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.Hepatitis C virus (HCV) infection is a major cause of chronic hepatitis and cirrhosis and may lead to 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 (6). HCV is an enveloped RNA virus belonging to the genus Hepacivirus of the Flaviviridae family. Its genome is a 9.6-kb single-stranded RNA of positive polarity with a 5Ј untranslated region (UTR) that functions as an internal ribosome entry site, a single long open reading frame encoding a polyprotein of approximately 3,000 amino acids (aa) and a 3Ј UTR (1). This polypeptide is posttranslationally cleaved by host cell peptidases to yield three structural proteins and by viral proteases, which generate the five nonstructural proteins. The structural proteins, which are located in the amino-terminal region of the polyprotein, include the core protein and the envelope glycoproteins E1 and E2. The nonstructural proteins (NS) 2 to 5B are separated from the structural proteins by the short hydrophobic polypeptide p7, the function of which is unknown (1). By analogy to related positive-strand RNA viruses, replication occurs by means of a negative-strand RNA intermediate and is catalyzed by the NS proteins, which form a cytoplasmic membrane-associated replicase complex.Several entire cloned HCV genomes are able to initiate infection when introduced directly into chimpanzee livers (11). However, the transfection of cell lines with constructs containing these genomes does not result in HCV replication in vitro (1). Similarly, the use of infected sera to infect cell lines or primary cell cultures has yielded disappointing results because infection and replication are very inefficient (2). Molecular studies of the HCV infectious cycle in the host cell and the development of specific anti-HCV agents have been considerably hampered by the inability to achieve propagation of the virus in cultured cells in vitro. A significant advance in HCV research was recently made with the development of subgenomic HCV RNAs consisting of sequences encoding nonstructural proteins flanked by the 5Ј and 3Ј UTRs, which selfreplicate in hepatoma cells (4,18). However, this model cannot be used to address the structural features of the virion or its assembly pathway. In addition, viral particles are difficult to observe by electron microscopy in the plasma or liver tissues of infected patients. A number of attempts have bee...

Characterization of GB Virus B Polyprotein Processing Reveals the Existence of a Novel 13-kDa Protein with Partial Homology to Hepatitis C Virus p7 Protein

“…The capsid protein (C) and two envelope glycoproteins (E1 and E2) are believed to be components of the virion. Studies of these structural proteins have for the most part relied on heterologous expression systems, such as recombinant baculoviruses in insect cells (4), or recombinant vaccinia or Sindbis viruses (5) and Semliki Forest virus-derived vectors (6) in eukaryotic cells. The results of these studies support the hypothesis that the HCV glycoproteins are retained in the endoplasmic reticulum.…”

Producción y purificación de la proteína core del virus de la hepatitis C en el sistema de expresión del baculovirus para ensayos biológicos.

“…En este estudio, se detectaron las tres isoformas de Core del VHC en la fracción de proteínas asociadas con membranas celulares, luego de infectar células de insecto con el baculovirus recombinante; esta localización corrobora lo descrito previamente en la literatura (36).…”

“…De otra parte, se ha intentado producir la proteína Core en diferentes sistemas eucarióticos como levaduras (34), células de insectos (35) y células de mamíferos (36,37), con relativo éxito. En el caso de la producción en células de insecto, los artículos publicados en la literatura corresponden a la expresión simultánea de las proteínas estructurales Core, E1 y E2 del VHC (35,(38)(39)(40).…”

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.…”