Replication-Competent Recombinant Vesicular Stomatitis Virus Encoding Hepatitis C Virus Envelope Proteins

Abstract

Hepatitis C virus (HCV) is the major causative agent of blood-borne chronic non-A, non-B hepatitis, infecting at least 3% of the world's population. The majority of HCV-infected individuals develop chronic hepatitis that eventually progresses to liver cirrhosis and hepatocellular carcinoma (36). HCV is an enveloped single-stranded plus-sense RNA virus belonging to the genus Hepacivirus in the Flaviviridae family, which also includes members of the genus Flavivirus, such as yellow fever virus, dengue virus, and West Nile virus, and of the genus Pestivirus, such as bovine viral diarrhea virus and classical swine fever virus. The genome of HCV encodes a polyprotein of approximately 3,000 amino acids, which is subsequently processed into at least 10 viral proteins. The HCV envelope glycoproteins E1 and E2 are cleaved from the polyprotein by host signal peptidases and play a crucial role in the initiation of infection through interaction with cell surface receptor(s) in the HCV life cycle (17,38).A number of cellular components have been shown to participate in HCV adsorption and/or internalization, including human CD81 (hCD81) (52), low-density lipoprotein receptor (LDLr) (1), human scavenger receptor class B type I (SR-BI) (57), dendritic cell-specific intercellular adhesion molecule-3-grabbing nonintegrin (DC-SIGN), liver/lymph node-specific intercellular adhesion molecule-3-grabbing nonintegrin (L-SIGN or DC-SIGNR) (21, 34), glycosaminoglycans (2), and a tight junction component, claudin-1 (18). Recently, an in vitro cell culture system was developed for HCV of the genotype 2a JFH1 strain (HCVcc) isolated from a fulminant HCV patient (32,63,68). However, a robust cell culture system for HCV of the 1a and 1b genotypes, the most prevalent genotypes in the world, has not yet been successfully developed, except for the cell culture system of H77 or H77-S strain (1a genotype) (26,65). Furthermore, it is currently not possible to obtain a sufficient amount of HCV particles for biological and physiochemical studies due to the low viral load in the sera of hepatitis C patients and the low yield of HCV particles in cell culture. Thus, the relative contribution of these receptor candidates in HCV attachment and entry remains unclear (44).As surrogate systems for the investigation of HCV infection mechanisms, HCV-like particles (HCV-LP) produced in insect or mammalian cells by recombinant baculovirus vectors have been developed (7,37). Although the binding of HCV-LP to the target cells has been well characterized, HCV-LP are not suitable for the analysis of the HCV entry steps due to the absence of a clear distinction between binding and internalization. On the other hand, both murine leukemia virus (MLV)-and human immunodeficiency virus-based pseudotype retrovi-* Corresponding author. Mailing address:

Characterization of pH-Sensitive Molecular Switches That Trigger the Structural Transition of Vesicular Stomatitis Virus Glycoprotein from the Postfusion State toward the Prefusion State

“…To examine whether the mutant glycoproteins were able to sustain viral infection, we used a recombinant VSV (VSV⌬G-GFP) in which the G envelope gene was replaced by the green fluorescent protein (GFP) gene and which was pseudotyped with the VSV G glycoprotein (26,27). This pseudotyped recombinant was used to infect HEK cells (at an MOI of 3) either transfected or not transfected by a plasmid encoding WT or mutant glycoproteins.…”

“…VSV⌬G-GFP is a recombinant VSV which was derived from a full-length cDNA clone of the VSV genome (Indiana serotype) in which the coding region of the G protein was replaced by a modified version of the GFP gene and pseudotyped with the VSV G protein (26,27). VSV⌬G-GFP was propagated on HEK-293T cells that had been transfected with pCAGGS-VSV-G.…”

Contribution of Redox Status to Hepatitis C Virus E2 Envelope Protein Function and Antigenicity

“…CD81 Binding-Baculovirus-expressed E2 has been shown to be functionally analogous to E2 derived from mammalian cells (26), and E2 binding to CD81, which is central to the complex HCV entry process (27), represents the prevalent measure of a functional E2 conformation (19,28,29). Accordingly, the overall conformation of various E2 redox intermediates was monitored using CD81 binding in vitro.…”

Development of an Infectious Surrogate Hepatitis C Virus Based on a Recombinant Vesicular Stomatitis Virus Expressing Hepatitis C Virus Envelope Glycoproteins and Green Fluorescent Protein

“…However, 1 significant difference is that our infectious VSV/HCV actually replicates in and propagates on the hepatoma cells because it encodes E1/E2 in its genome, whereas previously described pseudotypes did not. Another group generated an rVSV encoding native E1/E2 and the p7 ion channel protein in place of G (31). Although this recombinant virus was similar to our infectious VSV/HCV, a major difference is that our recombinant virus does not encode or express p7, but rather expresses GFP.…”