The Linker Region of NS3 Plays a Critical Role in the Replication and Infectivity of Hepatitis C Virus

ABSTRACT Membrane association of the hepatitis C virus NS5A protein is required for viral replication. This association is dependent on an N-terminal amphipathic helix (AH) within NS5A and is restricted to a subset of host cell intracellular membranes. The mechanism underlying this specificity is not known, but it may suggest a novel strategy for developing specific antiviral therapy. Here we have probed the mechanistic details of NS5A AH-mediated binding to both cell-derived and model membranes by use of biochemical membrane flotation and quartz crystal microbalance (QCM) with dissipation. With both assays, we observed AH-mediated binding to model lipid bilayers. When cell-derived membranes were coated on the quartz nanosensor, however, significantly more binding was detected, and the QCM-derived kinetic measurements suggested the existence of an interacting receptor in the target membranes. Biochemical flotation assays performed with trypsin-treated cell-derived membranes exhibited reduced AH-mediated membrane binding, while membrane binding of control cytochrome b5 remained unaffected. Similarly, trypsin treatment of the nanosensor coated with cellular membranes abolished AH peptide binding to the cellular membranes but did not affect the binding of a control lipid-binding peptide. These results therefore suggest that a protein plays a critical role in mediating and stabilizing the binding of NS5A's AH to its target membrane. These results also demonstrate the successful development of a new nanosensor technology ideal both for studying the interaction between a protein and its target membrane and for developing inhibitors of that interaction.

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CRITICAL ROLE OF VAL/GLY86 HLA-DRB DIMORPHISM IN THE NEONATAL RESISTANCE OR SUSCEPTIBILITY TO MATERNAL HEPATITIS C VIRUS INFECTION

The Pediatric Infectious Disease Journal ◽

10.1097/00006454-199710000-00019 ◽

1997 ◽

Vol 16 (10) ◽

pp. 1001-1002 ◽

Cited By ~ 3

Author(s):

Miryam Martinetti ◽

Ilaria Pacati ◽

Cristina Daielli ◽

Laura Salvaneschi ◽

Anna Maccabruni

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Virus Infection ◽

Critical Role ◽

Hepatitis C Virus Infection

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Interactions between Viral Nonstructural Proteins and Host Protein hVAP-33 Mediate the Formation of Hepatitis C Virus RNA Replication Complex on Lipid Raft

Journal of Virology ◽

10.1128/jvi.78.7.3480-3488.2004 ◽

2004 ◽

Vol 78 (7) ◽

pp. 3480-3488 ◽

Cited By ~ 247

Author(s):

Lu Gao ◽

Hideki Aizaki ◽

Jian-Wen He ◽

Michael M. C. Lai

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Raft ◽

Critical Role ◽

Rna Replication ◽

Dominant Negative ◽

Host Protein ◽

Hcv Rna ◽

Replication Complex ◽

Detergent Resistant Membranes

ABSTRACT The lipid raft membrane has been shown to be the site of hepatitis C virus (HCV) RNA replication. The mechanism of formation of the replication complex is not clear. We show here that the formation of the HCV RNA replication complex on lipid raft (detergent-resistant membranes) requires interactions among the HCV nonstructural (NS) proteins and may be initiated by the precursor of NS4B, which has the intrinsic property of anchoring to lipid raft membrane. In hepatocyte cell lines containing an HCV RNA replicon, most of the other NS proteins, including NS5A, NS5B, and NS3, were also localized to the detergent-resistant membranes. However, when individually expressed, only NS4B was associated exclusively with lipid raft. In contrast, NS5B and NS3 were localized to detergent-sensitive membrane and cytosolic fractions, respectively. NS5A was localized to both detergent-sensitive and -resistant membrane fractions. Furthermore, we show that a cellular vesicle membrane transport protein named hVAP-33 (the human homologue of the 33-kDa vesicle-associated membrane protein-associated protein), which binds to both NS5A and NS5B, plays a critical role in the formation of HCV replication complex. The hVAP-33 protein is partially associated with the detergent-resistant membrane fraction. The expression of dominant-negative mutants and small interfering RNA of hVAP-33 in HCV replicon cells resulted in the relocation of NS5B from detergent-resistant to detergent-sensitive membranes. Correspondingly, the amounts of both HCV RNA and proteins in the cells were reduced, indicating that hVAP-33 is critical for the formation of HCV replication complex and RNA replication. These results indicate that protein-protein interactions among the various HCV NS proteins and hVAP-33 are important for the formation of HCV replication complex.

