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

2009 ◽  
Vol 84 (4) ◽  
pp. 1666-1673 ◽  
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.

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

2008 ◽  
Vol 82 (15) ◽  
pp. 7624-7639 ◽  
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.

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

2015 ◽  
Vol 5 (1) ◽  
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.

scholarly journals Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp111

2003 ◽  
Vol 77 (18) ◽  
pp. 10131-10138 ◽  
Author(s):  
Emmanuelle Blanchard ◽  
Christophe Hourioux ◽  
Denys Brand ◽  
Malika Ait-Goughoulte ◽  
Alain Moreau ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Semliki Forest Virus ◽  
Core Protein ◽  
Virus Assembly ◽  
Signal Sequence ◽  
E1 Protein ◽  
Hcv Core Protein ◽  
Virus Like Particle ◽  
Genes Encoding

ABSTRACT In the absence of a hepatitis C virus (HCV) culture system, the use of a Semliki Forest virus replicon expressing genes encoding HCV structural proteins that assemble into HCV-like particles provides an opportunity to study HCV morphogenesis. Using this system, we showed that the HCV core protein constitutes the budding apparatus of the virus and that its targeting to the endoplasmic reticulum by means of the signal sequence of E1 protein is essential for budding. In addition, the aspartic acid at position 111 in the HCV core protein sequence was found to be crucial for virus assembly, demonstrating the usefulness of this system for mapping amino acids critical to HCV morphogenesis.

scholarly journals Adaptive mutations promote hepatitis C virus assembly by accelerating Core translocation to the endoplasmic reticulum

2020 ◽  
pp. jbc.RA120.016010
Author(s):  
Fuxiang Zheng ◽  
Ni Li ◽  
Yi Xu ◽  
Yuanping Zhou ◽  
Yi-Ping Li
Keyword(s):  
Endoplasmic Reticulum ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Assembly ◽  
Buoyant Density ◽  
Virus Production ◽  
Adaptive Mutations ◽  
Huh7 Cells ◽  
Density Analysis

The envelopment of hepatitis C virus (HCV) is believed to occur primarily in the endoplasmic reticulum (ER)-associated membrane, and the translocation of viral Core protein from lipid droplets (LDs) to the ER is essential for the envelopment of viral particles. However, the factors involved in are not completely understood. Herein, we identified eight adaptive mutations that enhanced virus spread and infectivity of genotype 1a clone TNcc in hepatoma Huh7 cells through long-term culture adaptation and reverse genetic study. Of eight mutations, I853V in NS2 and C2865F in NS5B were found to be minimal mutation sets that enabled an increase in virus production without apparently affecting RNA replication, thus suggesting its roles in the post-replication stage of the HCV life cycle. Using a protease K protection and confocal microscopy analysis, we demonstrated that C2865F and the combination of I853V/C2865F enhanced virus envelopment by facilitating Core translocation from LDs to the ER. Buoyant density analysis revealed that I853V/C2865F contributed to the release of virion with a density of ~1.10 g/ml. Moreover, we demonstrated that NS5B directly interacted with NS2 at the protease domain, and that mutations I853V, C2865F, I853V/C2865F enhanced the interaction. In addition, C2865F also enhanced the interaction between NS5B and Core. In conclusion, this study demonstrated that adaptive mutations in NS2 and NS5B promoted HCV envelopment by accelerating Core translocation from LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.

scholarly journals 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

2012 ◽  
Vol 87 (3) ◽  
pp. 1385-1399 ◽  
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.

scholarly journals Alanine Scanning of the Hepatitis C Virus Core Protein Reveals Numerous Residues Essential for Production of Infectious Virus

2007 ◽  
Vol 81 (19) ◽  
pp. 10220-10231 ◽  
Author(s):  
Catherine L. Murray ◽  
Christopher T. Jones ◽  
Jodie Tassello ◽  
Charles M. Rice
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Core Protein ◽  
Virus Production ◽  
Alanine Scanning Mutagenesis ◽  
Alanine Scanning ◽  
Infectious Virion ◽  
Hcv Core Protein

ABSTRACT Hepatitis C virus (HCV) is an important human pathogen affecting an estimated 3% of the world's population. Recent advances have enabled in vitro propagation of the virus and allow assembly and egress to be investigated for the first time. As a component of the virion, the HCV core protein likely functions primarily in infectious virus production, although little is known about the determinants of this activity. To investigate the roles of core in the viral life cycle, we performed a comprehensive deletion and alanine scanning mutagenesis study of this protein in the context of a genotype 2a reporter virus. We have confirmed that core protein is essential for infectious virion production and have identified numerous residues required for this role. The infectivity of several assembly-defective core mutants could be rescued by compensatory mutations identified in p7 and NS2, suggesting genetic interactions with core and highlighting the importance of these nonstructural proteins in infectious virion morphogenesis.

scholarly journals Compensatory Mutations in E1, p7, NS2, and NS3 Enhance Yields of Cell Culture-Infectious Intergenotypic Chimeric Hepatitis C Virus

2006 ◽  
Vol 81 (2) ◽  
pp. 629-638 ◽  
Author(s):  
MinKyung Yi ◽  
Yinghong Ma ◽  
Jeremy Yates ◽  
Stanley M. Lemon
Keyword(s):  
Cell Culture ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Infectious Virus ◽  
Cultured Cells ◽  
Virus Assembly ◽  
Infectious Hepatitis ◽  
Adaptive Mutations ◽  
Compensatory Mutations

ABSTRACT There is little understanding of mechanisms underlying the assembly and release of infectious hepatitis C virus (HCV) from cultured cells. Cells transfected with synthetic genomic RNA from a unique genotype 2a virus (JFH1) produce high titers of virus, while virus yields are much lower with a prototype genotype 1a RNA containing multiple cell culture-adaptive mutations (H77S). To characterize the basis for this difference in infectious particle production, we constructed chimeric genomes encoding the structural proteins of H77S within the background of JFH1. RNAs encoding polyproteins fused at the NS2/NS3 junction (“H-NS2/NS3-J”) and at a site of natural, intergenotypic recombination within NS2 [“H-(NS2)-J”] produced infectious virus. In contrast, no virus was produced by a chimera fused at the p7-NS2 junction. Chimera H-NS2/NS3-J virus (vH-NS2/NS3-J) recovered from transfected cultures contained compensatory mutations in E1 and NS3 that were essential for the production of infectious virus, while yields of infectious vH-(NS2)-J were enhanced by mutations within p7 and NS2. These compensatory mutations were chimera specific and did not enhance viral RNA replication or polyprotein processing; thus, they likely compensate for incompatibilities between proteins of different genotypes at sites of interactions essential for virus assembly and/or release. Mutations in p7 and NS2 acted additively and increased the specific infectivity of vH-(NS2)-J particles, while having less impact on the numbers of particles released. We conclude that interactions between NS2 and E1 and p7 as well as between NS2 and NS3 are essential for virus assembly and/or release and that each of these viral proteins plays an important role in this process.

scholarly journals Cellular vimentin content regulates the protein level of hepatitis C virus core protein and the hepatitis C virus production in cultured cells

Virology ◽  
2009 ◽  
Vol 383 (2) ◽  
pp. 319-327 ◽  
Author(s):  
Yuko Nitahara-Kasahara ◽  
Masayoshi Fukasawa ◽  
Fumiko Shinkai-Ouchi ◽  
Shigeko Sato ◽  
Tetsuro Suzuki ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Protein Level ◽  
Cultured Cells ◽  
Core Protein ◽  
Virus Production ◽  
Virus Core

scholarly journals Hepatitis C Virus p7 and NS2 Proteins Are Essential for Production of Infectious Virus

2007 ◽  
Vol 81 (16) ◽  
pp. 8374-8383 ◽  
Author(s):  
Christopher T. Jones ◽  
Catherine L. Murray ◽  
Dawnnica K. Eastman ◽  
Jodie Tassello ◽  
Charles M. Rice
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Infectious Virus ◽  
Early Stage ◽  
Virus Production ◽  
Health Concern ◽  
Cell Culture Systems ◽  
Catalytic Active Site

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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