Characterization of Pseudotype VSV Possessing HCV Envelope Proteins

Abstract

The genome of hepatitis C virus (HCV) encodes two envelope glycoproteins (E1 and E2), which are thought to be responsible for receptor binding and membrane fusion resulting in virus penetration. To investigate cell surface determinants important for HCV infection, we used a recombinant vesicular stomatitis virus (VSV) in which the glycoprotein gene was replaced with a reporter gene encoding green fluorescent protein (GFP) and produced HCV-VSV pseudotypes possessing chimeric HCV E1 or E2 glycoproteins, either individually or together. The infectivity of the pseudotypes was determined by quantifying the number of cells expressing the GFP reporter gene. Pseudotypes that contained both of the chimeric E1 and E2 proteins exhibited 10--20 times higher infectivity on HepG2 cells than the viruses possessing either of the glycoproteins individually. These results indicated that both E1 and E2 envelope proteins are required for maximal infection by HCV. The infectivity of the pseudotype virus was not neutralized by anti-VSV polyclonal antibodies. Bovine lactoferrin specifically inhibited the infection of the pseudotype virus. Treatment of HepG2 cells with Pronase, heparinase, and heparitinase but not with phospholipase C and sodium periodate reduced the infectivity. Therefore, cell surface proteins and some glycosaminoglycans play an important role in binding or entry of HCV into susceptible cells. The pseudotype VSV possessing the chimeric HCV glycoproteins might offer an efficient tool for future research on cellular receptors for HCV and for the development of prophylactics and therapeutics for hepatitis C.

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Absolute quantification of infectious particles can be obtained by TCID 50 (50% tissue culture infectious dose) [6,191] and FFU (focus forming unit) assays [7,192] which are both applicable for all infectious HCV constructs and rely on immunolabelling of HCV-infected cells. An alternative is the use of a reporter system, with 14 either a reporter cell line (see Table 1) or a reporter virus (see Figure 3).…”

“…A resembling protocol is routinely used in our laboratory, with some modifications in the immunolabelling procedure and TCID 50 Next, the FFU assay corresponds to the quantification of HCV-infected cell foci [7,192]. As for the TCID 50 Last, the use of reporter viruses, and in particular the luciferase reporter viruses, is certainly the most convenient and time-saving alternative [63]. The virus does not need to be titered and a single virus dilution (usually the undiluted viral supernatant) can be incubated with various inhibitor concentrations to obtain a dose-response curve.…”

“…Please note that we would like to incorporate in the main text a link for a TCID 50 calculator (Excel sheet). This document was obtained from Marco Binder, Heidelberg, who agreed to make it freely accessible on the Heidelberg website.…”

“…In addition to that, different viruses can be used as a scaffold for E1E2 glycoproteins, a concept called pseudotyping. The first HCV pseudotypes elaborated were based on modified influenza [48] or vesicular stomatitis virus viruses [49][50][51][52]. However, to our knowledge, except for one report [49], only chimeric glycoproteins, 5 consisting of the ectodomains from E1E2 and both the transmembrane domain and cytoplasmic tail from the heterologous virus, could be incorporated into the pseudovirus envelope.…”

“…This section provides standard protocols to titrate a virus stock using the three main infectivity assays introduced above (TCID 50 , FFU and luciferase assays). In all cases, 10 4 Huh-7.5 target cells are seeded per well in 96-well dishes 24h before infection.…”