scholarly journals NS2 is dispensable for efficient assembly of hepatitis C virus-like particles in a bipartite trans-encapsidation system

2014 ◽  
Vol 95 (11) ◽  
pp. 2427-2441 ◽  
Author(s):  
Matthew J. Bentham ◽  
Najat Marraiki ◽  
Christopher J. McCormick ◽  
David J. Rowlands ◽  
Stephen Griffin
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Early Stage ◽  
Infectious Hepatitis ◽  
Structural Proteins ◽  
Structural Protein ◽  
Subgenomic Replicon ◽  
Particle Assembly ◽  
Baculovirus Expression

Infectious hepatitis C virus (HCV) particle production in the genotype 2a JFH-1-based cell culture system involves non-structural proteins in addition to canonical virion components. NS2 has been proposed to act as a protein adaptor, co-ordinating the early stages of virion assembly. However, other studies have identified late-acting roles for this protein, making its precise involvement in infectious particle production unclear. Using a robust, bipartite trans-encapsidation system based upon baculovirus expression of HCV structural proteins, we have generated HCV-like particles (HCV-LP) in the absence of NS2 with overt similarity to wild-type virions. HCV-LP could transduce naive cells with trans-encapsidated subgenomic replicon RNAs and shared similar biochemical and biophysical properties with JFH-1 HCV. Both genotype 1b and JFH-1 intracellular HCV-LP were produced in the absence of NS2, whereas restoring NS2 to the JFH-1 system dramatically enhanced secreted infectivity, consistent with a late-acting role. Our system recapitulated authentic HCV particle assembly via trans-complementation of bicistronic, NS2-deleted, chimeric HCV, which is otherwise deficient in particle production. This closely resembled replicon-mediated NS2 trans-complementation, confirming that baculovirus expression of HCV proteins did not unduly affect particle production. Furthermore, this suggests that separation of structural protein expression from replicating HCV RNAs that are destined to be packaged alleviates an early stage requirement for NS2 during particle formation. This highlights our current lack of understanding of how NS2 mediates assembly, yet comparison of full-length and bipartite systems may provide further insight into this process.

scholarly journals Role of seipin in lipid droplet morphology and hepatitis C virus life cycle

2013 ◽  
Vol 94 (10) ◽  
pp. 2208-2214 ◽  
Author(s):  
Sophie Clément ◽  
Catherine Fauvelle ◽  
Emilie Branche ◽  
Vincent Kaddai ◽  
Stéphanie Conzelmann ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Outer Surface ◽  
Lipid Droplets ◽  
Particle Production ◽  
Critical Factor ◽  
Infectious Hepatitis ◽  
Particle Assembly ◽  
Droplet Morphology

Infectious hepatitis C virus (HCV) particle assembly starts at the surface of lipid droplets, cytoplasmic organelles responsible for neutral fat storage. We analysed the relationship between HCV and seipin, a protein involved in lipid droplet maturation. Although seipin overexpression did not affect the total mean volume occupied by lipid droplets nor the total triglyceride and cholesterol ester levels per cell, it caused an increase in the mean diameter of lipid droplets by 60 %, while decreasing their total number per cell. The latter two effects combined resulted in a 34 % reduction of the total outer surface area of lipid droplets per cell, with a proportional decrease in infectious viral particle production, probably due to a defect in particle assembly. These results suggest that the available outer surface of lipid droplets is a critical factor for HCV release, independent of the neutral lipid content of the cell.

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 Hepatitis C Virus Structural Proteins Assemble into Viruslike Particles in Insect Cells

1998 ◽  
Vol 72 (5) ◽  
pp. 3827-3836 ◽  
Author(s):  
Thomas F. Baumert ◽  
Susumu Ito ◽  
David T. Wong ◽  
T. Jake Liang
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Insect Cells ◽  
Vaccine Development ◽  
Cultured Cells ◽  
Recombinant Baculovirus ◽  
Structural Proteins ◽  
Particle Assembly ◽  
Viruslike Particles ◽  
Hcv Structural Proteins

ABSTRACT Hepatitis C virus (HCV) is a leading cause of chronic hepatitis in the world. The study of HCV has been hampered by the low level of viral particles in infected individuals, the inability to propagate efficiently the virus in cultured cells, and the lack of a convenient animal model. Due to these obstacles, neither the structure of the virus nor the prerequisites for its assembly have been clearly defined. In this report, we describe a model for the production and purification of HCV-like particles in insect cells using a recombinant baculovirus containing the cDNA of the HCV structural proteins. In insect cells, expressed HCV structural proteins assembled into enveloped viruslike particles (40 to 60 nm in diameter) in large cytoplasmic cisternae, presumably derived from the endoplasmic reticulum. Biophysical characterization of viruslike particles by CsCl and sucrose gradient centrifugation revealed biophysical properties similar to those of putative virions isolated from infected humans. The results suggested that HCV core and envelope proteins without p7 were sufficient for viral particle formation. Analysis of particle-associated nucleic acids demonstrated that HCV RNAs were selectively incorporated into the particles over non-HCV transcripts. The synthesis of HCV-like particles in insect cells may provide an important tool to determine the structural requirements for HCV particle assembly as well as to study viral genome encapsidation and virus-host interactions. The described system may also represent a potential approach toward vaccine development.

scholarly journals Repression of Interferon Regulatory Factor 1 by Hepatitis C Virus Core Protein Results in Inhibition of Antiviral and Immunomodulatory Genes

2006 ◽  
Vol 81 (1) ◽  
pp. 202-214 ◽  
Author(s):  
Anna R. Ciccaglione ◽  
Emilia Stellacci ◽  
Cinzia Marcantonio ◽  
Valentina Muto ◽  
Michele Equestre ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Core Protein ◽  
Interferon Regulatory Factor ◽  
Structural Proteins ◽  
Transcriptional Level ◽  
Structural Protein ◽  
Regulatory Factor ◽  
The Core ◽  
Interferon Regulatory Factor 1

ABSTRACT Hepatitis C virus (HCV) proteins are known to interfere at several levels with both innate and adaptive responses of the host. A key target in these effects is the interferon (IFN) signaling pathway. While the effects of nonstructural proteins are well established, the role of structural proteins remains controversial. We investigated the effect of HCV structural proteins on the expression of interferon regulatory factor 1 (IRF-1), a secondary transcription factor of the IFN system responsible for inducing several key antiviral and immunomodulatory genes. We found substantial inhibition of IRF-1 expression in cells expressing the entire HCV replicon. Suppression of IRF-1 synthesis was mainly mediated by the core structural protein and occurred at the transcriptional level. The core protein in turn exerted a transcriptional repression of several interferon-stimulated genes, targets of IRF-1, including interleukin-15 (IL-15), IL-12, and low-molecular-mass polypeptide 2. These data recapitulate in a unifying mechanism, i.e., repression of IRF-1 expression, many previously described pathogenetic effects of HCV core protein and suggest that HCV core-induced IRF-1 repression may play a pivotal role in establishing persistent infection by dampening an effective immune response.

scholarly journals Domain III of NS5A contributes to both RNA replication and assembly of hepatitis C virus particles

2009 ◽  
Vol 90 (6) ◽  
pp. 1329-1334 ◽  
Author(s):  
Mair Hughes ◽  
Stephen Griffin ◽  
Mark Harris
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Critical Role ◽  
Rna Replication ◽  
Subgenomic Replicon ◽  
Virus Particles ◽  
C Terminus ◽  
Genotype 2 ◽  
Domain Iii

The hepatitis C virus (HCV) NS5A protein plays a critical role in viral RNA replication and has recently been shown to play a role in particle production in the infectious genotype 2a HCV clone (JFH-1). Here, we show that alanine substitutions of serines 2428/2430 within the C-terminal domain III of NS5A do not affect subgenomic replicon RNA replication but do reduce particle production. In contrast, substitution of serines 2390/2391 had no effect on either RNA replication or particle production. Relative to genotype 1, all genotype 2 HCV isolates contain a 19 residue insertion near the C terminus of domain III which, when deleted (▵2408–2426), resulted in a delay to both RNA replication and particle production. None of these mutations affected the ratio of basal to hyperphosphorylated NS5A, suggesting that serines between residues 2390 and 2430 are not phosphorylated. We propose that although domain III is dispensable for RNA replication, it nevertheless influences this process.

scholarly journals Detergent-Resistant Membrane Association of NS2 and E2 during Hepatitis C Virus Replication

2015 ◽  
Vol 89 (8) ◽  
pp. 4562-4574 ◽  
Author(s):  
Saravanabalaji Shanmugam ◽  
Dhanaranjani Saravanabalaji ◽  
MinKyung Yi
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Particle Production ◽  
Core Protein ◽  
Virus Assembly ◽  
Hcv Rna ◽  
Dependent Manner ◽  
Short Term Treatment ◽  
Particle Assembly ◽  
Detergent Resistant Membranes

