Rab18 is required for viral assembly of hepatitis C virus through trafficking of the core protein to lipid droplets

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.

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Interfering with hepatitis C virus assembly in vitro using affinity peptides directed towards core protein

Canadian Journal of Microbiology ◽

10.1139/w2012-009 ◽

2012 ◽

Vol 58 (4) ◽

pp. 475-482

Author(s):

Jean-Baptiste Duvignaud ◽

Nathalie Majeau ◽

Priscilla Delisle ◽

Normand Voyer ◽

Stéphane M. Gagné ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Self Assembly ◽

Core Protein ◽

Virus Assembly ◽

Viral Assembly ◽

Structural Protein ◽

Ns5a Protein ◽

The Core

Viral assembly is a crucial key step in the life cycle of every virus. In the case of Hepatitis C virus (HCV), the core protein is the only structural protein to interact directly with the viral genomic RNA. Purified recombinant core protein is able to self-assemble in vitro into nucleocapsid-like particles upon addition of a structured RNA, providing a robust assay with which to study HCV assembly. Inhibition of self-assembly of the C170 core protein (first 170 amino acids) was tested using short peptides derived from the HCV core, from HCV NS5A protein, and from diverse proteins (p21 and p73) known to interact with HCV core protein. Interestingly, peptides derived from the core were the best inhibitors. These peptides are derived from regions of the core predicted to be involved in the interaction between core subunits during viral assembly. We also demonstrated that a peptide derived from the C-terminal end of NS5A protein moderately inhibits the assembly process.

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Hepatitis C Virus NS5A Colocalizes with the Core Protein on Lipid Droplets and Interacts with Apolipoproteins

Virology ◽

10.1006/viro.2001.1225 ◽

2002 ◽

Vol 292 (2) ◽

pp. 198-210 ◽

Cited By ~ 208

Author(s):

Stephanie T. Shi ◽

Stephen J. Polyak ◽

Hong Tu ◽

Deborah R. Taylor ◽

David R. Gretch ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Lipid Droplets ◽

Core Protein ◽

The Core

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Immunoglobulin M reactivity towards the inununologically active region sp75 of the core protein of hepatitis C virus (HCV) in chronic HCV infection

Journal of Medical Virology ◽

10.1002/jmv.1890390412 ◽

1993 ◽

Vol 39 (4) ◽

pp. 325-332 ◽

Cited By ~ 28

Author(s):

U. B. Hellström ◽

S. P. E. Sylvan ◽

R. H. Decker ◽

A. Sönnerborg

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Active Region ◽

Core Protein ◽

Immunoglobulin M ◽

Hcv Infection ◽

The Core ◽

Chronic Hcv Infection ◽

Chronic Hcv

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Proteasomal Turnover of Hepatitis C Virus Core Protein Is Regulated by Two Distinct Mechanisms: a Ubiquitin-Dependent Mechanism and a Ubiquitin-Independent but PA28γ-Dependent Mechanism

Journal of Virology ◽

10.1128/jvi.01690-08 ◽

2008 ◽

Vol 83 (5) ◽

pp. 2389-2392 ◽

Cited By ~ 37

Author(s):

Ryosuke Suzuki ◽

Kohji Moriishi ◽

Kouichirou Fukuda ◽

Masayuki Shirakura ◽

Koji Ishii ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Loss Of Function ◽

Proteasome Activator ◽

Binding Partner ◽

The Core ◽

Virus Core ◽

Lysine Residues ◽

Dependent Mechanism

ABSTRACT We have previously reported on the ubiquitylation and degradation of hepatitis C virus core protein. Here we demonstrate that proteasomal degradation of the core protein is mediated by two distinct mechanisms. One leads to polyubiquitylation, in which lysine residues in the N-terminal region are preferential ubiquitylation sites. The other is independent of the presence of ubiquitin. Gain- and loss-of-function analyses using lysineless mutants substantiate the hypothesis that the proteasome activator PA28γ, a binding partner of the core, is involved in the ubiquitin-independent degradation of the core protein. Our results suggest that turnover of this multifunctional viral protein can be tightly controlled via dual ubiquitin-dependent and -independent proteasomal pathways.

