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Gurung D, Danielson JA, Tasnim A, Zhang JT, Zou Y, Liu JY. Proline Isomerization: From the Chemistry and Biology to Therapeutic Opportunities. BIOLOGY 2023; 12:1008. [PMID: 37508437 PMCID: PMC10376262 DOI: 10.3390/biology12071008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/02/2023] [Revised: 06/27/2023] [Accepted: 07/07/2023] [Indexed: 07/30/2023]
Abstract
Proline isomerization, the process of interconversion between the cis- and trans-forms of proline, is an important and unique post-translational modification that can affect protein folding and conformations, and ultimately regulate protein functions and biological pathways. Although impactful, the importance and prevalence of proline isomerization as a regulation mechanism in biological systems have not been fully understood or recognized. Aiming to fill gaps and bring new awareness, we attempt to provide a wholistic review on proline isomerization that firstly covers what proline isomerization is and the basic chemistry behind it. In this section, we vividly show that the cause of the unique ability of proline to adopt both cis- and trans-conformations in significant abundance is rooted from the steric hindrance of these two forms being similar, which is different from that in linear residues. We then discuss how proline isomerization was discovered historically followed by an introduction to all three types of proline isomerases and how proline isomerization plays a role in various cellular responses, such as cell cycle regulation, DNA damage repair, T-cell activation, and ion channel gating. We then explore various human diseases that have been linked to the dysregulation of proline isomerization. Finally, we wrap up with the current stage of various inhibitors developed to target proline isomerases as a strategy for therapeutic development.
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Affiliation(s)
- Deepti Gurung
- Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, USA
- Department of Cell and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA
| | - Jacob A Danielson
- Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, USA
| | - Afsara Tasnim
- Department of Bioengineering, University of Toledo College of Engineering, Toledo, OH 43606, USA
| | - Jian-Ting Zhang
- Department of Cell and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA
| | - Yue Zou
- Department of Cell and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA
| | - Jing-Yuan Liu
- Department of Medicine, University of Toledo College of Medicine, Toledo, OH 43614, USA
- Department of Cell and Cancer Biology, University of Toledo College of Medicine, Toledo, OH 43614, USA
- Department of Bioengineering, University of Toledo College of Engineering, Toledo, OH 43606, USA
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Huynh TTX, Pham TX, Lee GH, Lee JB, Lee SG, Tark D, Lim YS, Hwang SB. Amuvatinib Blocks SARS-CoV-2 Infection at the Entry Step of the Viral Life Cycle. Microbiol Spectr 2023; 11:e0510522. [PMID: 36995225 PMCID: PMC10269473 DOI: 10.1128/spectrum.05105-22] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/10/2023] [Accepted: 03/14/2023] [Indexed: 03/31/2023] Open
Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease 2019 (COVID-19). SARS-CoV-2 propagation is mediated by the protein interaction between viral proteins and host cells. Tyrosine kinase has been implicated in viral replication, and hence, it has become a target for developing antiviral drugs. We have previously reported that receptor tyrosine kinase inhibitor blocks the replication of hepatitis C virus (HCV). In the present study, we investigated two receptor tyrosine kinase-specific inhibitors, amuvatinib and imatinib, for their potential antiviral efficacies against SARS-CoV-2. Treatment with either amuvatinib or imatinib displays an effective inhibitory activity against SARS-CoV-2 propagation without an obvious cytopathic effect in Vero E6 cells. Notably, amuvatinib exerts a stronger antiviral activity than imatinib against SARS-CoV-2 infection. Amuvatinib blocks SARS-CoV-2 infection with a 50% effective concentration (EC50) value ranging from ~0.36 to 0.45 μM in Vero E6 cells. We further demonstrate that amuvatinib inhibits SARS-CoV-2 propagation in human lung Calu-3 cells. Using pseudoparticle infection assay, we verify that amuvatinib blocks SARS-CoV-2 at the entry step of the viral life cycle. More specifically, amuvatinib inhibits SARS-CoV-2 infection at the binding-attachment step. Moreover, amuvatinib exhibits highly efficient antiviral activity against emerging SARS-CoV-2 variants. Importantly, we demonstrate that amuvatinib inhibits SARS-CoV-2 infection by blocking ACE2 cleavage. Taken together, our data suggest that amuvatinib may provide a potential therapeutic agent for the treatment of COVID-19. IMPORTANCE Tyrosine kinase has been implicated in viral replication and has become an antiviral drug target. Here, we chose two well-known receptor tyrosine kinase inhibitors, amuvatinib and imatinib, and evaluated their drug potencies against SARS-CoV-2. Surprisingly, amuvatinib displays a stronger antiviral activity than imatinib against SARS-CoV-2. Amuvatinib blocks SARS-CoV-2 infection by inhibiting ACE2 cleavage and the subsequent soluble ACE2 receptor. All these data suggest that amuvatinib may be a potential therapeutic agent in SARS-CoV-2 prevention for those experiencing vaccine breakthroughs.
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Affiliation(s)
- Trang T. X. Huynh
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Thuy X. Pham
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Gun-Hee Lee
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Jae-Bong Lee
- Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Sung-Geun Lee
- Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, South Korea
- Ilsong Institute of Life Science, Hallym University, Seoul, South Korea
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3
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McPhail JA, Burke JE. Molecular mechanisms of PI4K regulation and their involvement in viral replication. Traffic 2023; 24:131-145. [PMID: 35579216 DOI: 10.1111/tra.12841] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/06/2022] [Revised: 03/07/2022] [Accepted: 03/30/2022] [Indexed: 11/28/2022]
Abstract
Lipid phosphoinositides are master signaling molecules in eukaryotic cells and key markers of organelle identity. Because of these important roles, the kinases and phosphatases that generate phosphoinositides must be tightly regulated. Viruses can manipulate this regulation, with the Type III phosphatidylinositol 4-kinases (PI4KA and PI4KB) being hijacked by many RNA viruses to mediate their intracellular replication through the formation of phosphatidylinositol 4-phosphate (PI4P)-enriched replication organelles (ROs). Different viruses have evolved unique approaches toward activating PI4K enzymes to form ROs, through both direct binding of PI4Ks and modulation of PI4K accessory proteins. This review will focus on PI4KA and PI4KB and discuss their roles in signaling, functions in membrane trafficking and manipulation by viruses. Our focus will be the molecular basis for how PI4KA and PI4KB are activated by both protein-binding partners and post-translational modifications, with an emphasis on understanding the different molecular mechanisms viruses have evolved to usurp PI4Ks. We will also discuss the chemical tools available to study the role of PI4Ks in viral infection.
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Affiliation(s)
- Jacob A McPhail
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada
| | - John E Burke
- Department of Biochemistry and Microbiology, University of Victoria, Victoria, British Columbia, Canada.,Department of Biochemistry and Molecular Biology, The University of British Columbia, Vancouver, British Columbia, Canada
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4
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Hepatitis C Virus-Lipid Interplay: Pathogenesis and Clinical Impact. Biomedicines 2023; 11:biomedicines11020271. [PMID: 36830808 PMCID: PMC9953247 DOI: 10.3390/biomedicines11020271] [Citation(s) in RCA: 14] [Impact Index Per Article: 7.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/16/2022] [Revised: 01/13/2023] [Accepted: 01/16/2023] [Indexed: 01/20/2023] Open
Abstract
Hepatitis C virus (HCV) infection represents the major cause of chronic liver disease, leading to a wide range of hepatic diseases, including cirrhosis and hepatocellular carcinoma. It is the leading indication for liver transplantation worldwide. In addition, there is a growing body of evidence concerning the role of HCV in extrahepatic manifestations, including immune-related disorders and metabolic abnormalities, such as insulin resistance and steatosis. HCV depends on its host cells to propagate successfully, and every aspect of the HCV life cycle is closely related to human lipid metabolism. The virus circulates as a lipid-rich particle, entering the hepatocyte via lipoprotein cell receptors. It has also been shown to upregulate lipid biosynthesis and impair lipid degradation, resulting in significant intracellular lipid accumulation (steatosis) and circulating hypocholesterolemia. Patients with chronic HCV are at increased risk for hepatic steatosis, dyslipidemia, and cardiovascular disease, including accelerated atherosclerosis. This review aims to describe different aspects of the HCV viral life cycle as it impacts host lipoproteins and lipid metabolism. It then discusses the mechanisms of HCV-related hepatic steatosis, hypocholesterolemia, and accelerated atherosclerosis.
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Lu Y, He P, Zhang Y, Ren Y, Zhang L. The emerging roles of retromer and sorting nexins in the life cycle of viruses. Virol Sin 2022; 37:321-330. [PMID: 35513271 PMCID: PMC9057928 DOI: 10.1016/j.virs.2022.04.014] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Accepted: 04/12/2022] [Indexed: 02/06/2023] Open
Abstract
Retromer and sorting nexins (SNXs) transport cargoes from endosomes to the trans-Golgi network or plasma membrane. Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses, including members of Coronaviridae, Flaviviridae and Retroviridae. Key components of retromer/SNXs, such as Vps35, Vps26, SNX5 and SNX27, can affect multiple steps of the viral life cycle, including facilitating the entry of viruses into cells, participating in viral replication, and promoting the assembly of virions. Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus, which will shed mechanistic insights into controlling virus infection.
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Affiliation(s)
- Yue Lu
- Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan, 250013, China; Department of Pathogen Biology, School of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China; Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China
| | - Ping He
- Department of Pathogen Biology, School of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China; Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China
| | - Yuxuan Zhang
- Department of Pathogen Biology, School of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China; Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China
| | - Yongwen Ren
- Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan, 250013, China; Department of Pathogen Biology, School of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China; Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China
| | - Leiliang Zhang
- Department of Clinical Laboratory Medicine, The First Affiliated Hospital of Shandong First Medical University & Shandong Provincial Qianfoshan Hospital, Jinan, 250013, China; Department of Pathogen Biology, School of Basic Medical Sciences, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China; Medical Science and Technology Innovation Center, Shandong First Medical University & Shandong Academy of Medical Sciences, Jinan, 250117, China.
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6
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Lim YS, Nguyen MT, Pham TX, Huynh TT, Park EM, Choi DH, Kang SM, Tark D, Hwang SB. Hepatitis C Virus Nonstructural 5A Protein Interacts with Telomere Length Regulation Protein: Implications for Telomere Shortening in Patients Infected with HCV. Mol Cells 2022; 45:148-157. [PMID: 34949741 PMCID: PMC8926864 DOI: 10.14348/molcells.2021.0167] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2021] [Revised: 10/17/2021] [Accepted: 10/27/2021] [Indexed: 11/27/2022] Open
Abstract
Hepatitis C virus (HCV) is a major cause of chronic liver disease and is highly dependent on cellular proteins for viral propagation. Using protein microarray analysis, we identified 90 cellular proteins as HCV nonstructural 5A (NS5A) interacting partners, and selected telomere length regulation protein (TEN1) for further study. TEN1 forms a heterotrimeric complex with CTC and STN1, which is essential for telomere protection and maintenance. Telomere length decreases in patients with active HCV, chronic liver disease, and hepatocellular carcinoma. However, the molecular mechanism of telomere length shortening in HCV-associated disease is largely unknown. In the present study, protein interactions between NS5A and TEN1 were confirmed by immunoprecipitation assays. Silencing of TEN1 reduced both viral RNA and protein expression levels of HCV, while ectopic expression of the siRNA-resistant TEN1 recovered the viral protein level, suggesting that TEN1 was specifically required for HCV propagation. Importantly, we found that TEN1 is re-localized from the nucleus to the cytoplasm in HCV-infected cells. These data suggest that HCV exploits TEN1 to promote viral propagation and that telomere protection is compromised in HCV-infected cells. Overall, our findings provide mechanistic insight into the telomere shortening in HCV-infected cells.