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Ligand Bioactive Conformation Plays a Critical Role in the Design of Drugs That Target the Hepatitis C Virus NS3 Protease

Journal of Medicinal Chemistry ◽

10.1021/jm401338c ◽

2013 ◽

Vol 57 (5) ◽

pp. 1777-1789 ◽

Cited By ~ 26

Author(s):

Steven R. LaPlante ◽

Herbert Nar ◽

Christopher T. Lemke ◽

Araz Jakalian ◽

Norman Aubry ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Critical Role ◽

Bioactive Conformation ◽

Ns3 Protease

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Hepatitis C Virus Modelling In Vitro and In Vivo

10.32920/ryerson.14649642.v1 ◽

2021 ◽

Author(s):

Kenneth Blahut

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Infected Cell ◽

Antiviral Therapy ◽

Critical Role ◽

Free Virus ◽

Cell Infection ◽

Local Modes ◽

Direct Cell

Experiments have shown that hepatitis C virus (HCV) infections in vitro disseminate both distally via the release and diﬀusion of cell-free virus through the medium, and locally via direct, cell-to-cell transmission. To determine the relative contribution of each mode of infection to HCV dissemination, we developed an agent-based model (ABM) that explicitly incorporates both distal and local modes of infection. The ABM tracks the concentration of extracellular infectious virus in the supernatant and the number of intracellular HCV RNA segments within each infected cell over the course of simulated in vitro HCV infections. By constraining our ABM parameters using experimental data, we found that direct, cell-to-cell infection accounts for 98% (85%–100%, 95% credible region) of infection events, making it the dominant mode of HCV dissemination in vitro. Yet, while HCV spread via cell-free virus contributes little to the total number of infection events in vitro, it plays a critical role in enhancing cell-to-cell HCV dissemination by providing access to distant, uninfected areas, away from the already established large infection foci. Mathematical modelling of HCV load decay under antiviral therapy has allowed for the determination of antiviral eﬃcacy and other important parameters. Current mathematical models (MMs) of HCV infection are based on a set of ordinary diﬀerential equations (ODEs) and assume that infectious cell lifespans are exponentially distributed over time, meaning that every infected cell has an equal probability of dying at any time. Here, we introduce a new MM which: (1) allows for a realistic eclipse delay between the moment of cell infection and the release of new virus; and (2) considers both exponential and (log)normal distributed durations for infectious cell lifespan, wherein cells are assumed to continuously producing virus. Application of this MM to HCV load data for patients undergoing antiviral therapy leads to diﬀerent conclusions when predicting parameter values.

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Interferon Resistance of Hepatitis C Virus Genotype 1b: Relationship to Nonstructural 5A Gene Quasispecies Mutations

Journal of Virology ◽

10.1128/jvi.72.4.2795-2805.1998 ◽

1998 ◽

Vol 72 (4) ◽

pp. 2795-2805 ◽

Cited By ~ 140

Author(s):

Jean-Michel Pawlotsky ◽

Georgios Germanidis ◽

Avidan U. Neumann ◽

Muriel Pellerin ◽

Pierre-Olivier Frainais ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Amino Acid Sequence ◽

