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.The human hepatitis B virus (HBV) is the prototype of the mammalian Hepadnaviridae (genus Orthohepadnavirus), a family of small hepatotropic DNA viruses causing acute and chronic liver disease (16). Despite the existence of an effective HBV vaccine, HBV remains a major health problem worldwide, as there is no generally effective treatment for the estimated 350 million chronic carriers who have a high risk of liver cirrhosis and hepatocellular carcinoma. A thorough understanding of HBV morphogenesis and life cycle is thus required for the development of innovative antiviral treatments. The virion (or Dane particle) is a spherical particle, 42 nm in diameter, consisting of an icosahedral nucleocapsid of approximately 30 nm in diameter and an envelope composed of three surface proteins and, presumably, lipids of host cell origin. The nucleocapsid and envelope are synthesized and mature separately in different cellular compartments, subsequently interacting to form the virion (5, 26, 34). The three HBV envelope proteins are encoded by a single open reading frame, using three in-frame start codons (21). The large surface protein (L, or p39) is the translation product of the entire open reading frame (389 to 400 amino acid [aa] residues, depending on HBV genotype). The middle surface protein (M, or p30) lacks the N-terminal 119 aa of L (the pre-S1 sequence), and the small surface protein (S, or p24) lacks the N-terminal 55 aa of M (the pre-S2 sequence). These proteins are synthesized at the endoplasmic reticulum (ER) membrane and have a complex transmembra...

SummaryAfter 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.

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.

The development of a prophylactic vaccine against hepatitis C virus (HCV) has become an important medical priority, because 3-4 million new HCV infections are thought to occur each year worldwide. Hepatitis B virus (HBV) is another major human pathogen, but infections with this virus can be prevented with a safe, efficient vaccine, based on the remarkable ability of the envelope protein (S) of this virus to self-assemble into highly immunogenic subviral particles. Chimeric HBV-HCV envelope proteins in which the N-terminal transmembrane domain of S was replaced with the transmembrane domain of the HCV envelope proteins (E1 or E2) were efficiently coassembled with the wild-type HBV S protein into subviral particles. These chimeric particles presented the full-length E1 and E2 proteins from a genotype 1a virus in an appropriate conformation for formation of the E1-E2 heterodimer. Produced in stably transduced Chinese hamster ovary cells and used to immunize New Zealand rabbits, these particles induced a strong specific antibody (Ab) response against the HCV and HBV envelope proteins in immunized animals. Sera containing anti-E1 or anti-E2 Abs elicited by these particles neutralized infections with HCV pseudoparticles and cellcultured viruses derived from different heterologous 1a, 1b, 2a, and 3 strains. Moreover, the anti-hepatitis B surface response induced by these chimeric particles was equivalent to the response induced by a commercial HBV vaccine. Conclusions: Our results provide support for approaches based on the development of bivalent HBV-HCV prophylactic vaccine candidates potentially able to prevent initial infection with either of these two hepatotropic viruses. (HEPATOLOGY 2013;57:1303-1313 C hronic hepatitis C virus (HCV) infection is a major public health problem affecting more than 170 million people worldwide. 1 Three to four million new infections are thought to occur each year and a prevalence of 10%-30% has been reported in countries in which this virus is highly endemic, including Egypt, which has the highest HCV prevalence in the world. 2 HCV infection is one of the leading causes of chronic liver disease; it is associated with a high risk of cirrhosis and hepatocellular carcinoma and is the major indication for liver transplantation in industrialized countries. Provided it is detected sufficiently early, progression to severe disease can be prevented by treatment with a combination of pegylated interferon (IFN)-a and ribavirin, in some cases supplemented with recently approved nonstructural protein 3/4A protease inhibitors. 3 This triple therapy yields a sustained virologic response, but is very expensive and may be associated with drug-drug interactions and severe side effects. Therefore, the development of a prophylactic vaccine against HCV is a major medical priority. However, the development of such a vaccine

Hepatitis C virus (HCV) core protein, expressed with a Semliki Forest virus replicon, self-assembles into HCV-like particles (HCV-LP) at the endoplasmic reticulum (ER) membrane, providing an opportunity to study HCV assembly and morphogenesis by electron microscopy. This model was used to investigate whether the processing of the HCV core protein by the signal peptide peptidase (SPP) is required for the HCV-LP assembly. Several mutants were designed as there are conflicting reports concerning the cleavage of mutant proteins by SPP. Production of the only core mutant protein that escaped SPP processing led to the formation of multiple layers of electrondense ER membrane, with no evidence of HCV-LP assembly. These data shed light on the HCV core residues involved in SPP cleavage and suggest that this cleavage is essential for HCV assembly.The hepatitis C virus (HCV) genome contains approximately 9600 nt. At both the 59 and 39 ends, untranslated regions flank a single open reading frame encoding a single polyprotein precursor of about 3000 aa. This precursor is co-and post-translationally processed by cellular and viral proteases, to yield three mature structural proteins [one core (C) and two envelope (E1 and E2) proteins] and six nonstructural proteins involved in polyprotein processing and viral RNA replication (Grakoui et al., 1993). The hydrophobic sequence at the junction between the HCV core protein and the envelope glycoprotein E1 (aa 170-191 in the polyprotein) functions as a signal sequence and targets the nascent polyprotein to the endoplasmic reticulum (ER) membrane, inducing the translocation of E1 into the lumen of the ER (Lo et al., 1995). Cleavage by a signal peptidase in the ER lumen releases the N-terminal end of E1, leaving the 191 aa core protein anchored by the signal peptide (McLauchlan, 2000). This 191 aa polypeptide (p23) is an immature form of the core protein and is further processed by an intramembrane presenilin-type aspartic protease SPP (for signal peptide peptidase) (Weihofen et al., 2002). The site of the SPP cleavage is unclear but probably lies between residues 173 and 179, resulting in the elimination from the mature core protein of all or part of the Nterminal hydrophobic domain containing the signal peptide of E1 (McLauchlan et al., 2002). The resulting mature HCV core protein (p21) is thought to constitute the HCV capsid and therefore to be an integral component of the virion.Indeed, this mature form of the HCV core protein is the predominant form detected in virus particles purified from the sera of patients with chronic HCV infection (Yasui et al., 1998). However, processing of the HCV core protein by SPP has also been shown to release the core protein from the ER membrane, leading to its trafficking to zones of the ER in which lipid droplets are produced (McLauchlan et al., 2002). During its trafficking, the proline-rich, central hydrophobic domain 2 of the HCV core protein (aa 119-173) is thought to remain anchored in the cytoplasmic phospholipid monolayer of the ER and is ...

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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.

SummaryHepatitis 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 selfassembly 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.