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Affiliation(s)
- Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Men T.N. Nguyen
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
| | - Thuy X. Pham
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Trang T.X. Huynh
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Eun-Mee Park
- Center for Immunology and Pathology, National Institute of Health, Korea Center for Disease Control & Prevention, Cheongju 28159, Korea
| | - Dong Hwa Choi
- Biocenter, Gyeonggido Business & Science Accelerator, Suwon 16229, Korea
| | - Sang Min Kang
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
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7
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Bulankina AV, Richter RM, Welsch C. Regulatory Role of Phospholipids in Hepatitis C Virus Replication and Protein Function. Pathogens 2022; 11:102. [PMID: 35056049 PMCID: PMC8779051 DOI: 10.3390/pathogens11010102] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/28/2021] [Revised: 01/12/2022] [Accepted: 01/13/2022] [Indexed: 11/16/2022] Open
Abstract
Positive-strand RNA viruses such as hepatitis C virus (HCV) hijack key factors of lipid metabolism of infected cells and extensively modify intracellular membranes to support the viral lifecycle. While lipid metabolism plays key roles in viral particle assembly and maturation, viral RNA synthesis is closely linked to the remodeling of intracellular membranes. The formation of viral replication factories requires a number of interactions between virus proteins and host factors including lipids. The structure-function relationship of those proteins is influenced by their lipid environments and lipids that selectively modulate protein function. Here, we review our current understanding on the roles of phospholipids in HCV replication and of lipid-protein interactions in the structure-function relationship of the NS5A protein. NS5A is a key factor in membrane remodeling in HCV-infected cells and is known to recruit phosphatidylinositol 4-kinase III alpha to generate phosphatidylinositol 4-phosphate at the sites of replication. The dynamic interplay between lipids and viral proteins within intracellular membranes is likely key towards understanding basic mechanisms in the pathobiology of virus diseases, the mode of action of specific antiviral agents and related drug resistance mechanisms.
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Affiliation(s)
- Anna V. Bulankina
- Department of Internal Medicine 1, Goethe University Hospital Frankfurt, 60590 Frankfurt, Germany; (A.V.B.); (R.M.R.)
- Research Group “Molecular Evolution & Adaptation”, 60590 Frankfurt, Germany
| | - Rebecca M. Richter
- Department of Internal Medicine 1, Goethe University Hospital Frankfurt, 60590 Frankfurt, Germany; (A.V.B.); (R.M.R.)
- Research Group “Molecular Evolution & Adaptation”, 60590 Frankfurt, Germany
| | - Christoph Welsch
- Department of Internal Medicine 1, Goethe University Hospital Frankfurt, 60590 Frankfurt, Germany; (A.V.B.); (R.M.R.)
- Research Group “Molecular Evolution & Adaptation”, 60590 Frankfurt, Germany
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8
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Hasanshahi Z, Hashempour A, Ghasabi F, Moayedi J, Musavi Z, Dehghani B, Sharafi H, Joulaei H. First report on molecular docking analysis and drug resistance substitutions to approved HCV NS5A and NS5B inhibitors amongst Iranian patients. BMC Gastroenterol 2021; 21:443. [PMID: 34819046 PMCID: PMC8612383 DOI: 10.1186/s12876-021-01988-y] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/18/2021] [Accepted: 10/22/2021] [Indexed: 12/13/2022] Open
Abstract
Background NS5A and NS5B proteins of hepatitis C virus (HCV) are the main targets of compounds that directly inhibit HCV infections. However, the emergence of resistance-associated substitutions (RASs) may cause substantial reductions in susceptibility to inhibitors. Methods Viral load and genotyping were determined in eighty-seven naïve HCV-infected patients, and the amplified NS5A and NS5B regions were sequenced by Sanger sequencing. In addition, physicochemical properties, structural features, immune epitopes, and inhibitors-protein interactions of sequences were analyzed using several bioinformatics tools. Results Several amino acid residue changes were found in NS5A and NS5B proteins; however, we did not find any mutations related to resistance to the treatment in NS5B. Different phosphorylation and few glycosylation sites were assessed. Disulfide bonds were identified in both proteins that had a significant effect on the function and structure of HCV proteins. Applying reliable software to predict B-cell epitopes, 3 and 5 regions were found for NS5A and NS5B, respectively, representing a considerable potential to induce the humoral immune system. Docking analysis determined amino acids involved in the interaction of inhibitors and mentioned proteins may not decrease the drug efficiency. Conclusions Strong interactions between inhibitors, NS5A and NS5B proteins and the lack of efficient drug resistance mutations in the analyzed sequences may confirm the remarkable ability of NS5A and NS5B inhibitors to control HCV infection amongst Iranian patients. The results of bioinformatics analysis could unveil all features of both proteins, which can be beneficial for further investigations on HCV drug resistance and designing novel vaccines. Supplementary Information The online version contains supplementary material available at 10.1186/s12876-021-01988-y.
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Affiliation(s)
- Zahra Hasanshahi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Ava Hashempour
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran.
| | - Farzane Ghasabi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Javad Moayedi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Zahra Musavi
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Behzad Dehghani
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
| | - Heidar Sharafi
- Baqiyatallah Research Center for Gastroenterology and Liver Diseases, Baqiyatallah University of Medical Sciences, Tehran, Iran.,Middle East Liver Diseases (MELD) Center, Tehran, Iran
| | - Hassan Joulaei
- Shiraz HIV/AIDS Research Center, Institute of Health, Shiraz University of Medical Sciences, Shiraz, Iran
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9
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Lim YS, Nguyen LP, Lee GH, Lee SG, Lyoo KS, Kim B, Hwang SB. Asunaprevir, a Potent Hepatitis C Virus Protease Inhibitor, Blocks SARS-CoV-2 Propagation. Mol Cells 2021; 44:688-695. [PMID: 34518443 PMCID: PMC8490202 DOI: 10.14348/molcells.2021.0076] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/29/2021] [Revised: 07/19/2021] [Accepted: 07/20/2021] [Indexed: 11/27/2022] Open
Abstract
The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has become a global health concern. Various SARS-CoV-2 vaccines have been developed and are being used for vaccination worldwide. However, no therapeutic agents against coronavirus disease 2019 (COVID-19) have been developed so far; therefore, new therapeutic agents are urgently needed. In the present study, we evaluated several hepatitis C virus direct-acting antivirals as potential candidates for drug repurposing against COVID-19. Theses include asunaprevir (a protease inhibitor), daclatasvir (an NS5A inhibitor), and sofosbuvir (an RNA polymerase inhibitor). We found that asunaprevir, but not sofosbuvir and daclatasvir, markedly inhibited SARS-CoV-2-induced cytopathic effects in Vero E6 cells. Both RNA and protein levels of SARS-CoV-2 were significantly decreased by treatment with asunaprevir. Moreover, asunaprevir profoundly decreased virion release from SARS-CoV-2-infected cells. A pseudoparticle entry assay revealed that asunaprevir blocked SARS-CoV-2 infection at the binding step of the viral life cycle. Furthermore, asunaprevir inhibited SARS-CoV-2 propagation in human lung Calu-3 cells. Collectively, we found that asunaprevir displays broad-spectrum antiviral activity and therefore might be worth developing as a new drug repurposing candidate for COVID-19.
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Affiliation(s)
- Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Lap P. Nguyen
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
| | - Gun-Hee Lee
- Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Sung-Geun Lee
- Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Kwang-Soo Lyoo
- Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Bumseok Kim
- College of Veterinary Medicine, Jeonbuk National University, Iksan 54531, Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
- Ilsong Institute of Life Science, Hallym University, Seoul 07247, Korea
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10
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Ashlin TG, Blunsom NJ, Cockcroft S. Courier service for phosphatidylinositol: PITPs deliver on demand. Biochim Biophys Acta Mol Cell Biol Lipids 2021; 1866:158985. [PMID: 34111527 PMCID: PMC8266687 DOI: 10.1016/j.bbalip.2021.158985] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/24/2021] [Revised: 05/18/2021] [Accepted: 06/01/2021] [Indexed: 12/30/2022]
Abstract
Phosphatidylinositol is the parent lipid for the synthesis of seven phosphorylated inositol lipids and each of them play specific roles in numerous processes including receptor-mediated signalling, actin cytoskeleton dynamics and membrane trafficking. PI synthesis is localised to the endoplasmic reticulum (ER) whilst its phosphorylated derivatives are found in other organelles where the lipid kinases also reside. Phosphorylation of PI to phosphatidylinositol (4,5) bisphosphate (PI(4,5)P2) at the plasma membrane and to phosphatidylinositol 4-phosphate (PI4P) at the Golgi are key events in lipid signalling and Golgi function respectively. Here we review a family of proteins, phosphatidylinositol transfer proteins (PITPs), that can mobilise PI from the ER to provide the substrate to the resident kinases for phosphorylation. Recent studies identify specific and overlapping functions for the three soluble PITPs (PITPα, PITPβ and PITPNC1) in phospholipase C signalling, neuronal function, membrane trafficking, viral replication and in cancer metastases.
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Affiliation(s)
- Tim G Ashlin
- Dept. of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London WC1E 6JJ, UK
| | - Nicholas J Blunsom
- Dept. of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London WC1E 6JJ, UK
| | - Shamshad Cockcroft
- Dept. of Neuroscience, Physiology and Pharmacology, Division of Biosciences, University College London, London WC1E 6JJ, UK.
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11
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Li HC, Yang CH, Lo SY. Hepatitis C Viral Replication Complex. Viruses 2021; 13:v13030520. [PMID: 33809897 PMCID: PMC8004249 DOI: 10.3390/v13030520] [Citation(s) in RCA: 11] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2021] [Revised: 03/18/2021] [Accepted: 03/19/2021] [Indexed: 12/16/2022] Open
Abstract
The life cycle of the hepatitis C virus (HCV) can be divided into several stages, including viral entry, protein translation, RNA replication, viral assembly, and release. HCV genomic RNA replication occurs in the replication organelles (RO) and is tightly linked to ER membrane alterations containing replication complexes (proteins NS3 to NS5B). The amplification of HCV genomic RNA could be regulated by the RO biogenesis, the viral RNA structure (i.e., cis-acting replication elements), and both viral and cellular proteins. Studies on HCV replication have led to the development of direct-acting antivirals (DAAs) targeting the replication complex. This review article summarizes the viral and cellular factors involved in regulating HCV genomic RNA replication and the DAAs that inhibit HCV replication.