Critical Role ◽

Patient Specific ◽

Amino Acid Substitutions ◽

Virus Genotype ◽

Ns5a Protein ◽

Genotype 1B

ABSTRACT A 40-amino-acid sequence located in the nonstructural 5A (NS5A) protein of hepatitis C virus genotype 1b (HCV-1b) was recently suggested to be the interferon sensitivity-determining region (ISDR), because HCV-1b strains with an ISDR amino acid sequence identical to that of the prototype strain HCV-J were found to be resistant to alpha interferon (IFN-α) whereas strains with amino acid substitutions were found to be sensitive (N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, N. Izumi, F. Marumo, and C. Sato, J. Clin. Invest. 96:224–230, 1995; N. Enomoto, I. Sakuma, Y. Asahina, M. Kurosaki, T. Murakami, C. Yamamoto, Y. Ogura, N. Izumi, F. Marumo, and C. Sato, N. Engl. J. Med. 334:77–81, 1996). We used single-strand conformation polymorphism (SSCP) analysis, combined with cloning and sequencing strategies, to characterize NS5A quasispecies in HCV-1b-infected patients and determine the relationships between pre- and posttreatment NS5A quasispecies mutations and the IFN-α sensitivity of HCV-1b. The serine residues involved in phosphorylation of NS5A protein were highly conserved both in the various patients and in quasispecies in a given patient, suggesting that phosphorylation is important in NS5A protein function. A hot spot for amino acid substitutions was found at positions 2217 to 2218; it could be the result of either strong selection pressure or tolerance to these amino acid replacements. The proportion of synonymous mutations was significantly higher than the proportion of nonsynonymous mutations, suggesting that genetic variability in the region studied was the result of high mutation rates and viral replication kinetics rather than of positive selection. Sustained HCV RNA clearance was associated with low viral load and low nucleotide sequence entropy, suggesting (i) that the replication kinetics when treatment is started plays a critical role in HCV-1b sensitivity to IFN-α and (ii) that HCV-1b resistance to IFN-α could be conferred by numerous and/or related mutations that could be patient specific and located at different positions throughout the viral genome and could allow escape variants to be selected by IFN-α-stimulated immune responses. No NS5A sequence appeared to be intrinsically resistant or sensitive to IFN-α, but the HCV-J sequence was significantly more frequent in nonresponder quasispecies than in sustained virological responder quasispecies, suggesting that the balance between NS5A quasispecies sequences in infected patients could have a subtle regulatory influence on HCV replication.

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Critical Role of Virion-Associated Cholesterol and Sphingolipid in Hepatitis C Virus Infection

Journal of Virology ◽

10.1128/jvi.02530-07 ◽

2008 ◽

Vol 82 (12) ◽

pp. 5715-5724 ◽

Cited By ~ 139

Author(s):

Hideki Aizaki ◽

Kenichi Morikawa ◽

Masayoshi Fukasawa ◽

Hiromichi Hara ◽

Yasushi Inoue ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Cell Attachment ◽

Critical Role ◽

Molar Ratio ◽

Complete Life Cycle ◽

Rna Accumulation ◽

Hydrolysis Of ◽

Hcv Structural Proteins

ABSTRACT In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.

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Hepatitis C: Nutrition Care Canadian Guidelines for Health Care Providers

Canadian Journal of Dietetic Practice and Research ◽

10.3148/64.3.2003.139 ◽

2003 ◽

Vol 64 (3) ◽

pp. 139-141 ◽

Cited By ~ 4

Keyword(s):

Health Care ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Health Care Providers ◽

