Hepatitis C Virus Morphogenesis Research Articles

Proximity labeling of host factor ANXA3 in HCV infection reveals a novel LARP1 function in viral entry

Hepatitis C virus (HCV) infection is tightly connected to the lipid metabolism with lipid droplets (LDs) serving as assembly sites for progeny virions. A previous LD proteome analysis identified annexin A3 (ANXA3) as an important HCV host factor that is enriched at LDs in infected cells and required for HCV morphogenesis. To further characterize ANXA3 function in HCV, we performed proximity labeling using ANXA3-BioID2 as bait in HCV-infected cells. Two of the top proteins identified proximal to ANXA3 during HCV infection were the La-related protein 1 (LARP1) and the ADP ribosylation factor-like protein 8B (ARL8B), both of which have been previously described to act in HCV particle production. In follow-up experiments ARL8B functioned as a pro-viral HCV host factor without localizing to LDs and thus likely independent of ANXA3. In contrast, LARP1 interacts with HCV core protein in an RNA-dependent manner and is translocated to LDs by core protein. Knockdown of LARP1 decreased HCV spreading without altering HCV RNA replication or viral titers. Unexpectedly, entry of HCV particles and E1/E2-pseudotyped lentiviral particles was reduced by LARP1 depletion, whereas particle production was not altered. Using a recombinant vesicular stomatitis virus (VSV)ΔG entry assay, we showed that LARP1 depletion also decreased entry of VSV with VSV, MERS, and CHIKV glycoproteins. Therefore, our data expand the role of LARP1 as an HCV host factor that is most prominently involved in the early steps of infection, likely contributing to endocytosis of viral particles through the pleiotropic effect LARP1 has on the cellular translatome.

Guanylate-binding protein 1 acts as a pro-viral factor for the life cycle of hepatitis C virus.

Viral infections trigger the expression of interferons (IFNs) and interferon stimulated genes (ISGs), which are crucial to modulate an antiviral response. The human guanylate binding protein 1 (GBP1) is an ISG and exhibits antiviral activity against several viruses. In a previous study, GBP1 was described to impair replication of the hepatitis C virus (HCV). However, the impact of GBP1 on the HCV life cycle is still enigmatic. To monitor the expression and subcellular distribution of GBP1 and HCV we performed qPCR, Western blot, CLSM and STED microscopy, virus titration and reporter gene assays. In contrast to previous reports, we observed that HCV induces the expression of GBP1. Further, to induce GBP1 expression, the cells were stimulated with IFNγ. GBP1 modulation was achieved either by overexpression of GBP1-Wt or by siRNA-mediated knockdown. Silencing of GBP1 impaired the release of viral particles and resulted in intracellular HCV core accumulation, while overexpression of GBP1 favored viral replication and release. CLSM and STED analyses revealed a vesicular distribution of GBP1 in the perinuclear region. Here, it colocalizes with HCV core around lipid droplets, where it acts as assembly platform and thereby favors HCV morphogenesis and release. Collectively, our results identify an unprecedented function of GBP1 as a pro-viral factor. As such, it is essential for viral assembly and release acting through tethering factors involved in HCV morphogenesis onto the surface of lipid droplets.

Adaptive mutations promote hepatitis C virus assembly by accelerating core translocation to the endoplasmic reticulum

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 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 the 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, and 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 the LDs to the ER and reinforced the interaction between NS2 and NS5B. The findings facilitate our understanding of the assembly of HCV morphogenesis.

The ATGL lipase cooperates with ABHD5 to mobilize lipids for hepatitis C virus assembly.

Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/β hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis.

HCV Interplay with Lipoproteins: Inside or Outside the Cells?

Hepatitis C virus (HCV) infection is a major public health issue leading to chronic liver diseases. HCV particles are unique owing to their particular lipid composition, namely the incorporation of neutral lipids and apolipoproteins. The mechanism of association between HCV virion components and these lipoproteins factors remains poorly understood as well as its impact in subsequent steps of the viral life cycle, such as entry into cells. It was proposed that the lipoprotein biogenesis pathway is involved in HCV morphogenesis; yet, recent evidence indicated that HCV particles can mature and evolve biochemically in the extracellular medium after egress. In addition, several viral, cellular and blood components have been shown to influence and regulate this specific association. Finally, this specific structure and composition of HCV particles was found to influence entry into cells as well as their stability and sensitivity to neutralizing antibodies. Due to its specific particle composition, studying the association of HCV particles with lipoproteins remains an important goal towards the rational design of a protective vaccine.

Functional microRNA screen uncovers O-linked N-acetylglucosamine transferase as a host factor modulating hepatitis C virus morphogenesis and infectivity

ObjectiveInfection of human hepatocytes by the hepatitis C virus (HCV) is a multistep process involving both viral and host factors. microRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate gene...

Functional Study of the C-Terminal Part of the Hepatitis C Virus E1 Ectodomain.

In the hepatitis C virus (HCV) envelope glycoproteins E1 and E2, which form a heterodimer, E2 is the receptor binding protein and the major target of neutralizing antibodies, whereas the function of E1 remains less characterized. To investigate E1 functions, we generated a series of mutants in the conserved residues of the C-terminal region of the E1 ectodomain in the context of an infectious clone. We focused our analyses on two regions of interest. The first region is located in the middle of the E1 glycoprotein (between amino acid [aa] 270 and aa 291), which contains a conserved hydrophobic sequence and was proposed to constitute a putative fusion peptide. The second series of mutants was generated in the region from aa 314 to aa 342 (the aa314-342 region), which has been shown to contain two α helices (α2 and α3) by nuclear magnetic resonance studies. Of the 22 generated mutants, 20 were either attenuated or noninfectious. Several mutations modulated the virus's dependence on claudin-1 and the scavenger receptor BI coreceptors for entry. Most of the mutations in the putative fusion peptide region affected virus assembly. Conversely, mutations in the α-helix aa 315 to 324 (315-324) residues M318, W320, D321, and M322 resulted in a complete loss of infectivity without any impact on E1E2 folding and on viral assembly. Further characterization of the W320A mutant in the HCVpp model indicated that the loss of infectivity was due to a defect in viral entry. Together, these results support a role for E1 in modulating HCV interaction with its coreceptors and in HCV assembly. They also highlight the involvement of α-helix 315-324 in a late step of HCV entry.IMPORTANCE HCV is a major public health problem worldwide. The virion harbors two envelope proteins, E1 and E2, which are involved at different steps of the viral life cycle. Whereas E2 has been extensively characterized, the function of E1 remains poorly defined. We characterized here the function of the putative fusion peptide and the region containing α helices of the E1 ectodomain, which had been previously suggested to be important for virus entry. We could confirm the importance of these regions for the virus infectivity. Interestingly, we found several residues modulating the virus's dependence on several HCV receptors, thus highlighting the role of E1 in the interaction of the virus with cellular receptors. Whereas mutations in the putative fusion peptide affected HCV infectivity and morphogenesis, several mutations in the α2-helix region led to a loss of infectivity with no effect on assembly, indicating a role of this region in virus entry.

Characterization of apolipoprotein C1 in hepatitis C virus infection and morphogenesis

Previous studies have shown that apolipoprotein C1 (apoC1)-specific antibodies precipitated hepatitis C virus (HCV) and neutralized HCV infectivity, suggesting that apoC1 is a HCV component. However, the importance of apoC1 in the HCV life cycle has not been experimentally examined. In the present study, we sought to determine the role of apoC1 in the HCV infection and morphogenesis by knocking out the apoC1 gene using the CRISPR/Cas9 system. Strikingly, apoC1 gene knockout markedly enhanced apoE expression. As a result, apoC1 gene knockout per se didn’t significantly affect HCV infection or morphogenesis, probably ascribing to its redundant functions with apoE. However, knockout of apoC1 gene potentiated the impairment of HCV infection and/or morphogenesis by apoE-specific small interfering RNAs. Additionally, a recombinant apoC1 protein efficiently blocked HCV infection. Collectively, these findings suggest that apoC1 and apoE have redundant functions in the HCV infection and morphogenesis.

Genetic Analysis of Serum-Derived Defective Hepatitis C Virus Genomes Revealed Novel Viral cis Elements for Virus Replication and Assembly

ABSTRACT Defective viral genomes (DVGs) of hepatitis C virus (HCV) exist, but their biological significances have not been thoroughly investigated. Here, we analyzed HCV DVGs circulating in patient sera that possess deletions in the structural protein-encoding region. About 30% of 41 HCV clinical isolates possess DVGs that originated from the full-length genome in the same patients. No correlation between DVGs, viremia, and alanine aminotransferase (ALT) levels was found. Sequencing analysis of DVGs revealed the existence of deletion hot spots, with upstream sites in E1 and downstream sites in E2 and NS2. Interestingly, the coding sequences for the core protein and the C-terminal protease domain of NS2 were always intact in DVGs despite the fact that both proteins are dispensable for HCV genome replication. Mechanistic studies showed that transmembrane segment 3 (TMS3) of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. Moreover, we identified a highly conserved secondary structure (SL750) within the core domain 2-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed novel viral cis elements that play important roles in virus replication and assembly. IMPORTANCE HCV DVGs have been identified in vivo and in vitro , but their biogenesis and physiological significances remain elusive. In addition, a conventional packaging signal has not yet been identified on the HCV RNA genome, and mechanisms underlying the specificity in the encapsidation of the HCV genome into infectious particles remain to be uncovered. Here, we identified new viral cis elements critical for the HCV life cycle by determining genetic constraints that define the boundary of serum-derived HCV DVGs. We found that transmembrane segment 3 of NS2, located immediately upstream of its protease domain, was required for the cleavage of NS2-NS3 and the replication of DVGs. We identified a highly conserved secondary structure (SL750) within the core-coding region that is critical for HCV genome packaging. In summary, our analysis of serum-derived HCV DVGs revealed previously unexpected novel cis elements critical for HCV replication and morphogenesis.

Functional Analysis of Hepatitis C Virus (HCV) Envelope Protein E1 Using a trans -Complementation System Reveals a Dual Role of a Putative Fusion Peptide of E1 in both HCV Entry and Morphogenesis

Hepatitis C virus (HCV) is an enveloped RNA virus belonging to the Flaviviridae family. It infects mainly human hepatocytes and causes chronic liver diseases, including cirrhosis and cancer. HCV encodes two envelope proteins, E1 and E2, that form a heterodimer and mediate virus entry. While E2 has been extensively studied, less has been done so for E1, and its role in the HCV life cycle still needs to be elucidated. Here we developed a new cell culture model for HCV infection based on the trans-complementation of E1. Virus production of the HCV genome lacking the E1-encoding sequence can be efficiently rescued by the ectopic expression of E1 in trans The resulting virus, designated HCVΔE1, can propagate in packaging cells expressing E1 but results in only single-cycle infection in naive cells. By using the HCVΔE1 system, we explored the role of a putative fusion peptide (FP) of E1 in HCV infection. Interestingly, we found that the FP not only contributes to HCV entry, as previously reported, but also may be involved in virus morphogenesis. Finally, we identified amino acid residues in FP that are critical for biological functions of E1. In summary, our work not only provides a new cell culture model for studying HCV but also provides some insights into understanding the role of E1 in the HCV life cycle.IMPORTANCE Hepatitis C virus (HCV), an enveloped RNA virus, encodes two envelope proteins, E1 and E2, that form a heterodimeric complex to mediate virus entry. Compared to E2, the biological functions of E1 in the virus life cycle are not adequately investigated. Here we developed a new cell culture model for single-cycle HCV infection based on the trans-complementation of E1. The HCV genome lacking the E1-encoding sequence can be efficiently rescued for virus production by the ectopic expression of E1 in trans This new model renders a unique system to dissect functional domains and motifs in E1. Using this system, we found that a putative fusion peptide in E1 is a multifunctional structural element contributing to both HCV entry and morphogenesis. Our work has provided a new cell culture model to study HCV and provides insights into understanding the biological roles of E1 in the HCV life cycle.

ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production.

Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

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.

NS2 proteins of GB virus B and hepatitis C virus share common protease activities and membrane topologies.

GB virus B (GBV-B), which is hepatotropic in experimentally infected small New World primates, is a member of the Hepacivirus genus but phylogenetically relatively distant from hepatitis C virus (HCV). To gain insights into the role and specificity of hepaciviral nonstructural protein 2 (NS2), which is required for HCV polyprotein processing and particle morphogenesis, we investigated whether NS2 structural and functional features are conserved between HCV and GBV-B. We found that GBV-B NS2, like HCV NS2, has cysteine protease activity responsible for cleavage at the NS2/NS3 junction, and we experimentally confirmed the location of this junction within the viral polyprotein. A model for GBV-B NS2 membrane topology was experimentally established by determining the membrane association properties of NS2 segments fused to green fluorescent protein (GFP) and their nuclear magnetic resonance structures using synthetic peptides as well as by applying an N-glycosylation scanning approach. Similar glycosylation studies confirmed the HCV NS2 organization. Together, our data show that despite limited amino acid sequence similarity, GBV-B and HCV NS2 proteins share a membrane topology with 3 N-terminal transmembrane segments, which is also predicted to apply to other recently discovered hepaciviruses. Based on these data and using trans-complementation systems, we found that intragenotypic hybrid NS2 proteins with heterologous N-terminal membrane segments were able to efficiently trans-complement an assembly-deficient HCV mutant with a point mutation in the NS2 C-terminal domain, while GBV-B/HCV or intergenotypic NS2 chimeras were not. These studies indicate that virus- and genotype-specific intramolecular interactions between N- and C-terminal domains of NS2 are critically involved in HCV morphogenesis. Nonstructural protein 2 (NS2) of hepatitis C virus (HCV) is a multifunctional protein critically involved in polyprotein processing and virion morphogenesis. To gain insights into NS2 mechanisms of action, we investigated whether NS2 structural and functional features are conserved between HCV and GB virus B (GBV-B), a phylogenetically relatively distant primate hepacivirus. We showed that GBV-B NS2, like HCV NS2, carries cysteine protease activity. We experimentally established a model for GBV-B NS2 membrane topology and demonstrated that despite limited sequence similarity, GBV-B and HCV NS2 share an organization with three N-terminal transmembrane segments. We found that the role of HCV NS2 in particle assembly is genotype specific and relies on critical interactions between its N- and C-terminal domains. This first comparative analysis of NS2 proteins from two hepaciviruses and our structural predictions of NS2 from other newly identified mammal hepaciviruses highlight conserved key features of the hepaciviral life cycle.

Hepatitis C Virus p7 is Critical for Capsid Assembly and Envelopment

Hepatitis C virus (HCV) p7 is a membrane-associated ion channel protein crucial for virus production. To analyze how p7 contributes to this process, we dissected HCV morphogenesis into sub-steps including recruitment of HCV core to lipid droplets (LD), virus capsid assembly, unloading of core protein from LDs and subsequent membrane envelopment of capsids. Interestingly, we observed accumulation of slowly sedimenting capsid-like structures lacking the viral envelope in cells transfected with HCV p7 mutant genomes which possess a defect in virion production. Concomitantly, core protein was enriched at the surface of LDs. This indicates a defect in core/capsid unloading from LDs and subsequent membrane envelopment rather than defective trafficking of core to this cellular organelle. Protease and ribonuclease digestion protection assays, rate zonal centrifugation and native, two dimensional gel electrophoresis revealed increased amounts of high-order, non-enveloped core protein complexes unable to protect viral RNA in cells transfected with p7 mutant genomes. These results suggest accumulation of capsid assembly intermediates that had not yet completely incorporated viral RNA in the absence of functional p7. Thus, functional p7 is necessary for the final steps of capsid assembly as well as for capsid envelopment. These results support a model where capsid assembly is linked with membrane envelopment of nascent RNA-containing core protein multimers, a process coordinated by p7. In summary, we provide novel insights into the sequence of HCV assembly events and essential functions of p7.

Effect of deoxynojirimycin derivatives on morphogenesis of hepatitis C virus

Viral hepatitis C is a dangerous, widespread human disease. The choice of drugs for treatment of chronic hepatitis C virus (HCV) infection is limited, and prophylactic vaccines do not exist. Thus, the development of new antiviral strategies and substances is an issue of great importance. The targeting of viral morphogenesis might be used as an alternative approach to existing strategies of HCV blocking. The glycosylation of viral envelope proteins is an important step of viral particle morphogenesis, which determines the correct assembly of HCV virions. Derivatives of a glucose analog deoxynojirimycin (DNJ) act as an α-glucosidase inhibitor and can impair the assembly of structural proteins and HCV particle formation. In the present work, the effects of alkylated DNJ derivatives, N-pentyl-DNJ and N-benzyl-DNJ, on HCV morphogenesis were studied in a model system of insect cells that produce three viral structural proteins with the formation of virus-like particles. It was shown that DNJ derivatives impair the intracellular N-glycosylation of HCV envelope glycoproteins. At the concentration of 1 mM, these substances cause an increase in the levels of gpE1 and gpE2 glycoproteins and a decrease in their electrophoretic mobility, apparently due to the inhibition of α-glucosidase in the endoplasmic reticulum and the accumulation of hyperglycosylated N-glycans in HCV glycoproteins. The interaction of the latter with calnexin results in the formation of unproductive dimers and blocks the productive assembly of virus-like particles.