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Affiliation(s)
- Hui-Chun Li
- Department of Biochemistry, Tzu Chi University, Hualien 97004, Taiwan;
| | - Chee-Hing Yang
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
| | - Shih-Yen Lo
- Department of Laboratory Medicine and Biotechnology, Tzu Chi University, Hualien 97004, Taiwan;
- Department of Laboratory Medicine, Buddhist Tzu Chi General Hospital, Hualien 97004, Taiwan
- Correspondence: ; Tel.: +886-3-8565301 (ext. 2322)
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12
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Lim YS, Mai HN, Nguyen LP, Kang SM, Tark D, Hwang SB. Adenosylhomocysteinase like 1 interacts with nonstructural 5A and regulates hepatitis C virus propagation. J Microbiol 2020; 59:101-109. [PMID: 33355889 DOI: 10.1007/s12275-021-0470-8] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2020] [Revised: 10/21/2020] [Accepted: 11/03/2020] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) life cycle is highly dependent on cellular proteins for viral propagation. In order to identify the cellular factors involved in HCV propagation, we previously performed a protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼9,000 human cellular proteins immobilized in a microarray, adenosylhomocysteinase like 1 (AHCYL1) was among 90 proteins identified as NS5A interactors. Of these candidates, AHCYL1 was selected for further study. In the present study, we verified the physical interaction between NS5A and AHCYL1 by both in vitro pulldown and coimmunoprecipitation assays. Furthermore, HCV NS5A interacted with endogenous AHCYL1 in Jc1-infected cells. Both NS5A and AHCYL1 were colocalized in the cytoplasmic region in HCV-replicating cells. siRNAmediated knockdown of AHCYL1 abrogated HCV propagation. Exogenous expression of the siRNA-resistant AHCYL1 mutant, but not of the wild-type AHCYL1, restored HCV protein expression levels, indicating that AHCYL1 was required specifically for HCV propagation. Importantly, AHCYL1 was involved in the HCV internal ribosome entry site-mediated translation step of the HCV life cycle. Finally, we demonstrated that the proteasomal degradation pathway of AHCYL1 was modulated by persistent HCV infection. Collectively, these data suggest that HCV may modulate the AHCYL1 protein to promote viral propagation.
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Affiliation(s)
- Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea
| | - Han N Mai
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea.,Ilsong Institute of Life Science, Hallym University, Anyang, 14066, Republic of Korea
| | - Lap P Nguyen
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea
| | - Sang Min Kang
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea
| | - Soon B Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan, 54531, Republic of Korea. .,Ilsong Institute of Life Science, Hallym University, Anyang, 14066, Republic of Korea.
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Cortactin Interacts with Hepatitis C Virus Core and NS5A Proteins: Implications for Virion Assembly. J Virol 2020; 94:JVI.01306-20. [PMID: 32727880 DOI: 10.1128/jvi.01306-20] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/29/2020] [Accepted: 07/17/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) exploits cellular proteins to facilitate viral propagation. To identify the cellular factors involved in the HCV life cycle, we previously performed protein microarray assays using either HCV nonstructural 5A (NS5A) protein or core protein as a probe. Interestingly, cellular cortactin strongly interacted with both NS5A and core. Cortactin is an actin-binding protein critically involved in tumor progression by regulating the migration and invasion of cancerous cells. Protein interaction between cortactin and NS5A or core was confirmed by coimmunoprecipitation and immunofluorescence assays. We showed that cortactin interacted with NS5A and core via the N-terminal acidic domain of cortactin. Cortactin expression levels were not altered by HCV infection. Small interfering RNA (siRNA)-mediated knockdown of cortactin dramatically decreased HCV protein expression and infectivity levels, whereas overexpression of cortactin increased viral propagation. Ectopic expression of the siRNA-resistant cortactin recovered the viral infectivity, suggesting that cortactin was specifically required for HCV propagation. We further showed that cortactin was involved in the assembly step without affecting viral entry, HCV internal ribosome entry site (IRES)-mediated translation, and the replication steps of the HCV life cycle. Of note, silencing of cortactin markedly reduced both NS5A and core protein levels on the lipid droplets (LDs), and this effect was reversed by the overexpression of cortactin. Importantly, NS5A and core promoted cell migration by activating the phosphorylation of cortactin at tyrosine residues 421 and 466. Taken together, these data suggest that cortactin is not only involved in HCV assembly but also plays an important role in the cell migration.IMPORTANCE Cortactin is a cytoskeletal protein that regulates cell migration in response to a number of extracellular stimuli. The functional involvement of cortactin in the virus life cycle is not yet fully understood. The most significant finding is that cortactin strongly interacted with both hepatitis C virus (HCV) core and NS5A. Cortactin is involved in HCV assembly by tethering core and NS5A on the lipid droplets (LDs) with no effect on LD biogenesis. It was noteworthy that HCV NS5A and core activated cortactin by phosphorylation at tyrosines 421 and 466 to regulate cell migration. Collectively, our study shows that cortactin is a novel host factor involved in viral production and HCV-associated pathogenesis.
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Choi JW, Kim JW, Nguyen LP, Nguyen HC, Park EM, Choi DH, Han KM, Kang SM, Tark D, Lim YS, Hwang SB. Nonstructural NS5A Protein Regulates LIM and SH3 Domain Protein 1 to Promote Hepatitis C Virus Propagation. Mol Cells 2020; 43:469-478. [PMID: 32344996 PMCID: PMC7264479 DOI: 10.14348/molcells.2020.0018] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/14/2020] [Revised: 02/26/2020] [Accepted: 03/20/2020] [Indexed: 12/11/2022] Open
Abstract
Hepatitis C virus (HCV) propagation is highly dependent on cellular proteins. To identify the host factors involved in HCV propagation, we previously performed protein microarray assays and identified the LIM and SH3 domain protein 1 (LASP-1) as an HCV NS5A-interacting partner. LASP-1 plays an important role in the regulation of cell proliferation, migration, and protein-protein interactions. Alteration of LASP-1 expression has been implicated in hepatocellular carcinoma. However, the functional involvement of LASP1 in HCV propagation and HCV-induced pathogenesis has not been elucidated. Here, we first verified the protein interaction of NS5A and LASP-1 by both in vitro pulldown and coimmunoprecipitation assays. We further showed that NS5A and LASP-1 were colocalized in the cytoplasm of HCV infected cells. NS5A interacted with LASP-1 through the proline motif in domain I of NS5A and the tryptophan residue in the SH3 domain of LASP-1. Knockdown of LASP-1 increased HCV replication in both HCV-infected cells and HCV subgenomic replicon cells. LASP-1 negatively regulated viral propagation and thereby overexpression of LASP-1 decreased HCV replication. Moreover, HCV propagation was decreased by wild-type LASP-1 but not by an NS5A binding-defective mutant of LASP-1. We further demonstrated that LASP-1 was involved in the replication stage of the HCV life cycle. Importantly, LASP-1 expression levels were increased in persistently infected cells with HCV. These data suggest that HCV modulates LASP-1 via NS5A in order to regulate virion levels and maintain a persistent infection.
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Affiliation(s)
- Jae-Woong Choi
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 5453, Korea
- Ilsong Institute of Life Science, Hallym University, Anyang 14066, Korea
| | - Jong-Wook Kim
- Ilsong Institute of Life Science, Hallym University, Anyang 14066, Korea
| | - Lap P. Nguyen
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 5453, Korea
- Ilsong Institute of Life Science, Hallym University, Anyang 14066, Korea
| | - Huu C. Nguyen
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 5453, Korea
| | - Eun-Mee Park
- Center for Immunology and Pathology, National Institute of Health, Korea Center for Disease Control & Prevention, Cheongju 28159, Korea
| | - Dong Hwa Choi
- Biocenter, Gyeonggido Business & Science Accelerator, Suwon 16229, Korea
- Graduate School of East-West Medical Science, Kyung Hee University, Yongin 17104, Korea
| | - Kang Min Han
- Department of Pathology, Dongguk University Ilsan Hospital, Goyang 1032, Korea
| | - Sang Min Kang
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Dongseob Tark
- Laboratory for Infectious Disease Prevention, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 54531, Korea
| | - Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 5453, Korea
- Ilsong Institute of Life Science, Hallym University, Anyang 14066, Korea
| | - Soon B. Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Jeonbuk National University, Iksan 5453, Korea
- Ilsong Institute of Life Science, Hallym University, Anyang 14066, Korea
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Cai H, Yao W, Huang J, Xiao J, Chen W, Hu L, Mai R, Liang M, Chen D, Jiang N, Zhou L, Peng T. Apolipoprotein M, identified as a novel hepatitis C virus (HCV) particle associated protein, contributes to HCV assembly and interacts with E2 protein. Antiviral Res 2020; 177:104756. [PMID: 32119870 DOI: 10.1016/j.antiviral.2020.104756] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2019] [Revised: 01/18/2020] [Accepted: 02/25/2020] [Indexed: 02/08/2023]
Abstract
Hepatitis C virus (HCV) infection is a major cause of chronic liver diseases such as steatosis, cirrhosis, and hepatocellular carcinoma. HCV particles have been found to associate with apolipoproteins, and apolipoproteins not only participate in the HCV life cycle, but also help HCV escape recognition by the host immune system, which pose challenges for the development of both HCV treatments and vaccines. However, no study has reported on the comprehensive identification of apolipoprotein associations with HCV particles. In the present study, we performed proteome analysis by affinity purification coupled with mass spectrometry (AP-MS) to comprehensively identify the apolipoprotein associations with HCV particles, and ApoM was first identified by AP-MS besides the previously reported ApoE, ApoB, ApoA-I and ApoC-I. Additionally, three assays further confirmed that ApoM was a novel virus particle associated protein. We also showed that ApoM was required for HCV production, especially for the assembly/release step of HCV life cycle. Furthermore, ApoM interacted with the HCV E2 protein. Finally, HCV infection reduced ApoM expression both in vitro and in vivo. Collectively, our study demonstrates that ApoM, identified as a novel HCV particle associated protein, contributes to HCV assembly/release and interacts with HCV E2 protein. It provides new insights on how HCV and the host apolipoproteins are reciprocally influenced and lays a basis for research in developing innovative antiviral strategies.
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Affiliation(s)
- Hua Cai
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Wenxia Yao
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China.
| | - Jingxian Huang
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Jing Xiao
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Wenli Chen
- Department of Infectious Diseases, Guangdong Provincial People's Hospital, Guangzhou, China
| | - Longbo Hu
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Runming Mai
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Mengdi Liang
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Di Chen
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China
| | - Nan Jiang
- The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Li Zhou
- The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | - Tao Peng
- Guangzhou Hoffmann Institute of Immunology, College of Basic Sciences, The Fifth Affiliated Hospital of Guangzhou Medical University, Guangzhou Medical University, Guangzhou, China.
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Nguyen LP, Tran SC, Suetsugu S, Lim YS, Hwang SB. PACSIN2 Interacts with Nonstructural Protein 5A and Regulates Hepatitis C Virus Assembly. J Virol 2020; 94:e01531-19. [PMID: 31801866 PMCID: PMC7022371 DOI: 10.1128/jvi.01531-19] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/06/2019] [Accepted: 11/26/2019] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) is a major etiologic agent of chronic liver diseases. HCV is highly dependent on cellular machinery for viral propagation. Using protein microarray analysis, we previously identified 90 cellular proteins as nonstructural 5A (NS5A) interacting partners. Of these, protein kinase C and casein kinase substrate in neurons protein 2 (PACSIN2) was selected for further study. PACSIN2 belongs to the PACSIN family, which is involved in the formation of caveolae. Protein interaction between NS5A and PACSIN2 was confirmed by pulldown assay and further verified by both coimmunoprecipitation and immunofluorescence assays. We showed that PACSIN2 interacted with domain I of NS5A and the Fer-CIP4 homology (FCH)-Bin/amphiphysin/Rvs (F-BAR) region of PACSIN2. Interestingly, NS5A specifically attenuated protein kinase C alpha (PKCα)-mediated phosphorylation of PACSIN2 at serine 313 by interrupting PACSIN2 and PKCα interaction. In fact, mutation of the serine 313 to alanine (S313A) of PACSIN2 increased protein interaction with NS5A. Silencing of PACSIN2 decreased both viral RNA and protein expression levels of HCV. Ectopic expression of the small interfering RNA (siRNA)-resistant PACSIN2 recovered the viral infectivity, suggesting that PACSIN2 was specifically required for HCV propagation. PACSIN2 was involved in viral assembly without affecting other steps of the HCV life cycle. Indeed, overexpression of PACSIN2 promoted NS5A and core protein (core) interaction. We further showed that inhibition of PKCα increased NS5A and core interaction, suggesting that phosphorylation of PACSIN2 might influence HCV assembly. Moreover, PACSIN2 was required for lipid droplet formation via modulating extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Taken together, these data indicate that HCV modulates PACSIN2 via NS5A to promote virion assembly.IMPORTANCE PACSIN2 is a lipid-binding protein that triggers the tubulation of the phosphatidic acid-containing membranes. The functional involvement of PACSIN2 in the virus life cycle has not yet been demonstrated. We showed that phosphorylation of PACSIN2 displayed a negative effect on NS5A and core interaction. The most significant finding is that NS5A prevents PKCα from binding to PACSIN2. Therefore, the phosphorylation level of PACSIN2 is decreased in HCV-infected cells. We showed that HCV NS5A interrupted PKCα-mediated PACSIN2 phosphorylation at serine 313, thereby promoting NS5A-PACSIN2 interaction. We further demonstrated that PACSIN2 modulated lipid droplet formation through ERK1/2 phosphorylation. These data provide evidence that PACSIN2 is a proviral cellular factor required for viral propagation.