Scientific Evidence ◽

Critical Role ◽

Care Providers ◽

Nutrition Care ◽

On Line ◽

Accepted Practice

Nutrition plays a critical role in the management of hepatitis C. Dietitians of Canada has developed comprehensive, evidencebased guidelines to familiarize health care providers with effective nutrition care for the growing number of Canadians infected with the hepatitis C virus. The complete guidelines and two supporting educational fact sheets are available for downloading from http://www.dietitians.ca/resources/HepatitisC_Guidelines.htm . The guidelines and fact sheets are available in both English and French. The guidelines contain the full text, practice essentials, references, and extensive appendices with practical tools to assist educators in promoting nutrition to persons infected with the hepatitis C virus. Reprinting or photocopying of the document is encouraged provided the source is acknowledged. In addition, an on-line education course is available for health care providers and is available on www.dieteticsatwork.ca . These guidelines are directed to all health care providers who are in a position to offer nutrition-related advice and guidance to persons infected with the hepatitis C virus, in all stages of the disease. A national advisory committee comprised of leading authorities in Canada steered the development of the guidelines. The guidelines are based on the best information available at the time of publication; where scientific evidence was not available, best-accepted practice is presented.

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Hepatitis C Virus Proteins Interact with the Endosomal Sorting Complex Required for Transport (ESCRT) Machinery via Ubiquitination To Facilitate Viral Envelopment

mBio ◽

10.1128/mbio.01456-16 ◽

2016 ◽

Vol 7 (6) ◽

Cited By ~ 19

Author(s):

Rina Barouch-Bentov ◽

Gregory Neveu ◽

Fei Xiao ◽

Melanie Beer ◽

Elena Bekerman ◽

...

Keyword(s):

Plasma Membrane ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Critical Role ◽

Complex Component ◽

Escrt Machinery ◽

Late Domain ◽

Protein Ubiquitination ◽

Entry Points ◽

Late Domains

ABSTRACTEnveloped viruses commonly utilize late-domain motifs, sometimes cooperatively with ubiquitin, to hijack theendosomalsortingcomplexrequired fortransport (ESCRT) machinery for budding at the plasma membrane. However, the mechanisms underlying budding of viruses lacking defined late-domain motifs and budding into intracellular compartments are poorly characterized. Here, we map a network of hepatitis C virus (HCV) protein interactions with the ESCRT machinery using a mammalian-cell-based protein interaction screen and reveal nine novel interactions. We identify HRS (hepatocyte growth factor-regulated tyrosine kinase substrate), an ESCRT-0 complex component, as an important entry point for HCV into the ESCRT pathway and validate its interactions with the HCV nonstructural (NS) proteins NS2 and NS5A in HCV-infected cells. Infectivity assays indicate that HRS is an important factor for efficient HCV assembly. Specifically, by integrating capsid oligomerization assays, biophysical analysis of intracellular viral particles by continuous gradient centrifugations, proteolytic digestion protection, and RNase digestion protection assays, we show that HCV co-opts HRS to mediate a late assembly step, namely, envelopment. In the absence of defined late-domain motifs, K63-linked polyubiquitinated lysine residues in the HCV NS2 protein bind the HRS ubiquitin-interacting motif to facilitate assembly. Finally, ESCRT-III and VPS/VTA1 components are also recruited by HCV proteins to mediate assembly. These data uncover involvement of ESCRT proteins in intracellular budding of a virus lacking defined late-domain motifs and a novel mechanism by which HCV gains entry into the ESCRT network, with potential implications for other viruses.IMPORTANCEViruses commonly bud at the plasma membrane by recruiting the host ESCRT machinery via conserved motifs termed late domains. The mechanism by which some viruses, such as HCV, bud intracellularly is, however, poorly characterized. Moreover, whether envelopment of HCV and other viruses lacking defined late domains is ESCRT mediated and, if so, what the entry points into the ESCRT pathway are remain unknown. Here, we report the interaction network of HCV with the ESCRT machinery and a critical role for HRS, an ESCRT-0 complex component, in HCV envelopment. Viral protein ubiquitination was discovered to be a signal for HRS binding and HCV assembly, thereby functionally compensating for the absence of late domains. These findings characterize how a virus lacking defined late domains co-opts ESCRT to bud intracellularly. Since the ESCRT machinery is essential for the life cycle of multiple viruses, better understanding of this virus-host interplay may yield targets for broad-spectrum antiviral therapies.

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