Functional role of hepatitis C virus chimeric glycoproteins in the infectivity of pseudotyped virus

Abstract

The putative envelope glycoproteins of hepatitis C virus (HCV) likely play an important role in the initiation of viral infection. Available information suggests that the genomic regions encoding the putative envelope glycoproteins, when expressed as recombinant proteins in mammalian cells, largely accumulate in the endoplasmic reticulum. In this study, genomic regions which include the putative ectodomain of the E1 (amino acids 174 to 359) and E2 (amino acids 371 to 742) glycoproteins were appended to the transmembrane domain and cytoplasmic tail of vesicular stomatitis virus (VSV) G protein. This provided a membrane anchor signal and the VSV incorporation signal at the carboxy termini of the E1 and E2 glycoproteins. The chimeric gene constructs exhibited expression of the recombinant proteins on the cell surface in a transient expression assay. When infected with a temperature-sensitive VSV mutant (ts045) and grown at the nonpermissive temperature (40.5 degrees C), cells transiently expressing the E1 or E2 chimeric glycoprotein generated VSV/HCV pseudotyped virus. The resulting pseudotyped virus generated from E1 or E2 surprisingly exhibited the ability to infect mammalian cells and sera derived from chimpanzees immunized with the homologous HCV envelope glycoproteins neutralized pseudotyped virus infectivity. Results from this study suggested a potential functional role for both the E1 and E2 glycoproteins in the infectivity of VSV/HCV pseudotyped virus in mammalian cells. These observations further suggest the importance of using both viral glycoproteins in a candidate subunit vaccine and the potential for using a VSV/HCV pseudotyped virus to determine HCV neutralizing antibodies.

FIG. 1

Schematic presentation of the chimeric gene constructs used for expression of the HCV glycoproteins on the mammalian cell surface.

FIG. 2

In vitro translation and immunoprecipitation of E1 and E2 chimeric glycoproteins with VSV G by specific antibodies. (A) [³⁵S]methionine-labeled in vitro-translated E1 chimeric proteins in the presence of microsomal membrane were immunoprecipitated separately with a MAb to E1 (lane 1) and a polyclonal antiserum to VSV (lane 2) and analyzed by SDS-PAGE (10% gel). A negative control was similarly run with pcDNA3 vector and the polyclonal antiserum to VSV (lane 3). (B) An in vitro-translated E2 chimeric glycoprotein was tested by using a polyclonal antibody to VSV (lane 1) with vector DNA as the negative control (lane 2). Immunoprecipitates were analyzed by SDS-PAGE (7% gel). Arrowheads on the left indicate locations of the mature E1 or E2 chimeric proteins; sizes on the right are expressed in kilodaltons.

FIG. 3

Surface immunofluorescence of cells transiently expressing chimeric glycoproteins from the E1G and E2G gene constructs. HeLa cells were infected with recombinant vvT7 and transfected with E1G (A), E2G (B), or pcDNA3 vector DNA (C) and reacted with a MAb to E1 or E2 after 18 h of incubation. Cells in panel C were tested with a mixture of the MAbs to both E1 and E2.

FIG. 4

Serial dilutions of sera from chimpanzees L357, L559, L534, and 635 vaccinated with recombinant envelope glycoproteins of HCV were tested for neutralizing antibodies to VSV/HCV pseudotyped virus. Activities at different reciprocal dilutions of immunized sera to the E1 (□) and E2 (▪) glycoproteins are expressed against pseudotype plaque numbers. Preimmune chimpanzee sera were similarly tested for reactivity to the E1 (○) and E2 (•) glycoproteins as negative controls. Plaque numbers varied within ±5 in each independent experiment.