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Affiliation(s)
- Lap P Nguyen
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
| | - Si C Tran
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
| | - Shiro Suetsugu
- Laboratory of Molecular Medicine and Cell Biology, Graduate School of Biosciences, Nara Institute of Science and Technology, Ikoma, Japan
| | - Yun-Sook Lim
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
| | - Soon B Hwang
- Laboratory of RNA Viral Diseases, Korea Zoonosis Research Institute, Chonbuk National University, Iksan, South Korea
- National Research Laboratory of Hepatitis C Virus and Ilsong Institute of Life Science, Hallym University, Anyang, South Korea
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Phan TK, Bindra GK, Williams SA, Poon IK, Hulett MD. Combating Human Pathogens and Cancer by Targeting Phosphoinositides and Their Metabolism. Trends Pharmacol Sci 2019; 40:866-882. [DOI: 10.1016/j.tips.2019.09.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/15/2019] [Revised: 09/11/2019] [Accepted: 09/13/2019] [Indexed: 12/19/2022]
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Mansi, Kushwaha NK, Singh AK, Karim MJ, Chakraborty S. Nicotiana benthamiana phosphatidylinositol 4-kinase type II regulates chilli leaf curl virus pathogenesis. MOLECULAR PLANT PATHOLOGY 2019; 20:1408-1424. [PMID: 31475785 PMCID: PMC6792133 DOI: 10.1111/mpp.12846] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 09/08/2023]
Abstract
Geminiviruses are single-stranded DNA viruses that can cause significant losses in economically important crops. In recent years, the role of different kinases in geminivirus pathogenesis has been emphasized. Although geminiviruses use several host kinases, the role of phosphatidylinositol 4-kinase (PI4K) remains obscure. We isolated and characterized phosphatidylinositol 4-kinase type II from Nicotiana benthamiana (NbPI4KII) which interacts with the replication initiator protein (Rep) of a geminivirus, chilli leaf curl virus (ChiLCV). NbPI4KII-mGFP was localized into cytoplasm, nucleus or both. NbPI4KII-mGFP was also found to be associated with the cytoplasmic endomembrane systems in the presence of ChiLCV. Furthermore, we demonstrated that Rep protein directly interacts with NbPI4KII protein and influenced nuclear occurrence of NbPI4KII. The results obtained in the present study revealed that NbPI4KII is a functional protein kinase lacking lipid kinase activity. Downregulation of NbPI4KII expression negatively affects ChiLCV pathogenesis in N. benthamiana. In summary, NbPI4KII is a susceptible factor, which is required by ChiLCV for pathogenesis.
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Affiliation(s)
- Mansi
- Molecular Virology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
| | - Nirbhay Kumar Kushwaha
- Molecular Virology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
| | - Ashish Kumar Singh
- Molecular Virology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
| | - Mir Jishan Karim
- Molecular Virology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
| | - Supriya Chakraborty
- Molecular Virology Laboratory, School of Life SciencesJawaharlal Nehru UniversityNew DelhiIndia
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5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl)methyl]-L-proline Inhibits Hepatitis C Virus Entry. Sci Rep 2019; 9:7288. [PMID: 31086268 PMCID: PMC6514212 DOI: 10.1038/s41598-019-43783-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2019] [Accepted: 04/30/2019] [Indexed: 12/13/2022] Open
Abstract
Hepatitis C virus (HCV) is the major causative agent of chronic liver diseases, including liver cirrhosis and hepatocellular carcinoma. The recent development of highly effective direct-acting antivirals (DAAs) has revolutionized the treatment of HCV patients. However, these DAAs are exorbitantly expensive for the majority of HCV patients worldwide. Moreover, these drugs still show genotypic difference in cure rate and have some resistant-associated variants. Tylophorine, a natural compound derived from Tylophora indica plants, is known to have anti-inflammatory and anti-cancerous growth activities. In the present study, we showed that two tylophorine intermediates, 5-Oxo-1-[(2,3,6,7-tetramethoxy-9-phenanthrenyl) methyl]-L-proline (O859585) and 2,3,6,7-tetramethoxy-9-phenanthrenecarboxylic acid (T298875), displayed anti-HCV activity with an EC50 of 38.25 µM for T298875 and 29.11~35.3 µM for O859585 in various HCV genotypes. We demonstrated that O859585 efficiently blocked HCV attachment by neutralizing free viral particles without affecting other stages of the HCV life cycle and interferon stimulation. O859585 interrupted binding between HCV E2 and CD81. Of note, co-treatment of O859585 with either interferon alpha (IFNα) or sofosbuvir exerted either an additive or synergistic antiviral activity in HCV-infected cells with no measurable effect on cell viability. Most importantly, O859585 in combination with IFNα and sofosbuvir exhibited synergistic effects on anti-HCV activity in primary human hepatocytes. Collectively, these data suggest that O859585 may be a novel antiviral agent for HCV therapy.
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Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1. J Virol 2018; 92:JVI.01952-17. [PMID: 29367253 DOI: 10.1128/jvi.01952-17] [Citation(s) in RCA: 46] [Impact Index Per Article: 6.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2017] [Accepted: 01/19/2018] [Indexed: 01/25/2023] Open
Abstract
Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase IIIβ (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PI-transfer protein β. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.
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21
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Del Bel LM, Brill JA. Sac1, a lipid phosphatase at the interface of vesicular and nonvesicular transport. Traffic 2018; 19:301-318. [PMID: 29411923 DOI: 10.1111/tra.12554] [Citation(s) in RCA: 52] [Impact Index Per Article: 7.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/19/2017] [Revised: 02/02/2018] [Accepted: 02/02/2018] [Indexed: 12/14/2022]
Abstract
The lipid phosphatase Sac1 dephosphorylates phosphatidylinositol 4-phosphate (PI4P), thereby holding levels of this crucial membrane signaling molecule in check. Sac1 regulates multiple cellular processes, including cytoskeletal organization, membrane trafficking and cell signaling. Here, we review the structure and regulation of Sac1, its roles in cell signaling and development and its links to health and disease. Remarkably, many of the diverse roles attributed to Sac1 can be explained by the recent discovery of its requirement at membrane contact sites, where its consumption of PI4P is proposed to drive interorganelle transfer of other cellular lipids, thereby promoting normal lipid homeostasis within cells.
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Affiliation(s)
- Lauren M Del Bel
- Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
| | - Julie A Brill
- Cell Biology Program, The Hospital for Sick Children, Toronto, Ontario, Canada.,Department of Molecular Genetics, University of Toronto, Toronto, Ontario, Canada
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Phosphorylation of Serine 225 in Hepatitis C Virus NS5A Regulates Protein-Protein Interactions. J Virol 2017; 91:JVI.00805-17. [PMID: 28615203 PMCID: PMC5553161 DOI: 10.1128/jvi.00805-17] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/15/2017] [Accepted: 06/03/2017] [Indexed: 12/31/2022] Open
Abstract
Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is a phosphoprotein that plays key, yet poorly defined, roles in both virus genome replication and virion assembly/release. It has been proposed that differential phosphorylation could act as a switch to regulate the various functions of NS5A; however, the mechanistic details of the role of this posttranslational modification in the virus life cycle remain obscure. We previously reported (D. Ross-Thriepland, J. Mankouri, and M. Harris, J Virol 89:3123–3135, 2015, doi:10.1128/JVI.02995-14) a role for phosphorylation at serine 225 (S225) of NS5A in the regulation of JFH-1 (genotype 2a) genome replication. A phosphoablatant (S225A) mutation resulted in a 10-fold reduction in replication and a perinuclear restricted distribution of NS5A, whereas the corresponding phosphomimetic mutation (S225D) had no phenotype. To determine the molecular mechanisms underpinning this phenotype we conducted a label-free proteomics approach to identify cellular NS5A interaction partners. This analysis revealed that the S225A mutation disrupted the interactions of NS5A with a number of cellular proteins, in particular the nucleosome assembly protein 1-like protein 1 (NAP1L1), bridging integrator 1 (Bin1, also known as amphiphysin II), and vesicle-associated membrane protein-associated protein A (VAP-A). These interactions were validated by immunoprecipitation/Western blotting, immunofluorescence, and proximity ligation assay. Importantly, small interfering RNA (siRNA)-mediated knockdown of NAP1L1, Bin1 or VAP-A impaired viral genome replication and recapitulated the perinuclear redistribution of NS5A seen in the S225A mutant. These results demonstrate that S225 phosphorylation regulates the interactions of NS5A with a defined subset of cellular proteins. Furthermore, these interactions regulate both HCV genome replication and the subcellular localization of replication complexes. IMPORTANCE Hepatitis C virus is an important human pathogen. The viral nonstructural 5A protein (NS5A) is the target for new antiviral drugs. NS5A has multiple functions during the virus life cycle, but the biochemical details of these roles remain obscure. NS5A is known to be phosphorylated by cellular protein kinases, and in this study, we set out to determine whether this modification is required for the binding of NS5A to other cellular proteins. We identified 3 such proteins and show that they interacted only with NS5A that was phosphorylated on a specific residue. Furthermore, these proteins were required for efficient virus replication and the ability of NS5A to spread throughout the cytoplasm of the cell. Our results help to define the function of NS5A and may contribute to an understanding of the mode of action of the highly potent antiviral drugs that are targeted to NS5A.
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Treatment with PTEN-Long protein inhibits hepatitis C virus replication. Virology 2017; 511:1-8. [PMID: 28783500 DOI: 10.1016/j.virol.2017.08.002] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/23/2017] [Revised: 07/26/2017] [Accepted: 08/02/2017] [Indexed: 01/15/2023]
Abstract
Hepatitis C virus (HCV) infection is a confirmed risk factor for hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) possesses tumor suppression function that is frequently defective in HCC tumors. PTEN-Long, a translation isoform of PTEN, functions in a cell non-autonomous manner. In this study, we demonstrated that intracellular overexpression of PTEN-Long inhibits HCV replication. More importantly, we showed that treatment with extracellular PTEN-Long protein inhibits HCV replication in a dose-dependent manner. Furthermore, we showed that PTEN-Long interacts with HCV core protein and this interaction is required for HCV replication inhibition by PTEN-Long. In summary, we demonstrated, for the first time, that PTEN-Long protein, an isoform of the canonical PTEN and in the form of extracellular protein treatment, inhibits HCV replication. Our study offers an opportunity for developing additional anti-HCV agents.
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Abstract
Hepatitis C virus (HCV) infection leads to severe liver diseases including hepatocellular carcinoma (HCC). Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumour suppressor, is frequently mutated or deleted in HCC tumors. PTEN has previously been demonstrated to inhibit HCV secretion. In this study, we determined the effects of PTEN on the other steps in HCV life cycle, including entry, translation, and replication. We showed that PTEN inhibits HCV entry through its lipid phosphatase activity. PTEN has no effect on HCV RNA translation. PTEN decreases HCV replication and the protein phosphatase activity of PTEN is essential for this function. PTEN interacts with the HCV core protein and requires R50 in domain I of HCV core and PTEN residues 1–185 for this interaction. This interaction is required for PTEN-mediated inhibition of HCV replication. This gives rise to a reduction in PTEN levels and intracellular lipid abundance, which may in turn regulate HCV replication. HCV core domain I protein increases the lipid phosphatase activity of PTEN in an in vitro assay, suggesting that HCV infection can also regulate PTEN. Taken together, our results demonstrated an important regulatory role of PTEN in the HCV life cycle.
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25
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Hepatitis C virus triggers Golgi fragmentation and autophagy through the immunity-related GTPase M. Proc Natl Acad Sci U S A 2017; 114:E3462-E3471. [PMID: 28389568 DOI: 10.1073/pnas.1616683114] [Citation(s) in RCA: 99] [Impact Index Per Article: 12.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Positive-stranded RNA viruses, such as hepatitis C virus (HCV), assemble their viral replication complexes by remodeling host intracellular membranes to a membranous web. The precise composition of these replication complexes and the detailed mechanisms by which they are formed are incompletely understood. Here we show that the human immunity-related GTPase M (IRGM), known to contribute to autophagy, plays a previously unrecognized role in this process. We show that IRGM is localized at the Golgi apparatus and regulates the fragmentation of Golgi membranes in response to HCV infection, leading to colocalization of Golgi vesicles with replicating HCV. Our results show that IRGM controls phosphorylation of GBF1, a guanine nucleotide exchange factor for Arf-GTPases, which normally operates in Golgi membrane dynamics and vesicle coating in resting cells. We also find that HCV triggers IRGM-mediated phosphorylation of the early autophagy initiator ULK1, thereby providing mechanistic insight into the role of IRGM in HCV-mediated autophagy. Collectively, our results identify IRGM as a key Golgi-situated regulator that links intracellular membrane remodeling by autophagy and Golgi fragmentation with viral replication.
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Awad A, Gassama-Diagne A. PI3K/SHIP2/PTEN pathway in cell polarity and hepatitis C virus pathogenesis. World J Hepatol 2017; 9:18-29. [PMID: 28105255 PMCID: PMC5220268 DOI: 10.4254/wjh.v9.i1.18] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/28/2016] [Revised: 09/10/2016] [Accepted: 11/02/2016] [Indexed: 02/06/2023] Open
Abstract
Hepatitis C virus (HCV) infects hepatocytes, polarized cells in the liver. Chronic HCV infection often leads to steatosis, fibrosis, cirrhosis and hepatocellular carcinoma, and it has been identified as the leading cause of liver transplantation worldwide. The HCV replication cycle is dependent on lipid metabolism and particularly an accumulation of lipid droplets in host cells. Phosphoinositides (PIs) are minor phospholipids enriched in different membranes and their levels are tightly regulated by specific PI kinases and phosphatases. PIs are implicated in a vast array of cellular responses that are central to morphogenesis, such as cytoskeletal changes, cytokinesis and the recruitment of downstream effectors to govern mechanisms involved in polarization and lumen formation. Important reviews of the literature identified phosphatidylinositol (PtdIns) 4-kinases, and their lipid products PtdIns(4)P, as critical regulators of the HCV life cycle. SH2-containing inositol polyphosphate 5-phosphatase (SHIP2), phosphoinositide 3-kinase (PI3K) and their lipid products PtdIns(3,4)P2 and PtdIns(3,4,5)P3, respectively, play an important role in the cell membrane and are key to the establishment of apicobasal polarity and lumen formation. In this review, we will focus on these new functions of PI3K and SHIP2, and their deregulation by HCV, causing a disruption of apicobasal polarity, actin organization and extracellular matrix assembly. Finally we will highlight the involvement of this pathway in the event of insulin resistance and nonalcoholic fatty liver disease related to HCV infection.
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Nonstructural 3 Protein of Hepatitis C Virus Modulates the Tribbles Homolog 3/Akt Signaling Pathway for Persistent Viral Infection. J Virol 2016; 90:7231-7247. [PMID: 27252525 DOI: 10.1128/jvi.00326-16] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/19/2016] [Accepted: 05/24/2016] [Indexed: 12/14/2022] Open
Abstract
UNLABELLED Hepatitis C virus (HCV) infection often causes chronic hepatitis, liver cirrhosis, and ultimately hepatocellular carcinoma. However, the mechanisms underlying HCV-induced liver pathogenesis are still not fully understood. By transcriptome sequencing (RNA-Seq) analysis, we recently identified host genes that were significantly differentially expressed in cell culture-grown HCV (HCVcc)-infected cells. Of these, tribbles homolog 3 (TRIB3) was selected for further characterization. TRIB3 was initially identified as a binding partner of protein kinase B (also known as Akt). TRIB3 blocks the phosphorylation of Akt and induces apoptosis under endoplasmic reticulum (ER) stress conditions. HCV has been shown to enhance Akt phosphorylation for its own propagation. In the present study, we demonstrated that both mRNA and protein levels of TRIB3 were increased in the context of HCV replication. We further showed that promoter activity of TRIB3 was increased by HCV-induced ER stress. Silencing of TRIB3 resulted in increased RNA and protein levels of HCV, whereas overexpression of TRIB3 decreased HCV replication. By employing an HCV pseudoparticle entry assay, we further showed that TRIB3 was a negative host factor involved in HCV entry. Both in vitro binding and immunoprecipitation assays demonstrated that HCV NS3 specifically interacted with TRIB3. Consequently, the association of TRIB3 and Akt was disrupted by HCV NS3, and thus, TRIB3-Akt signaling was impaired in HCV-infected cells. Moreover, HCV modulated TRIB3 to promote extracellular signal-regulated kinase (ERK) phosphorylation, activator protein 1 (AP-1) activity, and cell migration. Collectively, these data indicate that HCV exploits the TRIB3-Akt signaling pathway to promote persistent viral infection and may contribute to HCV-mediated pathogenesis. IMPORTANCE TRIB3 is a pseudokinase protein that acts as an adaptor in signaling pathways for important cellular processes. So far, the functional involvement of TRIB3 in virus-infected cells has not yet been demonstrated. We showed that both mRNA and protein expression levels of TRIB3 were increased in the context of HCV RNA replication. Gene silencing of TRIB3 increased HCV RNA and protein levels, and thus, overexpression of TRIB3 decreased HCV replication. TRIB3 is known to promote apoptosis by negatively regulating the Akt signaling pathway under ER stress conditions. Most importantly, we demonstrated that the TRIB3-Akt signaling pathway was disrupted by NS3 in HCV-infected cells. These data provide evidence that HCV modulates the TRIB3-Akt signaling pathway to establish persistent viral infection.
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Cell-death-inducing DFFA-like Effector B Contributes to the Assembly of Hepatitis C Virus (HCV) Particles and Interacts with HCV NS5A. Sci Rep 2016; 6:27778. [PMID: 27282740 PMCID: PMC4901263 DOI: 10.1038/srep27778] [Citation(s) in RCA: 19] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/02/2016] [Accepted: 05/23/2016] [Indexed: 12/12/2022] Open
Abstract
Hepatitis C virus (HCV) uses components of the very-low-density lipoprotein (VLDL) pathway for assembly/release. We previously reported that hepatocyte nuclear factor 4α (HNF4α) participates in HCV assembly/release through downstream factors those participate in VLDL assembly/secretion. Cell-death-inducing DFFA-like effector B (CIDEB) is an important regulator of the VLDL pathway. CIDEB is required for entry of HCV particles from cell culture (HCVcc), but the effects of CIDEB on the post-entry steps of the HCV lifecycle are unclear. In the present study, we determined that CIDEB is required for HCV assembly in addition to HCVcc entry. Furthermore, CIDEB interacts with the HCV NS5A protein, and the N terminus of CIDEB and the domain I of NS5A are involved in this interaction. Moreover, CIDEB silencing impairs the association of apolipoprotein E (ApoE) with HCV particles. Interestingly, CIDEB is also required for the post-entry stages of the dengue virus (DENV) life cycle. Collectively, these results indicate that CIDEB is a new host factor that is involved in HCV assembly, presumably by interacting with viral protein, providing new insight into the exploitation of the VLDL regulator CIDEB by HCV.
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Hepatitis C virus NS5A protein cooperates with phosphatidylinositol 4-kinase IIIα to induce mitochondrial fragmentation. Sci Rep 2016; 6:23464. [PMID: 27010100 PMCID: PMC4806301 DOI: 10.1038/srep23464] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Accepted: 03/07/2016] [Indexed: 12/22/2022] Open
Abstract
Hepatitis C virus (HCV) has long been observed to take advantage of the host mitochondria to support viral replication and assembly. The HCV core protein has been implicated to fragment host mitochondria. In this report, we have discovered that the non-structural protein 5A (NS5A) plays an instructive role in attaching ER with mitochondria, causing mitochondrial fragmentation. Dynamin-related protein 1(Drp1), a host protein essential to mitochondrial membrane fission, does not play a role in NS5A-induced mitochondrial fragmentation. Instead, phosphatidylinositol 4-kinase IIIα (PI4KA), which has been demonstrated to bind to NS5A and is required to support HCV life cycle, is required for NS5A to induce mitochondrial fragmentation. Both NS5A and core are required by HCV to fragment the mitochondria, as inhibiting either of their respective downstream proteins, PI4KA or Drp1, resulted in lengthening of mitochondria tubules in HCVcc-infected cells. By fragmenting the mitochondria, NS5A renders the cells more resistant to mitochondria mediated apoptosis. This finding indicates previously-ignored contribution of NS5A in HCV-induced mitochondria dysfunction.
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Eyre NS, Hampton-Smith RJ, Aloia AL, Eddes JS, Simpson KJ, Hoffmann P, Beard MR. Phosphorylation of NS5A Serine-235 is essential to hepatitis C virus RNA replication and normal replication compartment formation. Virology 2016; 491:27-44. [PMID: 26874015 DOI: 10.1016/j.virol.2016.01.018] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2015] [Revised: 01/21/2016] [Accepted: 01/23/2016] [Indexed: 01/09/2023]
Abstract
Hepatitis C virus (HCV) NS5A protein is essential for HCV RNA replication and virus assembly. Here we report the identification of NS5A phosphorylation sites Ser-222, Ser-235 and Thr-348 during an infectious HCV replication cycle and demonstrate that Ser-235 phosphorylation is essential for HCV RNA replication. Confocal microscopy revealed that both phosphoablatant (S235A) and phosphomimetic (S235D) mutants redistribute NS5A to large juxta-nuclear foci that display altered colocalization with known replication complex components. Using electron microscopy (EM) we found that S235D alters virus-induced membrane rearrangements while EM using 'APEX2'-tagged viruses demonstrated S235D-mediated enrichment of NS5A in irregular membranous foci. Finally, using a customized siRNA screen of candidate NS5A kinases and subsequent analysis using a phospho-specific antibody, we show that phosphatidylinositol-4 kinase III alpha (PI4KIIIα) is important for Ser-235 phosphorylation. We conclude that Ser-235 phosphorylation of NS5A is essential for HCV RNA replication and normal replication complex formation and is regulated by PI4KIIIα.