Cell Culture-Adaptive Mutations Promote Viral Protein-Protein Interactions and Morphogenesis of Infectious Hepatitis C Virus

Recent genetic studies suggested that viral nonstructural (NS) proteins play important roles in morphogenesis of flaviviruses, particularly hepatitis C virus (HCV). Adaptive and compensatory mutations occurring in different NS proteins were demonstrated to promote HCV production in cell culture. However, the underlying molecular mechanism of NS proteins in HCV morphogenesis is poorly understood. We have isolated a cell culture-adapted HCV of genotype 2a (JFH1) which grew to an infectious titer 3 orders of magnitude higher than that of wild-type virus. Sequence analysis identified a total of 16 amino acid mutations in core (C), E1, NS2, NS3, NS5A, and NS5B, with the majority of mutations clustered in NS5A. Reverse genetic analysis of these mutations individually or in different combinations demonstrated that amino acid mutations in NS2 and NS5A markedly enhanced HCV production. Additionally, mutations in C, E1, NS3, and NS5B synergistically promoted HCV production in the background of NS2 and NS5A mutations. Adaptive mutations in NS5A domains I, II, and III independently enhanced HCV production, suggesting that all three domains of NS5A are important for HCV morphogenesis. More importantly, adaptive mutations greatly enhanced physical interactions among HCV structural and NS proteins, as determined by studies with coimmunoprecipitation and mammalian two-hybrid assays. Collectively, these findings demonstrate that adaptive mutations can enhance specific protein-protein interactions among viral structural and NS proteins and therefore promote the assembly of infectious HCV particles.

Morphogenesis of Infectious Hepatitis C Virus Particles

More than 170 million individuals are currently infected with hepatitis C virus (HCV) worldwide and are at continuous risk of developing chronic liver disease. Since a cell culture system enabling relatively efficient propagation of HCV has become available, an increasing number of viral and host factors involved in HCV particle formation have been identified. Association of the viral Core, which forms the capsid with lipid droplets appears to be prerequisite for early HCV morphogenesis. Maturation and release of HCV particles is tightly linked to very-low-density lipoprotein biogenesis. Although expression of Core as well as E1 and E2 envelope proteins produces virus-like particles in heterologous expression systems, there is increasing evidence that non-structural viral proteins and p7 are also required for the production of infectious particles, suggesting that HCV genome replication and virion assembly are closely linked. Advances in our understanding of the various molecular mechanisms by which infectious HCV particles are formed are summarized.

Effects of hypolipidemic agent nordihydroguaiaretic acid on lipid droplets and hepatitis C virus.

Hepatitis C virus (HCV) relies on host lipid metabolic pathways for its replication, assembly, secretion, and entry. HCV induces de novo lipogenesis, inhibits β-oxidation, and lipoprotein export resulting in a lipid-enriched cellular environment critical for its proliferation. We investigated the effects of a hypolipidemic agent, nordihydroguaiaretic acid (NDGA), on host lipid/fatty acid synthesis and HCV life cycle. NDGA negated the HCV-induced alteration of host lipid homeostasis. NDGA decreased sterol regulatory element binding protein (SREBP) activation and enhanced expression of genes involved in β-oxidation. NDGA inhibited very low-density lipoprotein (VLDL) secretion by affecting mediators of VLDL biosynthesis. Lipid droplets (LDs), the neutral lipid storage organelles, play a key role in HCV morphogenesis. HCV induces accumulation and perinuclear distribution of LDs, whereas NDGA most notably reduced the overall number and increased the average size of LDs. The antiviral effects of NDGA resulted in reduced HCV replication and secretion. NDGA-mediated alterations of host lipid metabolism, LD morphology, and VLDL transport appear to negatively influence HCV proliferation.

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.

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.