Expression of the M gene of vesicular stomatitis virus cloned in various vaccinia virus vectors.
Initial attempts to clone the matrix (M) gene of vesicular stomatitis virus (VSV) in a vaccinia virus expression vector failed, apparently because the expressed M protein, and particularly a carboxy-terminus-distal two-thirds fragment, was lethal for the virus recombinant. Therefore, a transient eucaryotic expression system was used in which a cDNA clone of the VSV M protein mRNA was inserted into a region of plasmid pTF7 flanked by the promoter and terminator sequences for the T7 bacteriophage RNA polymerase. When CV-1 cells infected with recombinant vaccinia virus vTF1-6,2 expressing the T7 RNA polymerase were transfected with pTF7-M3, the cells produced considerable amounts of M protein reactive by Western blot (immunoblot) analysis with monoclonal antibodies directed to VSV M protein. Evidence for biological activity of the plasmid-expressed wild-type M protein was provided by marker rescue of the M gene temperature-sensitive mutant tsO23(III) at the restrictive temperature. Somewhat higher levels of M protein expression were obtained in CV-1 cells coinfected with a vaccinia virus-M gene recombinant under control of the T7 polymerase promoter along with T7 polymerase-expressing vaccinia virus vTF1-6,2.
Li
Y
Y
Department of Microbiology, University of Virginia School of Medicine, Charlottesville 22908.
Luo
L Z
LZ
Snyder
R M
RM
Wagner
R R
RR
D005814:Genes, Viral; D005822:Genetic Vectors; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000096:biosynthesis*; D013604:T-Phages / Q000235:genetics; D014616:Vaccinia virus / Q000235:genetics; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014763:Viral Matrix Proteins / Q000096:biosynthesis*
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins