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 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 A host cell RNA-binding protein, Staufen1, has a role in hepatitis C virus replication before virus assembly

2013 ◽  
Vol 94 (11) ◽  
pp. 2429-2436 ◽  
Author(s):  
Samantha L. Blackham ◽  
Michael J. McGarvey
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Rna Binding ◽  
Core Protein ◽  
Virus Assembly ◽  
Binding Protein ◽  
Replication Cycle ◽  
Hcv Rna ◽  
Huh7 Cells ◽  
Hiv 1

Staufen1 is a dsRNA-binding protein involved in the regulation of translation and the trafficking and degradation of cellular RNAs. Staufen1 has also been shown to stimulate translation of human immunodeficiency virus type 1 (HIV-1) RNA, regulate HIV-1 and influenza A virus assembly, and there is also indication that it can interact with hepatitis C virus (HCV) RNA. To investigate the role of Staufen1 in the HCV replication cycle, the effects of small interfering RNA knockout of Staufen1 on HCV strain JFH-1 replication and the intracellular distribution of the Staufen1 protein during HCV infection were examined. Silencing Staufen1 in HCV-infected Huh7 cells reduced virus secretion by around 70 %, intracellular HCV RNA levels by around 40 %, and core and NS3 proteins by around 95 and 45 %, respectively. Staufen1 appeared to be predominantly localized in the endoplasmic reticulum at the nuclear periphery in both uninfected and HCV-infected Huh7 cells. However, Staufen1 showed significant co-localization with NS3 and dsRNA, indicating that it may bind to replicating HCV RNA that is associated with the non-structural proteins. Staufen1 and HCV core protein localized very closely to one another during infection, but did not appear to overlap, indicating that Staufen1 may not bind to core protein or localize to the core-coated lipid droplets, suggesting that it may not be directly involved in HCV virus assembly. These findings indicate that Staufen1 is an important factor in HCV replication and that it might play a role early in the HCV replication cycle, e.g. in translation, replication or trafficking of the HCV genome, rather than in virion morphogenesis.

scholarly journals Cellular Determinants of Hepatitis C Virus Assembly, Maturation, Degradation, and Secretion

2007 ◽  
Vol 82 (5) ◽  
pp. 2120-2129 ◽  
Author(s):  
Pablo Gastaminza ◽  
Guofeng Cheng ◽  
Stefan Wieland ◽  
Jin Zhong ◽  
Wei Liao ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Buoyant Density ◽  
Infectious Hepatitis ◽  
Low Density ◽  
Very Low Density Lipoproteins ◽  
Independent Manner ◽  
Viral Egress ◽  
Vldl Assembly

ABSTRACT Intracellular infectious hepatitis C virus (HCV) particles display a distinctly higher buoyant density than do secreted virus particles, suggesting that the characteristic low density of extracellular HCV particles is acquired during viral egress. We took advantage of this difference to examine the determinants of assembly, maturation, degradation, and egress of infectious HCV particles. The results demonstrate that HCV assembly and maturation occur in the endoplasmic reticulum (ER) and post-ER compartments, respectively, and that both depend on microsomal transfer protein and apolipoprotein B, in a manner that parallels the formation of very-low-density lipoproteins (VLDL). In addition, they illustrate that only low-density particles are efficiently secreted and that immature particles are actively degraded, in a proteasome-independent manner, in a post-ER compartment of the cell. These results suggest that by coopting the VLDL assembly, maturation, degradation, and secretory machinery of the cell, HCV acquires its hepatocyte tropism and, by mimicry, its tendency to persist.

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 Efficient Virus Assembly, but Not Infectivity, Determines the Magnitude of Hepatitis C Virus-Induced Interferon Alpha Responses of Plasmacytoid Dendritic Cells

2014 ◽  
Vol 89 (6) ◽  
pp. 3200-3208 ◽  
Author(s):  
Elena Grabski ◽  
Ilka Wappler ◽  
Stephanie Pfaender ◽  
Eike Steinmann ◽  
Sibylle Haid ◽  
...  
Keyword(s):  
Dendritic Cells ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Virus Assembly ◽  
Plasmacytoid Dendritic Cells ◽  
Type I ◽  
Hepatitis C Virus Infection ◽  
Infected Cells ◽  
Interferon Responses

