2011] [Accepted: 10/18/2011] [Indexed: 12/26/2022]
Abstract
The human immunodeficiency virus type 1 (HIV-1) Rev protein is essential for the virus because it promotes nuclear export of alternatively processed mRNAs, and Rev is also linked to translation of viral mRNAs and genome encapsidation. Previously, the human DEAD-box helicase DDX1 was suggested to be involved in Rev functions, but this relationship is not well understood. Biochemical studies of DDX1 and its interactions with Rev and model RNA oligonucleotides were carried out to investigate the molecular basis for association of these components. A combination of gel-filtration chromatography and circular dichroism spectroscopy demonstrated that recombinant DDX1 expressed in Escherichia coli is a well-behaved folded protein. Binding assays using fluorescently labeled Rev and cell-based immunoprecipitation analysis confirmed a specific RNA-independent DDX1–Rev interaction. Additionally, DDX1 was shown to be an RNA-activated ATPase, wherein Rev-bound RNA was equally effective at stimulating ATPase activity as protein-free RNA. Gel mobility shift assays further demonstrated that DDX1 forms complexes with Rev-bound RNA. RNA silencing of DDX1 provided strong evidence that DDX1 is required for both Rev activity and HIV production from infected cells. Collectively, these studies demonstrate a clear link between DDX1 and HIV-1 Rev in cell-based assays of HIV-1 production and provide the first demonstration that recombinant DDX1 binds Rev and RNA and has RNA-dependent catalytic activity.
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Affiliation(s)
- Stephen P Edgcomb
- Department of Molecular Biology and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA 92037, USA
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Xin Z, Han W, Zhao Z, Xia Q, Yin B, Yuan J, Peng X. PCBP2 enhances the antiviral activity of IFN-α against HCV by stabilizing the mRNA of STAT1 and STAT2. PLoS One 2011; 6:e25419. [PMID: 22022391 PMCID: PMC3191149 DOI: 10.1371/journal.pone.0025419] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2011] [Accepted: 09/03/2011] [Indexed: 11/28/2022] Open
Abstract
Interferon-α (IFN-α) is a natural choice for the treatment of hepatitis C, but half of the chronically infected individuals do not achieve sustained clearance of hepatitis C virus (HCV) during treatment with IFN-α alone. The virus can impair IFN-α signaling and cellular factors that have an effect on the viral life cycles. We found that the protein PCBP2 is down-regulated in HCV-replicon containing cells (R1b). However, the effects and mechanisms of PCBP2 on HCV are unclear. To determine the effect of PCBP2 on HCV, overexpression and knockdown of PCBP2 were performed in R1b cells. Interestingly, we found that PCBP2 can facilitate the antiviral activity of IFN-α against HCV, although the RNA level of HCV was unaffected by either the overexpression or absence of PCBP2 in R1b cells. RIP-qRT-PCR and RNA half-life further revealed that PCBP2 stabilizes the mRNA of STAT1 and STAT2 through binding the 3′Untranslated Region (UTR) of these two molecules, which are pivotal for the IFN-α anti-HCV effect. RNA pull-down assay confirmed that there were binding sites located in the C-rich tracts in the 3′UTR of their mRNAs. Stabilization of mRNA by PCBP2 leads to the increased protein expression of STAT1 and STAT2 and a consistent increase of phosphorylated STAT1 and STAT2. These effects, in turn, enhance the antiviral effect of IFN-α. These findings indicate that PCBP2 may play an important role in the IFN-α response against HCV and may benefit the HCV clinical therapy.
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Affiliation(s)
- Zhongshuai Xin
- National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, China
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Hepatitis C virus co-opts Ras-GTPase-activating protein-binding protein 1 for its genome replication. J Virol 2011; 85:6996-7004. [PMID: 21561913 DOI: 10.1128/jvi.00013-11] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
We recently reported that Ras-GTPase-activating protein-binding protein 1 (G3BP1) interacts with hepatitis C virus (HCV) nonstructural protein (NS)5B and the 5' end of the HCV minus-strand RNA. In the current study we confirmed these observations using immunoprecipitation and RNA pulldown assays, suggesting that G3BP1 might be an HCV replication complex (RC) component. In replicon cells, transfected G3BP1 interacts with multiple HCV nonstructural proteins. Using immunostaining and confocal microscopy, we demonstrate that G3BP1 is colocalized with HCV RCs in replicon cells. Small interfering RNA (siRNA)-mediated knockdown of G3BP1 moderately reduces established HCV RNA replication in HCV replicon cells and dramatically reduces HCV replication-dependent colony formation and cell-culture-produced HCV (HCVcc) infection. In contrast, knockdown of G3BP2 has no effect on HCVcc infection. Transient replication experiments show that G3BP1 is involved in HCV genome amplification. Thus, G3BP1 is associated with HCV RCs and may be co-opted as a functional RC component for viral replication. These findings may facilitate understanding of the molecular mechanisms of HCV genome replication.
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Hepatitis C virus hijacks P-body and stress granule components around lipid droplets. J Virol 2011; 85:6882-92. [PMID: 21543503 DOI: 10.1128/jvi.02418-10] [Citation(s) in RCA: 142] [Impact Index Per Article: 10.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023] Open
Abstract
The microRNA miR-122 and DDX6/Rck/p54, a microRNA effector, have been implicated in hepatitis C virus (HCV) replication. In this study, we demonstrated for the first time that HCV-JFH1 infection disrupted processing (P)-body formation of the microRNA effectors DDX6, Lsm1, Xrn1, PATL1, and Ago2, but not the decapping enzyme DCP2, and dynamically redistributed these microRNA effectors to the HCV production factory around lipid droplets in HuH-7-derived RSc cells. Notably, HCV-JFH1 infection also redistributed the stress granule components GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), ataxin-2 (ATX2), and poly(A)-binding protein 1 (PABP1) to the HCV production factory. In this regard, we found that the P-body formation of DDX6 began to be disrupted at 36 h postinfection. Consistently, G3BP1 transiently formed stress granules at 36 h postinfection. We then observed the ringlike formation of DDX6 or G3BP1 and colocalization with HCV core after 48 h postinfection, suggesting that the disruption of P-body formation and the hijacking of P-body and stress granule components occur at a late step of HCV infection. Furthermore, HCV infection could suppress stress granule formation in response to heat shock or treatment with arsenite. Importantly, we demonstrate that the accumulation of HCV RNA was significantly suppressed in DDX6, Lsm1, ATX2, and PABP1 knockdown cells after the inoculation of HCV-JFH1, suggesting that the P-body and the stress granule components are required for the HCV life cycle. Altogether, HCV seems to hijack the P-body and the stress granule components for HCV replication.
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Comparison of alternative extraction methods for secretome profiling in human hepatocellular carcinoma cells. SCIENCE CHINA-LIFE SCIENCES 2011; 54:34-8. [DOI: 10.1007/s11427-010-4122-1] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/11/2010] [Accepted: 08/30/2010] [Indexed: 12/25/2022]
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