Genetically modified VSVNJ vector is capable of accommodating a large foreign gene insert and allows high level gene expression

Abstract

It is desirable to develop a RNA virus vector capable of accommodating large foreign genes for high level gene expression. Vesicular stomatitis virus (VSV) has been used as a gene expression vector, especially Indiana serotype (VSVInd), but less with New Jersey serotype (VSVNJ). Here, we report constructions of genetically modified rVSVNJ vector carrying various lengths of human hepatitis C virus (HCV) non-structural (NS) protein genes, level of inserted gene expression and characterization of rVSVNJ. We modified the M gene of VSVNJ by changing methionine to arginine at positions 48 and 51 (rVSVNJ-M) (Kim and Kang, 2007) for construction of rVSVNJ with various lengths of HCV non-structural genes. The NS polyprotein genes of HCV were inserted between the G and L genes of the rVSVNJ-M vector, and recombinant VSVNJ-M viruses with HCV gene inserts were recovered by the reverse genetics. The recombinant VSVNJ-M vector with the HCV NS genes express high levels of all different forms of the NS proteins. The electron microscopic examination showed that lengths of recombinant VSVNJ-M without gene of interests, VSVNJ-M with a gene of HCV NS3 and NS4A (VSVNJ-M-NS3/4A), VSVNJ-M with a gene of HCV NS4AB plus NS5AB (VSVNJ-M-NS4AB/5AB), and VSVNJ-M carrying a gene of HCV NS3, NS4AB, and NS5AB (VSVNJ-M-NS3/4AB/5AB) were 172±10.5nm, 201±12.5nm, 226±12.9nm, and 247±18.2nm, respectively. The lengths of recombinant VSVs increased approximately 10nm by insertion of 1kb of foreign genes. The diameter of these recombinant viruses also increased slightly by longer HCV gene inserts. Our results showed that the recombinant VSVNJ-M vector can accommodate as much as 6000 bases of the foreign gene. We compared the magnitude of the IFN induction in mouse fibroblast L(Y) cells infected with rVSVNJ wild type and rVSVNJ M mutant viruses and show that the rVSVNJ M mutant virus infection induced a higher level of the IFN-β compare to the wild type virus. In addition, we showed that the NS protein expression level in IFN-incompetent cells (Mouse-L) infected with rVSVNJ-M viruses was higher than in IFN-competent L(Y) cells. In addition, we confirmed that HCV NS protein genes were expressed and properly processed. We also confirmed that NS3 protein expressed from the rVSVNJ-M cleaves NS polyprotein at junctions and that NS4A plays an important role as a co-factor for NS3 protease to cleave at the NS4B/5A site and at the NS5A/5B site.