ABSTRACTWorldwide, approximately 160 million people are chronically infected with hepatitis C virus (HCV), seven distinct genotypes of which are discriminated. The hallmarks of HCV are its genetic variability and the divergent courses of hepatitis C progression in patients. We assessed whether intragenotypic HCV variations would differentially trigger host innate immunity. To this end, we stimulated human primary plasmacytoid dendritic cells (pDC) with crude preparations of different cell culture-derived genotype 2a HCV variants. Parental Japanese fulminant hepatitis C virus (JFH1) did not induce interferon alpha (IFN-α), whereas the intragenotypic chimera Jc1 triggered massive IFN-α responses. Purified Jc1 retained full infectivity but no longer induced IFN-α. Coculture of pDC with HCV-infected hepatoma cells retrieved the capacity to induce IFN-α, whereas Jc1-infected cells triggered stronger responses than JFH1-infected cells. Since the infectivity of virus particles did not seem to affect pDC activation, we next tested Jc1 mutants that were arrested at different stages of particle assembly. These experiments revealed that efficient assembly and core protein envelopment were critically needed to trigger IFN-α. Of note, sequences within domain 2 of the core that vitally affect virus assembly also crucially influenced the IFN-α responses of pDC. These data showed that viral determinants shaped host innate IFN-α responses to HCV.IMPORTANCEAlthough pegylated IFN-α plus ribavirin currently is the standard of care for the treatment of chronic hepatitis C virus infection, not much is known about the relevance of early interferon responses in the pathogenesis of hepatitis C virus infection. Here, we addressed whether intragenotypic variations of hepatitis C virus would account for differential induction of type I interferon responses mounted by primary blood-derived plasmacytoid dendritic cells. Surprisingly, a chimeric genotype 2a virus carrying the nonstructural genes of Japanese fulminant hepatitis C virus (JFH1) induced massive type I interferon responses, whereas the original genotype 2a JFH1 strain did not. Our detailed analyses revealed that, not the virus infectivity, but rather, the efficiency of virus assembly and core protein envelopment critically determined the magnitude of interferon responses. To our knowledge, this is the first example of hepatitis C virus-associated genetic variations that determine the magnitude of innate host responses.

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 A Conserved Proline between Domains II and III of Hepatitis C Virus NS5A Influences both RNA Replication and Virus Assembly

2009 ◽  
Vol 83 (20) ◽  
pp. 10788-10796 ◽  
Author(s):  
Mair Hughes ◽  
Sarah Gretton ◽  
Holly Shelton ◽  
David D. Brown ◽  
Christopher J. McCormick ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Virus Assembly ◽  
Infectious Clone ◽  
Consensus Sequence ◽  
Virus Production ◽  
Rna Replication ◽  
Subgenomic Replicon ◽  
Genotype 1B

ABSTRACT We previously demonstrated that two closely spaced polyproline motifs, with the consensus sequence Pro-X-X-Pro-X-Lys/Arg, located between residues 343 to 356 of NS5A, mediated interactions with cellular SH3 domains. The N-terminal motif (termed PP2.1) is only conserved in genotype 1 isolates, whereas the C-terminal motif (PP2.2) is conserved throughout all hepatitis C virus (HCV) isolates, although this motif was shown to be dispensable for replication of the genotype 1b subgenomic replicon. In order to investigate the potential role of these motifs in the viral life cycle, we have undertaken a detailed mutagenic analysis of these proline residues in the context of both genotype 1b (FK5.1) or 2a subgenomic replicons and the genotype 2a infectious clone, JFH-1. We show that the PP2.2 motif is dispensable for RNA replication of all subgenomic replicons and, furthermore, is not required for virus production in JFH-1. In contrast, the PP2.1 motif is only required for genotype 1b RNA replication. Mutation of proline 346 within PP2.1 to alanine dramatically attenuated genotype 1b replicon replication in three distinct genetic backgrounds, but the corresponding proline 342 was not required for replication of the JFH-1 subgenomic replicon. However, the P342A mutation resulted in both a delay to virus release and a modest (up to 10-fold) reduction in virus production. These data point to critical roles for these proline residues at multiple stages in the HCV life cycle; however, they also caution against extrapolation of data from culture-adapted replicons to infectious virus.

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