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 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.

773 HEPATITIS C VIRUS AND LIPID DROPLETS: ROLE OF SEIPIN IN LIPID DROPLET MATURATION AND VIRAL LIFE CYCLE

2011 ◽  
Vol 54 ◽  
pp. S311 ◽  
Author(s):  
S. Clement ◽  
C. Fauvelle ◽  
S. Pascarella ◽  
S. Conzelmann ◽  
V. Kaddai ◽  
...  
Keyword(s):  
Life Cycle ◽  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Viral Life Cycle

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 Disrupting the association of hepatitis C virus core protein with lipid droplets correlates with a loss in production of infectious virus

2007 ◽  
Vol 88 (8) ◽  
pp. 2204-2213 ◽  
Author(s):  
Steeve Boulant ◽  
Paul Targett-Adams ◽  
John McLauchlan
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Core Protein ◽  
Virus Production ◽  
Endoplasmic Reticulum Membrane ◽  
Wild Type ◽  
The Core ◽  
Phenylalanine Residue

In infected cells, hepatitis C virus (HCV) core protein is targeted to lipid droplets, which serve as intracellular storage organelles. Using a tissue culture system to generate infectious HCV, we have shown that the coating of lipid droplets by the core protein occurs in a time-dependent manner and coincides with higher rates of virus production. At earlier times, the protein was located at punctate sites in close proximity to the edge of lipid droplets. Investigations by using Z-stack analysis have shown that many lipid droplets contained a single punctate site that could represent positions where core transfers from the endoplasmic reticulum membrane to droplets. The effects of lipid droplet association on virus production were studied by introducing mutations into the domain D2, the C-terminal region of the core protein necessary for droplet attachment. Alteration of a phenylalanine residue that was crucial for lipid droplet association generated an unstable form of the protein that could only be detected in the presence of a proteasome inhibitor. Moreover, converting two proline residues in D2 to alanines blocked coating of lipid droplets by core, although the protein was directed to punctate sites that were indistinguishable from those observed at early times for wild-type core protein. Neither of these virus mutants gave rise to virus progeny. By contrast, mutation at a cysteine residue positioned 2 aa upstream of the phenylalanine residue did not affect lipid droplet localization and produced wild-type levels of infectious progeny. Taken together, our findings indicate that lipid droplet association by core is connected to virus production.

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 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 Lipid Droplet Binding of Hepatitis C Virus Core Protein Genotype 3

2012 ◽  
Vol 2012 ◽  
pp. 1-7 ◽  
Author(s):  
Guan Qiang ◽  
Ravi Jhaveri
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Fluorescent Protein ◽  
Core Protein ◽  
Genotype 1 ◽  
Genotype 3 ◽  
Core Domain ◽  
Domain 3

Background. Hepatitis C virus (HCV) genotype 3 is known to cause steatosis (fatty liver) that is more frequent and severe than other genotypes. We previously identified sequence elements within genotype 3 HCV Core domain 3 that were sufficient for lipid accumulation. Aims. We examined various genotype 3 Core domains for lipid droplet localization and compared the lipid droplet binding regions of domain 2 with a genotype 1 isolate. Methods. We generated HCV Core domain constructs fused with green fluorescent protein and performed immunofluorescence to visualize lipid droplets. Results. Constructs containing HCV Core domain 2 are appropriately localized to lipid droplets with varying degrees of efficiency. When compared to genotype 1, there are polymorphisms within domain 2 that do not appear to alter lipid droplet localization. Conclusions. In summary, the differences in a steatosis-associated HCV Core genotype 3 isolate do not appear to involve altered lipid droplet localization.

scholarly journals Suppression of Hepatitis C Virus Genome Replication and Particle Production by a Novel Diacylglycerol Acyltransferases Inhibitor

Molecules ◽  
2018 ◽  
Vol 23 (8) ◽  
pp. 2083
Author(s):  
Dahee Kim ◽  
Ja-Il Goo ◽  
Mi Kim ◽  
Sung-Jin Lee ◽  
Moonju Choi ◽  
...  
Keyword(s):  
Hepatitis C Virus ◽  
Hepatitis C ◽  
Lipid Droplets ◽  
Particle Production ◽  
Critical Role ◽  
Antiviral Effect ◽  
Virus Genome ◽  
Efficient Production ◽  
Genome Replication ◽  
Viral Genome Replication

Diacylglycerol acyltransferases (DGATs) play a critical role in the biosynthesis of endogenous triglycerides (TGs) and formation of lipid droplets (LDs) in the liver. In particular, one member of DGATs, DGAT-1 was reported to be an essential host factor for the efficient production of hepatitis C virus (HCV) particles. By utilizing our previously characterized three different groups of twelve DGAT inhibitors, we found that one of the DGAT inhibitors, a 2-((4-adamantylphenoxy) methyl)-N-(furan-2-ylmethyl)-1H-benzo[d]imidazole-5-carboxam (10j) is a potent suppressor of both HCV genome replication and particle production. 10j was able to induce inhibition of these two critical viral functions in a mutually separate manner. Abrogation of the viral genome replication by 10j led to a significant reduction in the viral protein expression as well. Interestingly, we found that its antiviral effect did not depend on the reduction of TG biosynthesis by 10j. This suggests that the inhibitory activity of 10j against DGATs may not be directly related with its antiviral action.

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