Rab18 Binds to Hepatitis C Virus NS5A and Promotes Interaction between Sites of Viral Replication and Lipid Droplets

Hepatitis C virus (HCV) is a single-stranded RNA virus that replicates on endoplasmic reticulum-derived membranes. HCV particle assembly is dependent on the association of core protein with cellular lipid droplets (LDs). However, it remains uncertain whether HCV assembly occurs at the LD membrane itself or at closely associated ER membranes. Furthermore, it is not known how the HCV replication complex and progeny genomes physically associate with the presumed sites of virion assembly at or near LDs. Using an unbiased proteomic strategy, we have found that Rab18 interacts with the HCV nonstructural protein NS5A. Rab18 associates with LDs and is believed to promote physical interaction between LDs and ER membranes. Active (GTP-bound) forms of Rab18 bind more strongly to NS5A than a constitutively GDP-bound mutant. NS5A colocalizes with Rab18-positive LDs in HCV-infected cells, and Rab18 appears to promote the physical association of NS5A and other replicase components with LDs. Modulation of Rab18 affects genome replication and possibly also the production of infectious virions. Our results support a model in which specific interactions between viral and cellular proteins may promote the physical interaction between membranous HCV replication foci and lipid droplets.     

Hepatitis C virus structural proteins and virus-like particles produced in recombinant baculovirus-infected insect cells

Three proteins, namely, the core protein C and envelope glycoproteins E1 and E2, are main structural proteins forming a hepatitis C virus (HCV) virion. The virus structure and assembly and the role of the structural proteins in virion morphogenesis remain unknown because of the lack of an efficient culture system for HCV to be grown in vitro. Highly efficient heterologous expression systems make it possible to obtain self-assembled, nonreplicating, genome-lacking particles that are morphologically similar to intact virions. Using recombinant baculoviruses expressing the HCV structural protein genes in insect cells, the individual HCV structural proteins were expressed to 25–35% of the total cell protein, and the CE1 and E1E2 heterodimers and HCV-like particles were obtained. It was demonstrated that the recombinant C, E1, and E2 proteins underwent posttranslational modification, the glycoproteins formed a noncovalent heterodimer, and HCV- like particles were located in endoplasmic reticulum membranes of infected cells. The formation of E1E2 dimers and HCV-like particles was used to study the effect of E1 glycosylation on the expression and processing of the coat proteins.     

Scavenger receptor class B is required for hepatitis C virus uptake and cross-presentation by human dendritic cells

Class B scavenger receptors (SR-Bs) bind lipoproteins and play an important role in lipid metabolism. Most recently, SR-B type I (SR-BI) and its splicing variant SR-BII have been found to mediate bacterial adhesion and cytosolic bacterial invasion in mammalian cells. In this study, we demonstrate that SR-BI is a key host factor required for hepatitis C virus (HCV) uptake and cross-presentation by human dendritic cells (DCs). Whereas monocytes and T and B cells were characterized by very low or undetectable SR-BI expression levels, human DCs demonstrated a high level of cell surface expression of SR-BI similar to that of primary human hepatocytes. Antibodies targeting the extracellular loop of SR-BI efficiently inhibited HCV-like particle binding, uptake, and cross-presentation by human DCs. Moreover, human high-density lipoprotein specifically modulated HCV-like particle binding to DCs, indicating an interplay of HCV with the lipid transfer function of SR-BI in DCs. Finally, we demonstrate that anti-SR-BI antibodies inhibit the uptake of cell culture-derived HCV (HCVcc) in DCs. In conclusion, these findings identify a novel function of SR-BI for viral antigen uptake and recognition and may have an important impact on the design of HCV vaccines and immunotherapeutic approaches aiming at the induction of efficient antiviral immune responses.     

Visualization and Analysis of Hepatitis C Virus Structural Proteins at Lipid Droplets by Super-Resolution Microscopy

Cytosolic lipid droplets are central organelles in the Hepatitis C Virus (HCV) life cycle. The viral capsid protein core localizes to lipid droplets and initiates the production of viral particles at lipid droplet–associated ER membranes. Core is thought to encapsidate newly synthesized viral RNA and, through interaction with the two envelope proteins E1 and E2, bud into the ER lumen. Here, we visualized the spatial distribution of HCV structural proteins core and E2 in vicinity of small lipid droplets by three-color 3D super-resolution microscopy. We observed and analyzed small areas of colocalization between the two structural proteins in HCV-infected cells with a diameter of approximately 100 nm that might represent putative viral assembly sites.     

Signal peptide peptidase-catalyzed cleavage of hepatitis C virus core protein is dispensable for virus budding but destabilizes the viral capsid

The capsid of hepatitis C virus (HCV) particles is considered to be composed of the mature form (p21) of core protein. Maturation to p21 involves cleavage of the transmembrane domain of the precursor form (p23) of core protein by signal peptide peptidase (SPP), a cellular protease embedded in the endoplasmic reticulum membrane. Here we have addressed whether SPP-catalyzed maturation to p21 is a prerequisite for HCV particle morphogenesis in the endoplasmic reticulum. HCV structural proteins were expressed by using recombinant Semliki Forest virus replicon in mammalian cells or recombinant baculovirus in insect cells, because these systems have been shown to allow the visualization of HCV budding events and the isolation of HCV-like particles, respectively. Inhibition of SPP-catalyzed cleavage of core protein by either an SPP inhibitor or HCV core mutations not only did not prevent but instead tended to facilitate the observation of viral buds and the recovery of virus-like particles. Remarkably, although maturation to p21 was only partially inhibited by mutations in insect cells, p23 was the only form of core protein found in HCV-like particles. Finally, newly developed assays demonstrated that p23 capsids are more stable than p21 capsids. These results show that SPP-catalyzed cleavage of core protein is dispensable for HCV budding but decreases the stability of the viral capsid. We propose a model in which p23 is the form of HCV core protein committed to virus assembly, and cleavage by SPP occurs during and/or after virus budding to predispose the capsid to subsequent disassembly in a new cell.     

The Replacement of 10 Non-Conserved Residues in the Core Protein of JFH-1 Hepatitis C Virus Improves Its Assembly and Secretion

Hepatitis C virus (HCV) assembly is still poorly understood. It is thought that trafficking of the HCV core protein to the lipid droplet (LD) surface is essential for its multimerization and association with newly synthesized HCV RNA to form the viral nucleocapsid. We carried out a mapping analysis of several complete HCV genomes of all genotypes, and found that the genotype 2 JFH-1 core protein contained 10 residues different from those of other genotypes. The replacement of these 10 residues of the JFH-1 strain sequence with the most conserved residues deduced from sequence alignments greatly increased virus production. Confocal microscopy of the modified JFH-1 strain in cell culture showed that the mutated JFH-1 core protein, C10M, was present mostly at the endoplasmic reticulum (ER) membrane, but not at the surface of the LDs, even though its trafficking to these organelles was possible. The non-structural 5A protein of HCV was also redirected to ER membranes and colocalized with the C10M core protein. Using a Semliki forest virus vector to overproduce core protein, we demonstrated that the C10M core protein was able to form HCV-like particles, unlike the native JFH-1 core protein. Thus, the substitution of a few selected residues in the JFH-1 core protein modified the subcellular distribution and assembly properties of the protein. These findings suggest that the early steps of HCV assembly occur at the ER membrane rather than at the LD surface. The C10M-JFH-1 strain will be a valuable tool for further studies of HCV morphogenesis.     

Assembly of hepatitis C virus particles

Infection with hepatitis C virus (HCV) is a major risk factor for chronic hepatitis, cirrhosis and hepatocellular carcinoma. Once robust cell culture systems for production of recombinant infectious HCV became available, evidence on molecular mechanisms underlying assembly and release of the virus particles began to accumulate. Recent studies have demonstrated that lipid droplets and viral nonstructural proteins play key roles in HCV morphogenesis. This review considers the current knowledge about maturation of HCV structural proteins and production of viral infectious particles.     

Maturation of hepatitis C virus core protein by signal peptide peptidase is required for virus production

Complete maturation of hepatitis C virus (HCV) core protein requires coordinate cleavage by signal peptidase and an intramembrane protease, signal peptide peptidase. We show that reducing the intracellular levels of signal peptide peptidase lowers the titer of infectious virus released from cells, indicating that it plays an important role in virus production. Proteolysis by the enzyme at a signal peptide between core and the E1 glycoprotein is needed to permit targeting of core to lipid droplets. From mutagenesis studies, introducing mutations into the core-E1 signal peptide delayed the appearance of signal peptide peptidase-processed core until between 48 and 72 h after the beginning of the infectious cycle. Accumulation of mature core at these times coincided with its localization to lipid droplets and a rise in titer of infectious HCV. Therefore, processing of core by signal peptide peptidase is a critical event in the virus life cycle. To study the stage in virus production that may be blocked by interfering with intramembrane cleavage of core, we examined the distribution of viral RNA in cells harboring the core-E1 signal peptide mutant. Results revealed that colocalization of core with HCV RNA required processing of the protein by signal peptide peptidase. Our findings provide new insights into the sequence requirements for proteolysis by signal peptide peptidase. Moreover, they offer compelling evidence for a function for an intramembrane protease to facilitate the association of core with viral genomes, thereby creating putative sites for assembly of nascent virus particles.     

Hepatitis C virus-like particle budding: role of the core protein and importance of its Asp111

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.     

Role of lipid metabolism in hepatitis C virus assembly and entry

HCV (hepatitis C virus) represents a major global health problem. A consistent body of evidence has been accumulating, suggesting a peculiar overlap between the HCV life cycle and lipid metabolism. This association becomes evident both for the clinical symptoms of HCV infection and the molecular mechanisms underlying the morphogenesis and entry process of this virus. The HCV core–lipid droplets association seems to be central to the HCV morphogenesis process. Moreover, the biogenesis pathway of very‐low‐density lipoproteins has been shown to be involved in HCV morphogenesis with MTP (microsomal triacylglycerol transfer protein), ApoB (apolipoprotein B) and ApoE (apolipoprotein E) as essential elements in the production of infectious HCV particles. HCV infectivity also correlates with the lipidation status of the particles. Furthermore, some HCV cellular receptors and the regulation of the entry process are also connected to lipoproteins and lipid metabolism. Specifically, lipoproteins modulate the entry process and the cholesterol transporter SR‐BI (scavenger receptor class B type I) is a cellular entry factor for HCV. The present review aims to summarize the advances in our understanding of the HCV–lipid metabolism association, which may open new therapeutic avenues.     

Yellow fever virus replicons as an expression system for hepatitis C virus structural proteins

Chimeric yellow fever virus (YF) RNAs were constructed in which the YF structural genes were replaced by the hepatitis C virus (HCV) structural genes or fusions between the YF and HCV structural genes. Interestingly, RNA replication required nucleotide complementarity between the 3'-located conserved sequence 1 and an RNA sequence located in the 5' end of the YF capsid sequence. The (chimeric-)HCV structural proteins were efficiently expressed and processed, and the native E1/E2 heterodimer was formed. However, no indication for the production of HCV-like particles was obtained.     

Hepatitis C virus-like particle morphogenesis

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.     

Development and characterization of a recombinant cDNA-based hepatitis C virus system

Invention of subgenomic HCV replicon a few years ago and recent success of in vitro production of infectious HCV have improved our knowledge of the HCV life cycle, replication, pathogenesis, and screening of anti-HCV therapeutics. However, the highly genotype-dependent nature of the in vitro HCV production system has limited its potential for HCV research. In this study, we constructed a recombinant DNA-based HCV system that contained EF-1alpha promoter-driven HCV genotype 1b with HCV E1/E2 deleted and replaced by GFP. We co-transfected this recombinant cDNA with HCV E1/E2 or VSV-G expression plasmid into 293T cells, and we showed HCV protein expression and processing and demonstrated production of HCV-like particles in culture supernatant of co-transfected cells. These results support potential use of this system for studies on expression and processing of the HCV polyprotein and assembly and release of HCV-like particles.     

Inhibition of Hepatitis C Virus Production by Aptamers against the Core Protein

Hepatitis C virus (HCV) core protein is essential for virus assembly. HCV core protein was expressed and purified. Aptamers against core protein were raised through the selective evolution of ligands by the exponential enrichment approach. Detection of HCV infection by core aptamers and the antiviral activities of aptamers were characterized. The mechanism of their anti-HCV activity was determined. The data showed that selected aptamers against core specifically recognize the recombinant core protein but also can detect serum samples from hepatitis C patients. Aptamers have no effect on HCV RNA replication in the infectious cell culture system. However, the aptamers inhibit the production of infectious virus particles. Beta interferon (IFN-β) and interferon-stimulated genes (ISGs) are not induced in virally infected hepatocytes by aptamers. Domains I and II of core protein are involved in the inhibition of infectious virus production by the aptamers. V31A within core is the major resistance mutation identified. Further study shows that the aptamers disrupt the localization of core with lipid droplets and NS5A and perturb the association of core protein with viral RNA. The data suggest that aptamers against HCV core protein inhibit infectious virus production by disrupting the localization of core with lipid droplets and NS5A and preventing the association of core protein with viral RNA. The aptamers for core protein may be used to understand the mechanisms of virus assembly. Core-specific aptamers may hold promise for development as early diagnostic reagents and potential therapeutic agents for chronic hepatitis C.     

Knockdown of autophagy-related gene decreases the production of infectious Hepatitis C virus particles

Hepatitis C virus (HCV) is a positive strand RNA virus, and classified within the Flaviridae family. It has been reported that Atg7-knockdown decreases the amount of HCV replicon RNA, when HCV JFH1 RNA and HCV subgenomic replicon were transfected into Huh7.5 cells. However, when infectious naïve HCV particles are directly infected into Huh7.5.1 cells, it is still unclear whether Atg7-knockdown decreases the production of intracellular HCV-related proteins, HCV mRNA and infectious HCV particles. When Atg7 protein in HCV-infected Huh7.5.1 cells was knocked down by RNA-interference, the levels of intracellular HCV core, NS3, NS5A proteins, HCV mRNA, and secreted albumin remained unchanged compared with those in the control HCV-infected cells. However, the level of infectious HCV particles released in the medium was decreased by the Atg7-knockdown. The similar results were obtained, when beclin1 was knocked down by RNA-interference. The colocalization of endogenous LC3-puncta with HCV core, HS5A proteins, and lipid droplets was also investigated. However, little endogenous LC3-puncta colocalized with HCV core, NS5A proteins or lipid droplets. These results suggested that autophagy contributed to the effective production of HCV particles, but little to the intracellular production of HCV-related proteins, HCV mRNA, and secretory pathway, in naïve HCV particles-infection system.     

Critical role of virion-associated cholesterol and sphingolipid in hepatitis C virus infection

In this study, we establish that cholesterol and sphingolipid associated with hepatitis C virus (HCV) particles are important for virion maturation and infectivity. In a recently developed culture system enabling study of the complete life cycle of HCV, mature virions were enriched with cholesterol as assessed by the molar ratio of cholesterol to phospholipid in virion and cell membranes. Depletion of cholesterol from the virus or hydrolysis of virion-associated sphingomyelin almost completely abolished HCV infectivity. Supplementation of cholesterol-depleted virus with exogenous cholesterol enhanced infectivity to a level equivalent to that of the untreated control. Cholesterol-depleted or sphingomyelin-hydrolyzed virus had markedly defective internalization, but no influence on cell attachment was observed. Significant portions of HCV structural proteins partitioned into cellular detergent-resistant, lipid-raft-like membranes. Combined with the observation that inhibitors of the sphingolipid biosynthetic pathway block virion production, but not RNA accumulation, in a JFH-1 isolate, our findings suggest that alteration of the lipid composition of HCV particles might be a useful approach in the design of anti-HCV therapy.     

Characterization of HCV-like particles produced in a human hepatoma cell line by a recombinant baculovirus

Although processing of the hepatitis C virus (HCV) polyprotein and characterization of each of its viral proteins have been described in detail, analysis of the structure and assembly of HCV particles has been hampered by the lack of a robust cell culture system to support efficient replication of HCV. In this study, we generated HCV-like particles (HCV-LP) using a recombinant baculovirus encoding structural and a part of non-structural proteins in a human hepatoma cell line. The HCV-LP exhibited a buoyant density of 1.17 g/ml in CsCl equilibrium gradient and particles of 40 to 50 nm in diameter. Binding of the HCV-LP to human hepatoma cells was partially inhibited by the treatment with anti-hCD81 antibody, in contrast to the hCD81-independent binding of HCV-LP produced in insect cells. These results indicate that HCV-LP generated in different types of cells exhibit different cellular tropism for binding to target cells.     

Assembly of infectious hepatitis C virus particles

A hallmark of the hepatitis C virus (HCV) replication cycle is its tight link with host cell lipid synthesis. This is best illustrated by the peculiar pathway used for the assembly of infectious HCV particles. Research in the past few years has shown that formation of HC-virions is closely connected to lipid droplets that could serve as an assembly platform. Moreover, HCV particle production appears to be strictly linked to very-low-density lipoproteins. In this review, we focus on new insights into the molecular aspects of the architecture and assembly of this unique type of virus particle.     

Essential role of domain III of nonstructural protein 5A for hepatitis C virus infectious particle assembly

Persistent infection with the hepatitis C virus (HCV) is a major risk factor for the development of liver cirrhosis and hepatocellular carcinoma. With an estimated about 3% of the world population infected with this virus, the lack of a prophylactic vaccine and a selective therapy, chronic hepatitis C currently is a main indication for liver transplantation. The establishment of cell-based replication and virus production systems has led to first insights into the functions of HCV proteins. However, the role of nonstructural protein 5A (NS5A) in the viral replication cycle is so far not known. NS5A is a membrane-associated RNA-binding protein assumed to be involved in HCV RNA replication. Its numerous interactions with the host cell suggest that NS5A is also an important determinant for pathogenesis and persistence. In this study we show that NS5A is a key factor for the assembly of infectious HCV particles. We specifically identify the C-terminal domain III as the primary determinant in NS5A for particle formation. We show that both core and NS5A colocalize on the surface of lipid droplets, a proposed site for HCV particle assembly. Deletions in domain III of NS5A disrupting this colocalization abrogate infectious particle formation and lead to an enhanced accumulation of core protein on the surface of lipid droplets. Finally, we show that mutations in NS5A causing an assembly defect can be rescued by trans-complementation. These data provide novel insights into the production of infectious HCV and identify NS5A as a major determinant for HCV assembly. Since domain III of NS5A is one of the most variable regions in the HCV genome, the results suggest that viral isolates may differ in their level of virion production and thus in their level of fitness and pathogenesis.