Christophe Hourioux

The genotype 3-specific hepatitis C virus core protein residue phenylalanine 164 increases steatosis in an in vitro cellular model

Background and aims: The prevalence and severity of liver steatosis are higher in patients infected with genotype 3 hepatitis C virus (HCV) than in patients infected with other genotypes. HCV core protein is known to affect lipid metabolism, inducing lipid droplet accumulation both in vitro and in vivo. An in vitro cellular model was used to investigate whether an HCV core protein with residues specific to genotype 3 increased this phenomenon. Methods: Sequence comparisons for HCV core protein domain II, which is known to interact with lipid droplets, identified the phenylalanine (F) residue at position 164 as the only residue specific to genotype 3. The area covered by lipid droplets in sections of cells producing a wild-type genotype 1a HCV core protein was compared with that in cells producing a Y164F mutant protein. Results: Cumulative lipid droplet area was significantly greater in sections of cells producing the Y164F mutant HCV core protein than in cells producing the wild-type protein (p<0.001). The frequency of cell sections containing more than 3 μm2 of lipid droplets, in particular, was higher for the mutant than for the wild-type protein. Conclusion: The data provide a molecular explanation for HCV genotype 3-specific lipid accumulation. This difference between genotypes may be due to phenylalanine having a higher affinity for lipids than tyrosine (Y). These observations provide useful information for further studies of the mechanisms involved in HCV-induced steatosis.

Morphogenesis of hepatitis B virus and its subviral envelope particles

After cell hijacking and intracellular amplification, non‐lytic enveloped viruses are usually released from the infected cell by budding across internal membranes or through the plasma membrane. The enveloped human hepatitis B virus (HBV) is an example of virus using an intracellular compartment to form new virions. Four decades after its discovery, HBV is still the primary cause of death by cancer due to a viral infection worldwide. Despite numerous studies on HBV genome replication little is known about its morphogenesis process. In addition to viral neogenesis, the HBV envelope proteins have the capability without any other viral component to form empty subviral envelope particles (SVPs), which are secreted into the blood of infected patients. A better knowledge of this process may be critical for future antiviral strategies. Previous studies have speculated that the morphogenesis of HBV and its SVPs occur through the same mechanisms. However, recent data clearly suggest that two different processes, including constitutive Golgi pathway or cellular machinery that generates internal vesicles of multivesicular bodies (MVB), independently form these two viral entities.

Hepatitis B Virus Subviral Envelope Particle Morphogenesis and Intracellular Trafficking

ABSTRACT Hepatitis B virus (HBV) is unusual in that its surface proteins (small [S], medium, and large [L]) are not only incorporated into the virion envelope but they also bud into empty subviral particles in great excess over virions. The morphogenesis of these subviral envelope particles remains unclear, but the S protein is essential and sufficient for budding. We show here that, in contrast to the presumed model, the HBV subviral particle formed by the S protein self-assembles into branched filaments in the lumen of the endoplasmic reticulum (ER). These long filaments are then folded and bridged for packing into crystal-like structures, which are then transported by ER-derived vesicles to the ER-Golgi intermediate compartment (ERGIC). Within the ERGIC, they are unpacked and relaxed, and their size and shape probably limits further progression through the secretory pathway. Such progression requires their conversion into spherical particles, which occurred spontaneously during the purification of these filaments by affinity chromatography. Small branched filaments are also formed by the L protein in the ER lumen, but these filaments are not packed into transport vesicles. They are transported less efficiently to the ERGIC, potentially accounting for the retention of the L protein within cells. These findings shed light on an important step in the HBV infectious cycle, as the intracellular accumulation of HBV subviral filaments may be directly linked to viral pathogenesis.

Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp111

ABSTRACT 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.

Hepatitis C virus core protein, lipid droplets and steatosis.

Summary.  Lipid droplets are intracellular organelles involved not only in lipid storage but also in cell signalling and the regulation of intracellular vesicular trafficking. Recent basic studies have suggested that interactions between hepatitis C virus (HCV) core protein and lipid droplets are required for the HCV infection cycle. In infected cells, the HCV core protein is associated with the surface of lipid droplets and the endoplasmic reticulum membranes closely surrounding these droplets, and its self‐assembly drives virion budding. This interaction also seems to be directly linked to a virus‐induced steatosis, which involves the deposition of triglycerides in the liver and contributes to the progression of fibrosis in patients with chronic hepatitis C. Many clinical studies have reported that virus‐induced steatosis is significantly more severe with HCV genotype 3 than with other genotypes, and this phenomenon has been modelled in recent basic studies based on the production of HCV core proteins of various genotypes in vitro. The association of HCV core protein with lipid droplets seems to play a central role in HCV pathogenesis and morphogenesis, suggesting that virus‐induced steatosis may be essential for the viral life cycle.