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A Disulfide-Bonded Dimer of the Core Protein of Hepatitis C Virus Is Important for Virus-Like Particle Production

Journal of Virology ◽

10.1128/jvi.00402-10 ◽

2010 ◽

Vol 84 (18) ◽

pp. 9118-9127 ◽

Cited By ~ 23

Author(s):

Yukihiro Kushima ◽

Takaji Wakita ◽

Makoto Hijikata

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Particle Production ◽

Rna Binding ◽

Core Protein ◽

Dominant Negative ◽

Proteinase K ◽

Dominant Negative Effect ◽

The Core ◽

Virus Like Particle

ABSTRACT Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

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Hepatitis C Virus-Induced Rab32 Aggregation and Its Implications for Virion Assembly

Journal of Virology ◽

10.1128/jvi.01662-16 ◽

2016 ◽

Vol 91 (3) ◽

Cited By ~ 4

Author(s):

Tu M. Pham ◽

Si C. Tran ◽

Yun-Sook Lim ◽

Soon B. Hwang

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Membrane Trafficking ◽

Core Protein ◽

Cell Types ◽

Viral Assembly ◽

Intracellular Membrane ◽

Virion Assembly ◽

Hcv Core Protein ◽

Infected Cells

ABSTRACT Hepatitis C virus (HCV) is highly dependent on cellular factors for viral propagation. Using high-throughput next-generation sequencing, we analyzed the host transcriptomic changes and identified 30 candidate genes which were upregulated in cell culture-grown HCV (HCVcc)-infected cells. Of these candidates, we selected Rab32 for further investigation. Rab32 is a small GTPase that regulates a variety of intracellular membrane-trafficking events in various cell types. In this study, we demonstrated that both mRNA and protein levels of Rab32 were increased in HCV-infected cells. Furthermore, we showed that HCV infection converted the predominantly expressed GTP-bound Rab32 to GDP-bound Rab32, contributing to the aggregation of Rab32 and thus making it less sensitive to cellular degradation machinery. In addition, GDP-bound Rab32 selectively interacted with HCV core protein and deposited core protein into the endoplasmic reticulum (ER)-associated Rab32-derived aggregated structures in the perinuclear region, which were likely to be viral assembly sites. Using RNA interference technology, we demonstrated that Rab32 was required for the assembly step but not for other stages of the HCV life cycle. Taken together, these data suggest that HCV may modulate Rab32 activity to facilitate virion assembly. IMPORTANCE Rab32, a member of the Ras superfamily of small GTPases, regulates various intracellular membrane-trafficking events in many cell types. In this study, we showed that HCV infection concomitantly increased Rab32 expression at the transcriptional level and altered the balance between GDP- and GTP-bound Rab32 toward production of Rab32-GDP. GDP-bound Rab32 selectively interacted with HCV core protein and enriched core in the ER-associated Rab32-derived aggregated structures that were probably necessary for viral assembly. Indeed, we showed that Rab32 was specifically required for the assembly of HCV. Collectively, our study identifies that Rab32 is a novel host factor essential for HCV particle assembly.

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The N-terminal half of the core protein of hepatitis C virus is sufficient for nucleocapsid formation

Journal of General Virology ◽

10.1099/vir.0.79775-0 ◽

2004 ◽

Vol 85 (4) ◽

pp. 971-981 ◽

Cited By ~ 54

Author(s):

Nathalie Majeau ◽

Valérie Gagné ◽

Annie Boivin ◽

Marilène Bolduc ◽

Josée-Anne Majeau ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Core Protein ◽

Host Cells ◽

Yeast Cells ◽

Main Function ◽

C Protein ◽

Multifunctional Protein ◽

The Core

The core (C) protein of hepatitis C virus (HCV) appears to be a multifunctional protein that is involved in many viral and cellular processes. Although its effects on host cells have been extensively discussed in the literature, little is known about its main function, the assembly and packaging of the viral genome. We have studied the in vitro assembly of several deleted versions of recombinant HCV C protein expressed in E. coli. We demonstrated that the 75 N-terminal residues of the C protein were sufficient to assemble and generate nucleocapsid-like particles (NLPs) in vitro. However, homogeneous particles of regular size and shape were observed only when NLPs were produced from at least the first 79 N-terminal amino acids of the C protein. This small protein unit fused to the endoplasmic reticulum-anchoring domain also generated NLPs in yeast cells. These data suggest that the N-terminal half of the C protein is important for formation of NLPs. Similarities between the HCV C protein and C proteins of other members of the Flaviviridae are discussed.

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