The N-terminal Helical Region of the Hepatitis C Virus p7 Ion Channel Protein Is Critical for Infectious Virus Production

ABSTRACT Hepatitis C virus (HCV) infection is a global health concern affecting an estimated 3% of the world's population. Recently, cell culture systems have been established, allowing recapitulation of the complete virus life cycle for the first time. Since the HCV proteins p7 and NS2 are not predicted to be major components of the virion, nor are they required for RNA replication, we investigated whether they might have other roles in the viral life cycle. Here we utilize the recently described infectious J6/JFH chimera to establish that the p7 and NS2 proteins are essential for HCV infectivity. Furthermore, unprocessed forms of p7 and NS2 were not required for this activity. Mutation of two conserved basic residues, previously shown to be important for the ion channel activity of p7 in vitro, drastically impaired infectious virus production. The protease domain of NS2 was required for infectivity, whereas its catalytic active site was dispensable. We conclude that p7 and NS2 function at an early stage of virion morphogenesis, prior to the assembly of infectious virus.

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Amino Acid Polymorphisms in Hepatitis C Virus Core Affect Infectious Virus Production and Major Histocompatibility Complex Class I Molecule Expression

Scientific Reports ◽

10.1038/srep13994 ◽

2015 ◽

Vol 5 (1) ◽

Cited By ~ 5

Author(s):

Megumi Tasaka-Fujita ◽

Nao Sugiyama ◽

Wonseok Kang ◽

Takahiro Masaki ◽

Asako Murayama ◽

...

Keyword(s):

Major Histocompatibility Complex ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Amino Acid ◽

Mhc Class I ◽

Infectious Virus ◽

Core Protein ◽

Virus Production ◽

Class I ◽

Histocompatibility Complex

Abstract Amino acid (aa) polymorphisms in the hepatitis C virus (HCV) genotype 1b core protein have been reported to be a potent predictor for poor response to interferon (IFN)-based therapy and a risk factor for hepatocarcinogenesis. We investigated the effects of these polymorphisms with genotype 1b/2a chimeric viruses that contained polymorphisms of Arg/Gln at aa 70 and Leu/Met at aa 91. We found that infectious virus production was reduced in cells transfected with chimeric virus RNA that had Gln at aa 70 (aa70Q) compared with RNA with Arg at aa 70 (aa70R). Using flow cytometry analysis, we confirmed that HCV core protein accumulated in aa70Q clone transfected cells and it caused a reduction in cell-surface expression of major histocompatibility complex (MHC) class I molecules induced by IFN treatment through enhanced protein kinase R phosphorylation. We could not detect any effects due to the polymorphism at aa 91. In conclusion, the polymorphism at aa 70 was associated with efficiency of infectious virus production and this deteriorated virus production in strains with aa70Q resulted in the intracellular accumulation of HCV proteins and attenuation of MHC class I molecule expression. These observations may explain the strain-associated resistance to IFN-based therapy and hepatocarcinogenesis of HCV.

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Identification of specific regions in hepatitis C virus core, NS2 and NS5A that genetically interact with p7 and co-ordinate infectious virus production

Journal of Viral Hepatitis ◽

10.1111/jvh.12004 ◽

2012 ◽

Vol 20 (4) ◽

pp. e66-e71 ◽

Cited By ~ 11

Author(s):

H. Gouklani ◽

C. Beyer ◽

H. Drummer ◽

E. J. Gowans ◽

H. J. Netter ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Virus Production ◽

Virus Core

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Genetic Analysis of the Carboxy-Terminal Region of the Hepatitis C Virus Core Protein

Journal of Virology ◽

10.1128/jvi.02043-09 ◽

2009 ◽

Vol 84 (4) ◽

pp. 1666-1673 ◽

Cited By ~ 27

Author(s):

Martina Kopp ◽

Catherine L. Murray ◽

Christopher T. Jones ◽

Charles M. Rice

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Core Protein ◽

Virus Assembly ◽

Signal Sequence ◽

Virus Production ◽

Terminal Region ◽

Dual Processing ◽

Chronic Hcv

ABSTRACT Hepatitis C virus (HCV) is a liver-tropic pathogen with severe health consequences for infected individuals. Chronic HCV infection can progress to cirrhosis and hepatocellular carcinoma and is a leading indicator for liver transplantation. The HCV core protein is an essential component of the infectious virus particle, but many aspects of its role remain undefined. The C-terminal region of the core protein acts as a signal sequence for the E1 glycoprotein and undergoes dual processing events during infectious virus assembly. The exact C terminus of the mature, virion-associated core protein is not known. Here, we performed genetic analyses to map the essential determinants of the HCV core C-terminal region, as well as to define the minimal length of the protein that can function for infectious virus production in trans.

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Characterization of Hepatitis C Virus Recombinants with Chimeric E1/E2 Envelope Proteins and Identification of Single Amino Acids in the E2 Stem Region Important for Entry

Journal of Virology ◽

10.1128/jvi.00684-12 ◽

2012 ◽

Vol 87 (3) ◽

pp. 1385-1399 ◽

Cited By ~ 20

Author(s):

Thomas H. R. Carlsen ◽

Troels K. H. Scheel ◽

Santseharay Ramirez ◽

Steven K. H. Foung ◽

Jens Bukh

Keyword(s):

Amino Acids ◽

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Core Protein ◽

Particle Density ◽

Virus Production ◽

Envelope Proteins ◽

Late Step

ABSTRACTThe hepatitis C virus (HCV) envelope proteins E1 and E2 play a key role in host cell entry and represent important targets for vaccine and drug development. Here, we characterized HCV recombinants with chimeric E1/E2 complexesin vitro. Using genotype 1a/2a JFH1-based recombinants expressing 1a core-NS2, we exchanged E2 with functional isolate sequences of genotypes 1a (alternative isolate), 1b, and 2a. While the 1a-E2 exchange did not impact virus viability, the 2a-E2 recombinant was nonviable. After E2 exchange from three 1b isolates, long delays were observed before spread of infection. For recovered 1b-E2 recombinants, single E2 stem region amino acid changes were identified at residues 706, 707, and 710. In reverse genetic studies, these mutations increased infectivity titers by ∼100-fold, apparently without influencing particle stability or cell binding although introducing slight decrease in particle density. In addition, the 1b-E2 exchange led to a decrease in secreted core protein of 25 to 50%, which was further reduced by the E2 stem region mutations. These findings indicated that compensatory mutations permitted robust infectious virus production, without increasing assembly/release. Studies of E1/E2 heterodimerization showed no differences in intracellular E1/E2 interaction for chimeric constructs with or without E2 stem region mutations. Interestingly, the E2 stem region mutations allowed efficient entry, which was verified in 1a-E1/1b-E2 HCV pseudoparticle assays. A CD81 inhibition assay indicated that the mutations influenced a late step of the HCV entry pathway. Overall, this study identified specific amino acids in the E2 stem region of importance for HCV entry and for production of infectious virus particles.

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NS3 Helicase Domains Involved in Infectious Intracellular Hepatitis C Virus Particle Assembly

Journal of Virology ◽

10.1128/jvi.00724-08 ◽

2008 ◽

Vol 82 (15) ◽

pp. 7624-7639 ◽

Cited By ~ 152

Author(s):

Yinghong Ma ◽

Jeremy Yates ◽

Yuqiong Liang ◽

Stanley M. Lemon ◽

MinKyung Yi

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Droplets ◽

Infectious Virus ◽

Core Protein ◽

Virus Assembly ◽

Virus Production ◽

Intermediate Step ◽

Particle Assembly ◽

Chimeric Rna

ABSTRACT A mutation within subdomain 1 of the hepatitis C virus (HCV) NS3 helicase (NS3-Q221L) (M. Yi, Y. Ma, J. Yates, and S. M. Lemon, J. Virol. 81:629-638, 2007) rescues a defect in production of infectious virus by an intergenotypic chimeric RNA (HJ3). Although NS3-Gln-221 is highly conserved across HCV genotypes, the Leu-221 substitution had no effect on RNA replication or NS3-associated enzymatic activities. However, while transfection of unmodified HJ3 RNA failed to produce either extracellular or intracellular infectious virus, transfection of HJ3 RNA containing the Q221L substitution (HJ3/QL) resulted in rapid accumulation of intracellular infectious particles with release into extracellular fluids. In the absence of the Q221L mutation, both NS5A and NS3 were recruited to core protein on the surface of lipid droplets, but there was no assembly of core into high-density, rapidly sedimenting particles. Further analysis demonstrated that a Q221N mutation minimally rescued virus production and led to a second-site I399V mutation in subdomain 2 of the helicase. Similarly, I399V alone allowed only low-level virus production and led to selection of an I286V mutation in subdomain 1 of the helicase which fully restored virus production, confirming the involvement of both major helicase subdomains in the assembly process. Thus, multiple mutations in the helicase rescue a defect in an early-intermediate step in virus assembly that follows the recruitment of NS5A to lipid droplets and precedes the formation of dense intracellular viral particles. These data reveal a previously unsuspected role for the NS3 helicase in early virion morphogenesis and provide a new perspective on HCV assembly.

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Regulation of Hepatitis C Virus Translation and Infectious Virus Production by the MicroRNA miR-122

Journal of Virology ◽

10.1128/jvi.00417-10 ◽

2010 ◽

Vol 84 (13) ◽

pp. 6615-6625 ◽

Cited By ~ 230

Author(s):

Rohit K. Jangra ◽

MinKyung Yi ◽

Stanley M. Lemon

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Infectious Virus ◽

Virus Production ◽

Hcv Rna ◽

Wild Type ◽

Entry Site ◽

Internal Ribosome Entry ◽

High Virus ◽

In Cells

ABSTRACT miR-122 is a liver-specific microRNA that positively regulates hepatitis C virus (HCV) RNA abundance and is essential for production of infectious HCV. Using a genetic approach, we show that its ability to enhance yields of infectious virus is dependent upon two miR-122-binding sites near the 5′ end of the HCV genome, S1 and S2. Viral RNA with base substitutions in both S1 and S2 failed to produce infectious virus in transfected cells, while virus production was rescued to near-wild-type levels in cells supplemented with a complementary miR-122 mutant. A comparison of mutants with substitutions in only one site revealed S1 to be dominant, as an S2 but not S1 mutant produced high virus yields in cells supplemented with wild-type miR-122. Translation of HCV RNA was reduced over 50% by mutations in either S1 or S2 and was partially rescued by transfection of the complementary miR-122 mutant. Unlike the case for virus replication, however, both sites function equally in regulating translation. We conclude that miR-122 promotes replication by binding directly to both sites in the genomic RNA and, at least in part, by stimulating internal ribosome entry site (IRES)-mediated translation. However, a comparison of the replication capacities of the double-binding-site mutant and an IRES mutant with a quantitatively equivalent defect in translation suggests that the decrement in translation associated with loss of miR-122 binding is insufficient to explain the profound defect in virus production by the double mutant. miR-122 is thus likely to act at an additional step in the virus life cycle.

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