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Affiliation(s)
- Nicholas S Eyre
- School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia.
| | - Rachel J Hampton-Smith
- School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia
| | - Amanda L Aloia
- School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia
| | - James S Eddes
- Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, Australia
| | - Kaylene J Simpson
- Victorian Centre for Functional Genomics, Peter MacCallum Cancer Centre, East Melbourne, Australia; The Sir Peter MacCallum Department of Oncology, University of Melbourne, Parkville, Australia
| | - Peter Hoffmann
- Adelaide Proteomics Centre, School of Biological Sciences, University of Adelaide, Adelaide, Australia; Institute for Photonics and Advanced Sensing (IPAS), University of Adelaide, Adelaide, Australia
| | - Michael R Beard
- School of Biological Sciences and Research Centre for Infectious Diseases, University of Adelaide, Adelaide, Australia; Centre for Cancer Biology, SA Pathology, Adelaide, Australia
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31
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Type III phosphatidylinositol 4 kinases: structure, function, regulation, signalling and involvement in disease. Biochem Soc Trans 2016; 44:260-6. [DOI: 10.1042/bst20150219] [Citation(s) in RCA: 30] [Impact Index Per Article: 3.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/03/2023]
Abstract
Many important cellular functions are regulated by the selective recruitment of proteins to intracellular membranes mediated by specific interactions with lipid phosphoinositides. The enzymes that generate lipid phosphoinositides therefore must be properly positioned and regulated at their correct cellular locations. Phosphatidylinositol 4 kinases (PI4Ks) are key lipid signalling enzymes, and they generate the lipid species phosphatidylinositol 4-phosphate (PI4P), which plays important roles in regulating physiological processes including membrane trafficking, cytokinesis and organelle identity. PI4P also acts as the substrate for the generation of the signalling phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3). PI4Ks also play critical roles in a number of pathological processes including mediating replication of a number of pathogenic RNA viruses, and in the development of the parasite responsible for malaria. Key to the regulation of PI4Ks is their regulation by a variety of both host and viral protein-binding partners. We review herein our current understanding of the structure, regulatory interactions and role in disease of the type III PI4Ks.
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32
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Yin P, Hong Z, Yang X, Chung RT, Zhang L. A role for retromer in hepatitis C virus replication. Cell Mol Life Sci 2016; 73:869-881. [PMID: 26298293 PMCID: PMC11108358 DOI: 10.1007/s00018-015-2027-7] [Citation(s) in RCA: 27] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2015] [Revised: 08/13/2015] [Accepted: 08/18/2015] [Indexed: 12/20/2022]
Abstract
Hepatitis C virus (HCV) has infected over 170 million people worldwide. Phosphatidylinositol 4-phosphate (PI4P) is the organelle-specific phosphoinositide enriched at sites of HCV replication. Whether retromer, a PI4P-related host transport machinery, unloads its cargo at HCV replication sites remains inconclusive. We sought to characterize the role of retromer in HCV replication. Here, we demonstrated the interaction between retromer subunit Vps35 and HCV NS5A protein by immunoprecipitation and GST pulldown. Vps35 colocalized with NS5A and PI4P in both OR6 replicon and JFH1 infected Huh 7.5.1 cells. HCV replication was inhibited upon silencing retromer subunits. CIMPR, a typical retromer cargo, participated in HCV replication. Our data suggest that retromer component Vps35 is recruited by NS5A to viral replication sites where PI4P unloads CIMPR. These findings demonstrate a dependence role of retromer in HCV replication and identify retromer as a potential therapeutic target against HCV.
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Affiliation(s)
- Peiqi Yin
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100176, China
| | - Zhi Hong
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100176, China
| | - Xiaojie Yang
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100176, China
| | - Raymond T Chung
- Gastrointestinal Division, Department of Medicine, Liver Center, Massachusetts General Hospital, Harvard Medical School, Boston, MA, 02114, USA
| | - Leiliang Zhang
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing, 100176, China.
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Muñoz-Moreno R, Galindo I, Cuesta-Geijo MÁ, Barrado-Gil L, Alonso C. Host cell targets for African swine fever virus. Virus Res 2015; 209:118-27. [DOI: 10.1016/j.virusres.2015.05.026] [Citation(s) in RCA: 13] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/21/2015] [Revised: 05/28/2015] [Accepted: 05/29/2015] [Indexed: 02/08/2023]
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34
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Boura E, Nencka R. Phosphatidylinositol 4-kinases: Function, structure, and inhibition. Exp Cell Res 2015; 337:136-45. [DOI: 10.1016/j.yexcr.2015.03.028] [Citation(s) in RCA: 95] [Impact Index Per Article: 9.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/04/2015] [Accepted: 03/12/2015] [Indexed: 02/07/2023]
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35
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Definition and expression in E. coli of large fragments from the human lipid kinase phosphatidylinositol 4-kinase type III alpha, and purification of a 1100-residue N-terminal module. Protein Expr Purif 2015; 114:121-7. [DOI: 10.1016/j.pep.2015.06.018] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/16/2015] [Accepted: 06/29/2015] [Indexed: 11/22/2022]
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36
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Colpitts CC, El-Saghire H, Pochet N, Schuster C, Baumert TF. High-throughput approaches to unravel hepatitis C virus-host interactions. Virus Res 2015; 218:18-24. [PMID: 26410623 DOI: 10.1016/j.virusres.2015.09.013] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2015] [Revised: 09/18/2015] [Accepted: 09/22/2015] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) remains a major global health burden, with more than 130 million individuals chronically infected and at risk for the development of hepatocellular carcinoma (HCC). The recent clinical licensing of direct-acting antivirals enables viral cure. However, limited access to therapy and treatment failure in patient subgroups warrants a continuing effort to develop complementary antiviral strategies. Furthermore, once fibrosis is established, curing HCV infection does not eliminate the risk for HCC. High-throughput approaches and screens have enabled the investigation of virus-host interactions on a genome-wide scale. Gain- and loss-of-function screens have identified essential host-dependency factors in the HCV viral life cycle, such as host cell entry factors or regulatory factors for viral replication and assembly. Network analyses of systems-scale data sets provided a comprehensive view of the cellular state following HCV infection, thus improving our understanding of the virus-induced responses of the target cell. Interactome, metabolomics and gene expression studies identified dysregulated cellular processes potentially contributing to HCV pathogenesis and HCC. Drug screens using chemical libraries led to the discovery of novel antivirals. Here, we review the contribution of high-throughput approaches for the investigation of virus-host interactions, viral pathogenesis and drug discovery.
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Affiliation(s)
- Che C Colpitts
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France
| | - Hussein El-Saghire
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France
| | - Nathalie Pochet
- Program in Translational NeuroPsychiatric Genomics, Brigham and Women's Hospital, Harvard Medical School, Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA, USA
| | - Catherine Schuster
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France
| | - Thomas F Baumert
- Inserm, U1110, Institut de Recherche sur les Maladies Virales et Hépatiques, 67000 Strasbourg, France; Université de Strasbourg, 67000 Strasbourg, France; Institut Hospitalo-Universitaire, PôleHépato-digestif, HôpitauxUniversitaires de Strasbourg, 67000 Strasbourg, France.
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Desrochers GF, Sherratt AR, Blais DR, Nasheri N, Ning Z, Figeys D, Goto NK, Pezacki JP. Profiling Kinase Activity during Hepatitis C Virus Replication Using a Wortmannin Probe. ACS Infect Dis 2015; 1:443-52. [PMID: 27617927 DOI: 10.1021/acsinfecdis.5b00083] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
To complete its life cycle, the hepatitis C virus (HCV) induces changes to numerous aspects of its host cell. As kinases act as regulators of many pathways utilized by HCV, they are likely enzyme targets for virally induced inhibition or activation. Herein, we used activity-based protein profiling (ABPP), which allows for the identification of active enzymes in complex protein samples and the quantification of their activity, to identify kinases that displayed differential activity in HCV-expressing cells. We utilized an ABPP probe, wortmannin-yne, based on the kinase inhibitor wortmannin, which contains a pendant alkyne group for bioconjugation using bioorthogonal chemistry. We observed changes in the activity of kinases involved in the mitogen-activated protein kinase pathway, apoptosis pathways, and cell cycle control. These results establish changes to the active kinome, as reported by wortmannin-yne, in the proteome of human hepatoma cells actively replicating HCV. The observed changes include kinase activity that affect viral entry, replication, assembly, and secretion, implying that HCV is regulating the pathways that it uses for its life cycle through modulation of the active kinome.
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Affiliation(s)
- Geneviève F. Desrochers
- Life Sciences Division, National Research Council of Canada, 100
Sussex Drive, Ottawa, Canada
| | - Allison R. Sherratt
- Life Sciences Division, National Research Council of Canada, 100
Sussex Drive, Ottawa, Canada
| | - David R. Blais
- Life Sciences Division, National Research Council of Canada, 100
Sussex Drive, Ottawa, Canada
| | - Neda Nasheri
- Life Sciences Division, National Research Council of Canada, 100
Sussex Drive, Ottawa, Canada
| | | | | | | | - John Paul Pezacki
- Life Sciences Division, National Research Council of Canada, 100
Sussex Drive, Ottawa, Canada
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38
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Identification of Host Cell Factors Associated with Astrovirus Replication in Caco-2 Cells. J Virol 2015; 89:10359-70. [PMID: 26246569 DOI: 10.1128/jvi.01225-15] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/12/2015] [Accepted: 07/28/2015] [Indexed: 01/25/2023] Open
Abstract
UNLABELLED Astroviruses are small, nonenveloped viruses with a single-stranded positive-sense RNA genome causing acute gastroenteritis in children and immunocompromised patients. Since positive-sense RNA viruses have frequently been found to replicate in association with membranous structures, in this work we characterized the replication of the human astrovirus serotype 8 strain Yuc8 in Caco-2 cells, using density gradient centrifugation and free-flow zonal electrophoresis (FFZE) to fractionate cellular membranes. Structural and nonstructural viral proteins, positive- and negative-sense viral RNA, and infectious virus particles were found to be associated with a distinct population of membranes separated by FFZE. The cellular proteins associated with this membrane population in infected and mock-infected cells were identified by tandem mass spectrometry. The results indicated that membranes derived from multiple cell organelles were present in the population. Gene ontology and protein-protein interaction network analysis showed that groups of proteins with roles in fatty acid synthesis and ATP biosynthesis were highly enriched in the fractions of this population in infected cells. Based on this information, we investigated by RNA interference the role that some of the identified proteins might have in the replication cycle of the virus. Silencing of the expression of genes involved in cholesterol (DHCR7, CYP51A1) and fatty acid (FASN) synthesis, phosphatidylinositol (PI4KIIIβ) and inositol phosphate (ITPR3) metabolism, and RNA helicase activity (DDX23) significantly decreased the amounts of Yuc8 genomic and antigenomic RNA, synthesis of the structural protein VP90, and virus yield. These results strongly suggest that astrovirus RNA replication and particle assembly take place in association with modified membranes potentially derived from multiple cell organelles. IMPORTANCE Astroviruses are common etiological agents of acute gastroenteritis in children and immunocompromised patients. More recently, they have been associated with neurological diseases in mammals, including humans, and are also responsible for different pathologies in birds. In this work, we provide evidence that astrovirus RNA replication and virus assembly occur in contact with cell membranes potentially derived from multiple cell organelles and show that membrane-associated cellular proteins involved in lipid metabolism are required for efficient viral replication. Our findings provide information to enhance our knowledge of astrovirus biology and provide information that might be useful for the development of therapeutic interventions to prevent virus replication.