Hepatitis C virus budding at lipid droplet-associated ER membrane visualized by 3D electron microscopy

The mechanisms underlying hepatitis C virus (HCV) morphogenesis remain elusive, but lipid droplets have recently been shown to be important organelles for virus production. We investigated the interaction between HCV-like particles and lipid droplets by three-dimensional reconstructions of serial ultrathin electron microscopy sections of cells producing the HCV core protein. The budding of HCV-like particles was mostly initiated at membranes close to the lipid droplets rather than at membranes directly apposed to the lipid droplets. This may have important implications for our understanding of the complex relationship between HCV and lipids and may make easier to dissect out the HCV life cycle.

Core protein domains involved in hepatitis C virus-like particle assembly and budding at the endoplasmic reticulum membrane.

Hepatitis C virus (HCV) core protein, expressed with a Semliki forest virus (SFV) replicon, self‐assembles into HCV‐like particles (HCV‐LPs) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV particle morphogenesis by electron microscopy. Various mutated HCV core proteins with engineered internal deletions were expressed with this system, to identify core domains required or dispensable for HCV‐LP assembly. The HCV core protein sequence was compared with its counterpart in GB virus B (GBV‐B), the virus most closely related to HCV, to identify conserved domains. GBV‐B and HCV display similar tropism for liver hepatocytes and their core proteins are organized similarly into three main domains (I, II and III), although GBV‐B core is smaller and lacks ∼35 amino acids (aa) in domain I. The deletion of short hydrophobic domains (aa 133–152 and 153–167 in HCV core) that appear highly conserved in domain II of both GBV‐B and HCV core proteins resulted in loss of HCV core ER anchoring and self‐assembly into HCV‐LPs. The deletion of short domains found within domain I of HCV core protein but not in the corresponding domain of GBV‐B core according to sequence alignment had contrasting effects. Amino acids 15–28 and 60–66 were shown to be dispensable for HCV‐LP assembly and morphogenesis, whereas aa 88–106 were required for this process. The production of GBV‐B core protein from a recombinant SFV vector was associated with specific ER ultrastructural changes, but did not lead to the morphogenesis of GBV‐B‐LPs, suggesting that different budding mechanisms occur in members of the Flaviviridae family.

Hepatitis C virus ultrastructure and morphogenesis.

Abstract Details of the ultrastructure of hepatitis C virus (HCV) virion remain unclear because it has proved extremely difficult to visualise virus particles from infected serum and tissues directly. In addition, although much is known about the viral genome, first cloned in 1989, little is known about HCV morphogenesis, due to the lack of an efficient in vitro culture system for HCV propagation. Virus‐like particles (VLPs) obtained by expressing genes encoding the HCV structural proteins in mammalian cells can be used as an alternative model for studying HCV morphogenesis. In particular, this HCV‐LP model has made it possible to demonstrate that HCV budding occurs at the ER membrane and that the core protein drives this process. The HCV‐LP model opens up new possibilities for the investigation of viral morphogenesis and virus‐host cell interactions, which may make it possible to establish the long‐awaited in vitro culture system for HCV.

Identification of the Glycoprotein 41™ Cytoplasmic Tail Domains of Human Immunodeficiency Virus Type 1 That Interact with Pr55Gag Particles

We investigated the protein/protein interactions that occur during human immunodeficiency virus (HIV-1) budding. We evaluated the binding to Pr55Gag particles of peptides mapping to the cytoplasmic tail of gp41TM and of host-cell proteins, in a cell-free, in vitro assay. Host-cell proteins and irrelevant viral envelope peptides did not bind. Peptides corresponding to a large central domain of the gp41TM cytoplasmic tail (93 residues) bound to Pr55Gag particles. This demonstrates that a Gag/Env interaction is responsible for the specific incorporation of the Env glycoprotein into nascent HIV-1 virions, and defines more accurately the gp41TM domain involved in this interaction.

Chimeric hepatitis B and C viruses envelope proteins can form subviral particles: implications for the design of new vaccine strategies

The hepatitis B virus (HBV) envelope protein (S) self-assembles into subviral particles used as commercial vaccines against hepatitis B. These particles are excellent carriers for foreign epitopes, which can be inserted into the external hydrophilic loop or at the N- or C-terminal end of the HBV S protein. We show here that the N-terminal transmembrane domain (TMD) of HBV S can be replaced by the TMDs of the hepatitis C virus (HCV) envelope proteins E1 and E2, to generate fusion proteins containing the entire HCV E1 or E2 sequence that are efficiently coassembled with the HBV S into particles. This demonstrates the remarkable tolerance of the HBV S protein to sequence substitutions conserving its subviral particle assembly properties. These findings may have implications for the design of new vaccine strategies based on the use of HBV subviral particles as carriers for various transmembrane proteins and produced using the same industrial procedures that are established for the HBV vaccine.