ABSTRACTPreviously, we demonstrated that the efficiency of hepatitis C virus (HCV) E2-p7 processing regulates p7-dependent NS2 localization to putative virus assembly sites near lipid droplets (LD). In this study, we have employed subcellular fractionations and membrane flotation assays to demonstrate that NS2 associates with detergent-resistant membranes (DRM) in a p7-dependent manner. However, p7 likely plays an indirect role in this process, since only the background level of p7 was detectable in the DRM fractions. Our data also suggest that the p7-NS2 precursor is not involved in NS2 recruitment to the DRM, despite its apparent targeting to this location. Deletion of NS2 specifically inhibited E2 localization to the DRM, indicating that NS2 regulates this process. Treatment of cells with methyl-β-cyclodextrin (MβCD) significantly reduced the DRM association of Core, NS2, and E2 and reduced infectious HCV production. Since disruption of the DRM localization of NS2 and E2, either due to p7 and NS2 defects, respectively, or by MβCD treatment, inhibited infectious HCV production, these proteins' associations with the DRM likely play an important role during HCV assembly. Interestingly, we detected the HCV replication-dependent accumulation of ApoE in the DRM fractions. Taking into consideration the facts that ApoE was shown to be a major determinant for infectious HCV particle production at the postenvelopment step and that the HCV Core protein strongly associates with the DRM, recruitment of E2 and ApoE to the DRM may allow the efficient coordination of Core particle envelopment and postenvelopment events at the DRM to generate infectious HCV production.IMPORTANCEThe biochemical nature of HCV assembly sites is currently unknown. In this study, we investigated the correlation between NS2 and E2 localization to the detergent-resistant membranes (DRM) and HCV particle assembly. We determined that although NS2's DRM localization is dependent on p7, p7 was not targeted to these membranes. We then showed that NS2 regulates E2 localization to the DRM, consistent with its role in recruiting E2 to the virus assembly sites. We also showed that short-term treatment with the cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) not only disrupted the DRM localization of Core, NS2, and E2 but also specifically inhibited intracellular virus assembly without affecting HCV RNA replication. Thus, our data support the role of the DRM as a platform for particle assembly process.

scholarly journals Novel Chimeric Genotype 1b/2a Hepatitis C Virus Suitable for High-Throughput Screening

2007 ◽  
Vol 52 (2) ◽  
pp. 666-674 ◽  
Author(s):  
Yingjia Zhang ◽  
Peter Weady ◽  
Rohit Duggal ◽  
Weidong Hao
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
High Throughput ◽  
High Throughput Screening ◽  
Luciferase Reporter ◽  
Structural Protein ◽  
Genotype 1 ◽  
Luciferase Reporter Gene ◽  
Subgenomic Replicon ◽  
Viral Particles

ABSTRACT A major obstacle in hepatitis C virus (HCV) research has been the lack of a permissive cell culture system that produces infectious viral particles. Significant breakthroughs have been achieved lately in establishing such culture systems. Yet to date, there are no reports of the applications of any of these systems in HCV drug screening. Here, we report the generation of two monocistronic, chimeric genotype 1 full-length HCV genome molecules. These molecules, C33J-Y835C-UBI and C33J-Y835C-FMDV2A, both contain the structural protein region from genotype 1 (subtype 1b, Con1) and the remaining region from the genotype 2a (JFH1) clone. Both contain the humanized Renilla luciferase reporter gene which is separated from the rest of the HCV open reading frame by two different cleavage sites. The viral RNAs replicated efficiently in transfected cells. Viral particles produced were infectious in naïve Huh7.5 cells, and the infectivity could be blocked by monoclonal antibody against a putative HCV entry cofactor, CD81. A pilot high-throughput screen of 900 unknown compounds was executed by both the genotype 2a subgenomic replicon system and the infectious system. Thirty-one compounds were identified as hits by both systems, whereas 78 compounds were identified as hits only for the infectious system, suggesting that the infectious system is capable of identifying inhibitors targeting the viral structural proteins and steps involving them in the viral life cycle. The infectious HCV system developed here provides a useful and versatile tool which should greatly facilitate the identification of HCV inhibitors currently not identified by the subgenomic replicon system.

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