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Abstract
UNLABELLED The life cycle of hepatitis C virus (HCV) is highly dependent on host cellular proteins for virus propagation. In order to identify the cellular factors involved in HCV propagation, we performed protein microarray assay using the HCV nonstructural 5A (NS5A) protein as a probe. Of ∼ 9,000 human cellular proteins immobilized in a microarray, approximately 90 cellular proteins were identified as NS5A interactors. Of these candidates, Pim1, a member of serine/threonine kinase family composed of three different isoforms (Pim1, Pim2, and Pim3), was selected for further study. Pim kinases share a consensus sequence which overlaps with kinase activity. Pim kinase activity has been implicated in tumorigenesis. In the present study, we verified the physical interaction between NS5A and Pim1 by both in vitro pulldown and coimmunoprecipitation assays. Pim1 interacted with NS5A through amino acid residues 141 to 180 of Pim1. We demonstrated that protein stability of Pim1 was increased by NS5A protein and this increase was mediated by protein interplay. Small interfering RNA (siRNA)-mediated knockdown or pharmacological inhibition of Pim kinase abrogated HCV propagation. By employing HCV pseudoparticle entry and single-cycle HCV infection assays, we further demonstrated that Pim kinase was involved in HCV entry at a postbinding step. These data suggest that Pim kinase may represent a new host factor for HCV entry. IMPORTANCE Pim1 is an oncogenic serine/threonine kinase. HCV NS5A protein physically interacts with Pim1 and contributes to Pim1 protein stability. Since Pim1 protein expression level is upregulated in many cancers, NS5A-mediated protein stability may be associated with HCV pathogenesis. Either gene silencing or chemical inhibition of Pim kinase abrogated HCV propagation in HCV-infected cells. We further showed that Pim kinase was specifically required at an early entry step of the HCV life cycle. Thus, we have identified Pim kinase not only as an HCV cell entry factor but also as a new anti-HCV therapeutic target.
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40
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Host cell kinases and the hepatitis C virus life cycle. BIOCHIMICA ET BIOPHYSICA ACTA-PROTEINS AND PROTEOMICS 2015; 1854:1657-62. [PMID: 25896387 DOI: 10.1016/j.bbapap.2015.04.011] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/07/2015] [Revised: 04/08/2015] [Accepted: 04/09/2015] [Indexed: 02/07/2023]
Abstract
Hepatitis C virus (HCV) infection relies on virus-host interactions with human hepatocytes, a context in which host cell kinases play critical roles in every step of the HCV life cycle. During viral entry, cellular kinases, including EGFR, EphA2 and PKA, regulate the localization of host HCV entry factors and induce receptor complex assembly. Following virion internalization, viral genomes replicate on endoplasmic reticulum-derived membranous webs. The formation of membranous webs depends on interactions between the HCV NS5a protein and PI4KIIIα. The phosphorylation status of NS5a, regulated by PI4KIIIα, CKI and other kinases, also acts as a molecular switch to virion assembly, which takes place on lipid droplets. The formation of lipid droplets is enhanced by HCV activation of IKKα. In view of the multiple crucial steps in the viral life cycle that are mediated by host cell kinases, these enzymes also represent complementary targets for antiviral therapy. This article is part of a Special Issue entitled: Inhibitors of Protein Kinases.
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Cyclophilin and NS5A inhibitors, but not other anti-hepatitis C virus (HCV) agents, preclude HCV-mediated formation of double-membrane-vesicle viral factories. Antimicrob Agents Chemother 2015; 59:2496-507. [PMID: 25666154 DOI: 10.1128/aac.04958-14] [Citation(s) in RCA: 45] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/07/2014] [Accepted: 02/02/2015] [Indexed: 12/14/2022] Open
Abstract
Although the mechanisms of action (MoA) of nonstructural protein 3 inhibitors (NS3i) and NS5B inhibitors (NS5Bi) are well understood, the MoA of cyclophilin inhibitors (CypI) and NS5A inhibitors (NS5Ai) are not fully defined. In this study, we examined whether CypI and NS5Ai interfere with hepatitis C virus (HCV) RNA synthesis of replication complexes (RCs) or with an earlier step of HCV RNA replication, the creation of double-membrane vesicles (DMVs) essential for HCV RNA replication. In contrast to NS5Bi, both CypI and NS5Ai do not block HCV RNA synthesis by way of RCs, suggesting that they exert their antiviral activity prior to the establishment of enzymatically active RCs. We found that viral replication is not a precondition for DMV formation, since the NS3-NS5B polyprotein or NS5A suffices to create DMVs. Importantly, only CypI and NS5Ai, but not NS5Bi, mir-122, or phosphatidylinositol-4 kinase IIIα (PI4KIIIα) inhibitors, prevent NS3-NS5B-mediated DMV formation. NS3-NS5B was unable to create DMVs in cyclophilin A (CypA) knockdown (KD) cells. We also found that the isomerase activity of CypA is absolutely required for DMV formation. This not only suggests that NS5A and CypA act in concert to build membranous viral factories but that CypI and NS5Ai mediate their early anti-HCV effects by preventing the formation of organelles, where HCV replication is normally initiated. This is the first investigation to examine the effect of a large panel of anti-HCV agents on DMV formation, and the results reveal that CypI and NS5Ai act at the same membranous web biogenesis step of HCV RNA replication, thus indicating a new therapeutic target of chronic hepatitis C.
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Serine phosphorylation of the hepatitis C virus NS5A protein controls the establishment of replication complexes. J Virol 2014; 89:3123-35. [PMID: 25552726 PMCID: PMC4337517 DOI: 10.1128/jvi.02995-14] [Citation(s) in RCA: 42] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022] Open
Abstract
The hepatitis C virus (HCV) nonstructural 5A (NS5A) protein is highly phosphorylated and involved in both virus genome replication and virion assembly. We and others have identified serine 225 in NS5A to be a phosphorylation site, but the function of this posttranslational modification in the virus life cycle remains obscure. Here we describe the phenotype of mutants with mutations at serine 225; this residue was mutated to either alanine (S225A; phosphoablatant) or aspartic acid (S225D; phosphomimetic) in the context of both the JFH-1 cell culture infectious virus and a corresponding subgenomic replicon. The S225A mutant exhibited a 10-fold reduction in genome replication, whereas the S225D mutant replicated like the wild type. By confocal microscopy, we show that, in the case of the S225A mutant, the replication phenotype correlated with an altered subcellular distribution of NS5A. This phenotype was shared by viruses with other mutations in the low-complexity sequence I (LCS I), namely, S229D, S232A, and S235D, but not by viruses with mutations that caused a comparable replication defect that mapped to domain II of NS5A (P315A, L321A). Together with other components of the genome replication complex (NS3, double-stranded RNA, and cellular lipids, including phosphatidylinositol 4-phosphate), the mutation in NS5A was restricted to a perinuclear region. This phenotype was not due to cell confluence or another environmental factor and could be partially transcomplemented by wild-type NS5A. We propose that serine phosphorylation within LCS I may regulate the assembly of an active genome replication complex. IMPORTANCE The mechanisms by which hepatitis C virus replicates its RNA genome remain poorly characterized. We show here that phosphorylation of the viral nonstructural protein NS5A at serine residues is important for the efficient assembly of a complex that is able to replicate the viral genome. This research implicates cellular protein kinases in the control of virus replication and highlights the need to further understand the interplay between the virus and the host cell in order to develop potential avenues for future antiviral therapy.
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NS5A inhibitors impair NS5A-phosphatidylinositol 4-kinase IIIα complex formation and cause a decrease of phosphatidylinositol 4-phosphate and cholesterol levels in hepatitis C virus-associated membranes. Antimicrob Agents Chemother 2014; 58:7128-40. [PMID: 25224012 DOI: 10.1128/aac.03293-14] [Citation(s) in RCA: 25] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
The hepatitis C virus (HCV) nonstructural (NS) protein 5A is a multifunctional protein that plays a central role in viral replication and assembly. Antiviral agents directly targeting NS5A are currently in clinical development. Although the elucidation of the mechanism of action (MOA) of NS5A inhibitors has been the focus of intensive research, a detailed understanding of how these agents exert their antiviral effect is still lacking. In this study, we observed that the downregulation of NS5A hyperphosphorylation is associated with the actions of NS5A inhibitors belonging to different chemotypes. NS5A is known to recruit the lipid kinase phosphatidylinositol 4-kinase IIIα (PI4KIIIα) to the HCV-induced membranous web in order to generate phosphatidylinositol 4-phosphate (PI4P) at the sites of replication. We demonstrate that treatment with NS5A inhibitors leads to an impairment in the NS5A-PI4KIIIα complex formation that is paralleled by a significant reduction in PI4P and cholesterol levels within the endomembrane structures of HCV-replicating cells. A similar decrease in PI4P and cholesterol levels was also obtained upon treatment with a PI4KIIIα-targeting inhibitor. In addition, both the NS5A and PI4KIIIα classes of inhibitors induced similar subcellular relocalization of the NS5A protein, causing the formation of large cytoplasmic NS5A-containing clusters previously reported to be one of the hallmarks of inhibition of the action of PI4KIIIα. Because of the similarities between the effects induced by treatment with PI4KIIIα or NS5A inhibitors and the observation that agents targeting NS5A impair NS5A-PI4KIIIα complex formation, we speculate that NS5A inhibitors act by interfering with the function of the NS5A-PI4KIIIα complex.
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Hong Z, Yang X, Yang G, Zhang L. Hepatitis C virus NS5A competes with PI4KB for binding to ACBD3 in a genotype-dependent manner. Antiviral Res 2014; 107:50-55. [PMID: 24792752 DOI: 10.1016/j.antiviral.2014.04.012] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2014] [Revised: 04/18/2014] [Accepted: 04/22/2014] [Indexed: 01/11/2023]
Abstract
Although genotype-dependency of PI4KB involved in HCV replication has been reported, the mechanism underlying that is unknown. In this study, we found that NS5A and PI4KB competed for association of acyl-coenzyme A binding domain containing protein 3 (ACBD3), which inhibited HCV replication. ACBD3 bind to GT1b NS5A with a higher affinity than to GT2a NS5A, which was consistent with higher co-localization between PI4KB and phosphatidylinositol 4-phosphate (PI4P) in GT1b HCV-infected cells than that in GT2a HCV-infected cells. These results suggested that NS5A could rob the preexisting ACBD3/PI4KB complex to form NS5A/ACBD3 complex and PI4KB could relocate to the viral RNA replication sites to facilitate HCV replication. Our findings not only revealed the anti-HCV function of ACBD3, but also shed mechanistic light on how ACBD3 was manipulated by NS5A from different GT of HCV.
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Affiliation(s)
- Zhi Hong
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100176, China
| | - Xiaojie Yang
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100176, China
| | - Guangbo Yang
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100176, China
| | - Leiliang Zhang
- MOH Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100176, China.
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Mapping of functional domains of the lipid kinase phosphatidylinositol 4-kinase type III alpha involved in enzymatic activity and hepatitis C virus replication. J Virol 2014; 88:9909-26. [PMID: 24920820 DOI: 10.1128/jvi.01063-14] [Citation(s) in RCA: 30] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
UNLABELLED The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIIIα) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIIIα is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIIIα in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIIIα mutants for subcellular localization, reconstitution of HCV replication in PI4KIIIα knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIIIα are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIIIα core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIIIα also were detrimental for enzymatic activity and for the ability of PI4KIIIα to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIIIα is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIIIα C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity. IMPORTANCE The lipid kinase PI4KIIIα is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIIIα mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication.
