Hepatitis C virus-like particle morphogenesis

Abstract

Although much is known about the hepatitis C virus (HCV) genome, first cloned in 1989, little is known about HCV structure and assembly due to the lack of an efficient in vitro culture system for HCV. Using a recombinant Semliki forest virus replicon expressing genes encoding HCV structural proteins, we observed for the first time the assembly of these proteins into HCV-like particles in mammalian cells. This system opens up new possibilities for the investigation of viral morphogenesis and virus-host cell interactions.

FIG. 1.

Organization of SFV and HCV RNA genomes and description of our SFV-HCV^dj DNA construct encoding HCV structural proteins. Flaviviruses differ from alphaviruses in genome structure (structural genes situated at the 5′ end of the genome) and in the absence of subgenomic RNA.

FIG. 2.

Production of HCV structural proteins in BHK-21 cells following electroporation. After linearization by digestion with SpeI, the SFV-HCV^dj vector was used as a template for the in vitro synthesis of recombinant RNA. For the negative control, recombinant β-Gal RNA, which encodes the β-Gal protein, was synthesized from the expression vector pSFV3 (Life Technologies). After electroporation and culture for 24 h, transfected cells were analyzed by immunofluorescence staining (A) and Western blotting (B) with monoclonal antibodies against HCV capsid protein (MAB8424 from Chemicon, Temecula, Calif.), E1 (A4; gift from Harry Greenberg) (7), and E2 (H52; gift from Jean Dubuisson) (7); size markers (SM) were from Bio-Rad (Hercules, Calif.).

FIG. 3.

Electron micrographs at low magnification of ultrathin sections of BHK-21 cells electroporated with the HCV^dj RNA (A) or β-Gal RNA (B). Bar, 500 nm. Arrows indicate the ER structures, normally distributed in the cytoplasm in panel B but forming areas of convoluted membranes in panel A. n, nucleus.

FIG. 4.

Electron micrographs at high magnification of ultrathin sections of BHK-21 cells electroporated with the HCV^dj RNA. Bars, 100 nm (A) and 50 nm (B, C, and D). Arrows indicate virus-like particles budding towards the ER lumen. The two particles in panel B may represent different steps for this phenomenon, from early (small arrow) to late (larger arrow) events. c, cytoplasm; lu, lumen.

FIG. 5.

Immunogold labeling of ultrathin section of BHK-21 cells electroporated with the HCV^dj RNA (A and C) and β-Gal RNA (B) and stained with anti-E1 (A and B) or anti-core (C) antibodies. Arrows indicate prebudding or budding structures in which gold labeling was particularly concentrated. Bars, 200 nm (A and B) and 50 nm (C). c, cytoplasm; lu, lumen.