Hepatitis C Virus-Like Particle Budding: Role of the Core Protein and Importance of Its Asp ¹¹¹

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 (HCV) infection is a major cause of chronic hepatitis and may lead to liver cirrhosis and hepatocellular carcinoma. With an estimated 170 million people worldwide chronically infected with HCV, this disease has emerged as a serious global health problem since the cloning of the viral genome in 1989 (8). Indeed, it has been predicted that chronic HCV infection will lead to an increase in the prevalence of hepatocellular carcinoma in the United States over the next 2 to 3 decades (39). HCV is a small, enveloped, positivestrand RNA virus belonging to the genus Hepacivirus of the Flaviviridae family. Its genome of approximately 9,600 nucleotides contains, at both the 5Ј and 3Ј ends, untranslated regions (UTRs) which flank a single open reading frame encoding a single polyprotein precursor of about 3,000 amino acids (aa) (13). The viral polyprotein can be broadly divided into two regions: the N-terminal one-third encodes the structural components of the virion, including the putative nucleocapsid or core protein and two envelope proteins (E1 and E2), and the remaining two-thirds encode the nonstructural proteins (22). These nonstructural proteins (NS2 through NS5B) have various functions, particularly in HCV genome replication, during the life cycle of the virus. They form a cytoplasmic membraneassociated complex similar to that formed by related positivestrand RNA viruses. The nonstructural proteins are separated from the structural proteins by the short hydrophobic polypeptide p7, the function of which is unknown (22). Translation to generate HCV polyprotein is initiated via an internal ribosome entry site located within the 5Ј UTR. During translation, the mature viral products are generated from the polyprotein by a series of cleavage events. The structural components are produced by cellular protease-mediated cleavages, whereas processing of the nonstructural proteins requires virus-encoded proteases (41). The core protein is produced at the N-terminal end of the polyprotein and is followed by the signal sequence of the E1 envelope glycoprotein. The signal sequence targets the nascent polypeptide chain to the endoplasmic reticulum (ER), allowing the translocation of E1 into the ER lumen, which is essential for the membrane-dependent processing of the core protein (23, 36). In the ER lumen, cleavage by a signal peptida...

Virus de l’hépatite C et gouttelettes lipidiques

“…Malheureusement, ces réplicons ne permettaient pas de produire du virus complet et d'étudier la morphogenèse virale. Cette étape du cycle infectieux n'a pu être abordée que grâce à des modèles de surexpression des protéines structurales du VHC, montrant notamment le rôle majeur de la protéine de core dans la morphogenèse de pseudovirions [3,4]. Finalement, l'année 2005 aura été un tournant dans la recherche sur le VHC, avec la mise au point d'un système cellulaire permettant de reproduire un cycle infectieux complet du VHC in vitro [5].…”

“…Elle est caractérisée par l'activation d'un système moléculaire complexe, la NADPH oxydase, qui induit une réduction de l'oxygène moléculaire en anion superoxyde (O 2 -. ) à l'origine d'autres formes réactives de l'oxygène telles que le peroxyde d'hydrogène (H 2 O 2 ), le radical hydroxyle (OH°) et l'acide hypochloreux (HOCl) [3,4].…”

Hepatitis C virus ultrastructure and morphogenesis

“…Unfortunately, its use is somewhat limited by the fact that most HCV-LPs seem to display abortive budding. EM observations show that only a few particles are fully released from the ER membrane, suggesting that some host-cell and/or viral factors may be required for the efficient, complete budding of these HCV-LPs (Blanchard et al, 2002;Blanchard et al, 2003a;Blanchard et al, 2003b). Nevertheless, this model provides an opportunity to identify such factors and should contribute to our understanding of HCV morphogenesis.…”

“…Although some subviral enveloped particles lacking cores have been described for other Flaviviridae such as the Japanese encephalitis virus (Konishi et al, 2001), HCV E1-E2 production did not result in the detection of particles by EM (unpublished data). However, production of the core protein alone resulted in the budding of VLPs towards the ER lumen, and the detection of electrondense areas lining the cytosolic side of the ER lumen (Blanchard et al, 2003b). The targeting of the HCV core protein to the ER by means of the signal sequence (SS) of E1 is necessary for this budding, as a construct encoding the core without the E1 SS did not generate VLPs.…”

Cellular models for the screening and development of anti-hepatitis C virus agents

“…Ezelle et al used a recombinant Vesicular Stomatitis Virus (VSV) vector expressing HCV core, E1, and E2 (genotype 1b) to generate HCV-LPs possessing properties similar to the ultrastructural properties of HCV virions (Ezelle et al, 2002). Both versions of SFV-and VSV-derived HCV-LPs were used to investigate the signal peptide peptidase processing of core protein, the interaction of structural proteins with the ER, and their role in HCV particle assembly and morphogenesis (Ait-Goughoulte et al, 2006;Blanchard et al, 2003a;Blanchard et al, 2003;Ezelle et al, 2002;Hourioux et al, 2007).…”