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Carpp LN, Rogers RS, Moritz RL, Aitchison JD. Quantitative proteomic analysis of host-virus interactions reveals a role for Golgi brefeldin A resistance factor 1 (GBF1) in dengue infection. Mol Cell Proteomics 2014; 13:2836-54. [PMID: 24855065 DOI: 10.1074/mcp.m114.038984] [Citation(s) in RCA: 43] [Impact Index Per Article: 3.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023] Open
Abstract
Dengue virus is considered to be the most important mosquito-borne virus worldwide and poses formidable economic and health care burdens on many tropical and subtropical countries. Dengue infection induces drastic rearrangement of host endoplasmic reticulum membranes into complex membranous structures housing replication complexes; the contribution(s) of host proteins and pathways to this process is poorly understood but is likely to be mediated by protein-protein interactions. We have developed an approach for obtaining high confidence protein-protein interaction data by employing affinity tags and quantitative proteomics, in the context of viral infection, followed by robust statistical analysis. Using this approach, we identified high confidence interactors of NS5, the viral polymerase, and NS3, the helicase/protease. Quantitative proteomics allowed us to exclude a large number of presumably nonspecific interactors from our data sets and imparted a high level of confidence to our resulting data sets. We identified 53 host proteins reproducibly associated with NS5 and 41 with NS3, with 13 of these candidates present in both data sets. The host factors identified have diverse functions, including retrograde Golgi-to-endoplasmic reticulum transport, biosynthesis of long-chain fatty-acyl-coenzyme As, and in the unfolded protein response. We selected GBF1, a guanine nucleotide exchange factor responsible for ARF activation, from the NS5 data set for follow up and functional validation. We show that GBF1 plays a critical role early in dengue infection that is independent of its role in the maintenance of Golgi structure. Importantly, the approach described here can be applied to virtually any organism/system as a tool for better understanding its molecular interactions.
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Affiliation(s)
- Lindsay N Carpp
- From the ‡Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109
| | - Richard S Rogers
- ‖Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109
| | - Robert L Moritz
- §Institute for Systems Biology, 401 Terry Ave N, Seattle, Washington 98109
| | - John D Aitchison
- From the ‡Seattle Biomedical Research Institute, 307 Westlake Avenue North, Suite 500, Seattle, Washington 98109; §Institute for Systems Biology, 401 Terry Ave N, Seattle, Washington 98109, ‖Institute for Systems Biology, 401 Terry Ave N, Seattle, WA 98109.
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Ke PY, Chen SSL. Autophagy in hepatitis C virus-host interactions: potential roles and therapeutic targets for liver-associated diseases. World J Gastroenterol 2014; 20:5773-93. [PMID: 24914338 PMCID: PMC4024787 DOI: 10.3748/wjg.v20.i19.5773] [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: 10/08/2013] [Revised: 01/14/2014] [Accepted: 03/04/2014] [Indexed: 02/06/2023] Open
Abstract
Autophagy is a lysosome-associated, degradative process that catabolizes cytosolic components to recycle nutrients for further use and maintain cell homeostasis. Hepatitis C virus (HCV) is a major cause of chronic hepatitis, which often leads to end-stage liver-associated diseases and is a significant burden on worldwide public health. Emerging lines of evidence indicate that autophagy plays an important role in promoting the HCV life cycle in host cells. Moreover, the diverse impacts of autophagy on a variety of signaling pathways in HCV-infected cells suggest that the autophagic process is required for balancing HCV-host cell interactions and involved in the pathogenesis of HCV-related liver diseases. However, the detailed molecular mechanism underlying how HCV activates autophagy to benefit viral growth is still enigmatic. Additionally, how the autophagic response contributes to disease progression in HCV-infected cells remains largely unknown. Hence, in this review, we overview the interplay between autophagy and the HCV life cycle and propose possible mechanisms by which autophagy may promote the pathogenesis of HCV-associated chronic liver diseases. Moreover, we outline the related studies on how autophagy interplays with HCV replication and discuss the possible implications of autophagy and viral replication in the progression of HCV-induced liver diseases, e.g., steatosis and hepatocellular carcinoma. Finally, we explore the potential therapeutics that target autophagy to cure HCV infection and its related liver diseases.
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Sherratt AR, Nasheri N, McKay CS, O'Hara S, Hunt A, Ning Z, Figeys D, Goto NK, Pezacki JP. A New Chemical Probe for Phosphatidylinositol Kinase Activity. Chembiochem 2014; 15:1253-6. [DOI: 10.1002/cbic.201402155] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2014] [Indexed: 12/13/2022]
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Qi H, Olson CA, Wu NC, Ke R, Loverdo C, Chu V, Truong S, Remenyi R, Chen Z, Du Y, Su SY, Al-Mawsawi LQ, Wu TT, Chen SH, Lin CY, Zhong W, Lloyd-Smith JO, Sun R. A quantitative high-resolution genetic profile rapidly identifies sequence determinants of hepatitis C viral fitness and drug sensitivity. PLoS Pathog 2014; 10:e1004064. [PMID: 24722365 PMCID: PMC3983061 DOI: 10.1371/journal.ppat.1004064] [Citation(s) in RCA: 53] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2013] [Accepted: 02/17/2014] [Indexed: 12/17/2022] Open
Abstract
Widely used chemical genetic screens have greatly facilitated the identification of many antiviral agents. However, the regions of interaction and inhibitory mechanisms of many therapeutic candidates have yet to be elucidated. Previous chemical screens identified Daclatasvir (BMS-790052) as a potent nonstructural protein 5A (NS5A) inhibitor for Hepatitis C virus (HCV) infection with an unclear inhibitory mechanism. Here we have developed a quantitative high-resolution genetic (qHRG) approach to systematically map the drug-protein interactions between Daclatasvir and NS5A and profile genetic barriers to Daclatasvir resistance. We implemented saturation mutagenesis in combination with next-generation sequencing technology to systematically quantify the effect of every possible amino acid substitution in the drug-targeted region (domain IA of NS5A) on replication fitness and sensitivity to Daclatasvir. This enabled determination of the residues governing drug-protein interactions. The relative fitness and drug sensitivity profiles also provide a comprehensive reference of the genetic barriers for all possible single amino acid changes during viral evolution, which we utilized to predict clinical outcomes using mathematical models. We envision that this high-resolution profiling methodology will be useful for next-generation drug development to select drugs with higher fitness costs to resistance, and also for informing the rational use of drugs based on viral variant spectra from patients. The emergence of drug resistance during antiviral treatment limits treatment options and poses challenges to pharmaceutical development. Meanwhile, the search for novel antiviral compounds with chemical genetic screens has led to the identification of antiviral agents with undefined drug mechanisms. Daclatasvir, an effective NS5A inhibitor, is one such example. In traditional methods to identify critical residues governing drug-protein interactions, wild type virus is passaged under drug treatment pressure, enabling the identification of resistant mutations evolved after multiple viral passages. However, this method only characterizes a fraction of the positively selected variants. Here we have simultaneously quantified the relative change in replication fitness as well as the relative sensitivity to Daclatasvir for all possible single amino acid mutations in the NS5A domain IA, thereby identifying the entire panel of positions that interact with the drug. Using mathematical models, we predicted which mutations pose the greatest risk of causing emergence of resistance under different scenarios of treatment compliance. The mutant fitness and drug-sensitivity profiles obtained can also inform the patient-specific use of Daclatasvir and may facilitate the development of second-generation drugs with a higher genetic barrier to resistance.
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Affiliation(s)
- Hangfei Qi
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America
| | - C Anders Olson
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Nicholas C Wu
- The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America
| | - Ruian Ke
- Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Claude Loverdo
- Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Virginia Chu
- Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Shawna Truong
- Department of Molecular, Cell and Developmental Biology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Roland Remenyi
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Zugen Chen
- Department of Human Genetics, University of California Los Angeles, Los Angeles, California, United States of America
| | - Yushen Du
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Sheng-Yao Su
- Institute of Information Science, Academia Sinica, Taipei, Taiwan; Institute of Biomedical Informatics, National Yang-Ming University, Taipei, Taiwan
| | - Laith Q Al-Mawsawi
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Ting-Ting Wu
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America
| | - Shu-Hua Chen
- Institute of Information Science, Academia Sinica, Taipei, Taiwan
| | - Chung-Yen Lin
- Institute of Information Science, Academia Sinica, Taipei, Taiwan
| | - Weidong Zhong
- Department of Infectious Diseases, Novartis Institutes for BioMedical Research, Emeryville, California, United States of America
| | - James O Lloyd-Smith
- Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, California, United States of America; Fogarty International Center, National Institutes of Health, Bethesda, Maryland, United States of America
| | - Ren Sun
- Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America; The Molecular Biology Institute, University of California Los Angeles, Los Angeles, California, United States of America; School of Medicine, Zhejiang University, Hangzhou, Zhejiang, China
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A complex comprising phosphatidylinositol 4-kinase IIIβ, ACBD3, and Aichi virus proteins enhances phosphatidylinositol 4-phosphate synthesis and is critical for formation of the viral replication complex. J Virol 2014; 88:6586-98. [PMID: 24672044 DOI: 10.1128/jvi.00208-14] [Citation(s) in RCA: 48] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
UNLABELLED Phosphatidylinositol 4-kinase IIIβ (PI4KB) is a host factor required for the replication of certain picornavirus genomes. We previously showed that nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB of Aichi virus (AiV), a picornavirus, interact with the Golgi protein, acyl-coenzyme A binding domain containing 3 (ACBD3), which interacts with PI4KB. These five viral proteins, ACBD3, PI4KB, and the PI4KB product phosphatidylinositol 4-phosphate (PI4P) colocalize to the AiV RNA replication sites (J. Sasaki et al., EMBO J. 31:754-766, 2012). We here examined the roles of these viral and cellular molecules in the formation of AiV replication complexes. Immunofluorescence microscopy revealed that treatment of AiV polyprotein-expressing cells with a small interfering RNA targeting ACBD3 abolished colocalization of the viral 2B, 2C, and 3A proteins with PI4KB. A PI4KB-specific inhibitor also prevented their colocalization. Virus RNA replication increased the level of cellular PI4P without affecting that of PI4KB, and individual expression of 2B, 2BC, 2C, 3A, or 3AB stimulated PI4P generation. These results suggest that the viral protein/ACBD3/PI4KB complex plays an important role in forming the functional replication complex by enhancing PI4P synthesis. Of the viral proteins, 3A and 3AB were shown to stimulate the in vitro kinase activity of PI4KB through forming a 3A or 3AB/ACBD3/PI4KB complex, whereas the ACBD3-mediated PI4KB activation by 2B and 2C remains to be demonstrated. IMPORTANCE The phosphatidylinositol 4-kinase PI4KB is a host factor required for the replication of certain picornavirus genomes. Aichi virus, a picornavirus belonging to the genus Kobuvirus, forms a complex comprising one of the viral nonstructural proteins 2B, 2BC, 2C, 3A, and 3AB, the Golgi protein ACBD3, and PI4KB to synthesize PI4P at the sites for viral RNA replication. However, the roles of this protein complex in forming the replication complex are unknown. This study showed that virus RNA replication and individual viral proteins enhance the level of cellular PI4P, and suggested that the viral protein/ACBD3/PI4KB complex plays an important role in forming a functional replication complex. Thus, the present study provides a new example of modulation of cellular lipid metabolism by viruses to support the replication of their genomes.
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