Studies on the expression of the structural proteins of hepatitis C virus

Abstract

Infection with hepatitis C virus (HCV) is generally subclinical in the acute phase, however, 80% of cases proceed to chronicity and there are currently 170 million carriers worldwide who may develop cirrhosis and/or hepatocellular carcinoma. At present there are no specific antivirals, but treatment with a combination of interferon and ribaravin results in viral clearance in 30-40% of individuals. The viral load in blood is so low that antigen cannot be easily detected and infection is generally determined by detection of anti-HCV or HCV RNA. Little is known about the HCV life cycle, as there is no reliable in vitro culture system, thus, existing knowledge on the function of the HCV proteins has been obtained using in vitro expression systems. Previous studies have shown that the HCV structural proteins core, El and E2 are cleaved from the N-terminal region of the polyprotein by a signalase in the endoplasmic reticulum (ER), and the cleavage products generally remain associated with this organelle. This study examined the effect of the structural proteins from an Australian HCV isolate when expressed in mammalian cells. The Semliki Forest virus (SFV) expression system was used for this purpose and high levels of the structural proteins were synthesised in BHK cells using this system. The core and envelope proteins were authentically cleaved and processed and exhibited the same pattern of cell localisation as reported previously. The level of structural proteins expressed by the HCV internal ribosome entry site (IRES)-directed translation was consistently lower compared with that from cap-directed translation, and this was likely due to the negative regulation of IRES-directed translation by the core protein, as previously reported. Immunoelectron microscopy of transfected cells revealed that the core and envelope proteins were localised to the membranes of HCV-induced vacuoles. Colabelling with antibodies against the structural proteins and protein disulphide isomerase (PDI), an ER resident protein, demonstrated that the core and envelope proteins co-localised in the membranes of induced vacuoles that were associated with ER-derived membranes. Thus, the HCV-induced vacuoles may have been induced by the accumulation of the HCV structural proteins in the ER in a situation analogous to events leading to virion assembly. Lysates of cells transfected with recombinant SFV RNA were examined for HCV virus­ like particle (VLP) formation. Rate-zonal sucrose gradient ultracentrifugation determined that the E2 protein expressed in this system had a density of 1.11-1.14 gm/cm3 and that a proportion of this protein represented native heterodimers, thought to be required for assembly. Electron microscopy of negatively stained samples revealed VLPs ranging from 40-80 nm in diameter that labelled with an antibody against the HCV envelope proteins. However, the VLPs were not purified from the cell lysates and consequently numerous contaminating particles were noted and the immunolabelling was not specific. Immunoprecipitation analysis of cell lysates sedimented through a sucrose cushion demonstrated that the core protein could be precipitated with an antibody to the envelope proteins. Therefore the core and E2 proteins interacted via El in a manner characteristic of VLPs. These data demonstrated that the HCV structural proteins colocalised within the ER membranes suggesting that viral assembly and budding occurs within these membranes. It would also appear that VLPs were assembled in the presence of the 5'UTR but that this process was inefficient.