Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies.
Bacterial fusion proteins containing portions of the Bunyamwera virus L protein were used as immunogens to prepare antisera in rabbits. Of five fusion proteins injected into rabbits, three yielded sera that reacted with the Bunyamwera virus L protein, detected by Western blotting or immunoprecipitation. Two of these antisera were specific for either the amino- or carboxy-terminal regions of the L protein. The specificity of these antisera was confirmed by their pattern of reactivity with full-length and truncated forms of the L protein. Plasmids containing the L gene cDNA under control of a bacteriophage T7 promoter were transfected into CV-1 cells which had previously been infected with a recombinant vaccinia virus, vTF7-3, that expresses T7 RNA polymerase. Antigenically authentic L protein was expressed. Using a nucleocapsid transfection assay developed previously, we showed that the transiently expressed L protein had RNA synthesis activity. Site-specific mutations were made in the L cDNA-containing plasmid to change certain amino acids in the putative polymerase domain of the L protein. The effects of these amino acid substitutions on the RNA synthesis activity of the L protein were monitored using the nucleocapsid transfection assay. These experiments showed that residues strictly conserved between the L proteins of different viruses in the family Bunyaviridae were obligatorily required for activity, whereas non-conserved residues could be substituted without abolishing RNA synthesis capability. Our results provide direct evidence for the functional significance of particular amino acids in the polymerase domain of a negative-strand virus RNA polymerase.
Jin
H
H
Institute of Virology, University of Glasgow, U.K.
Elliott
R M
RM
D000595:Amino Acid Sequence; D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis*; D001483:Base Sequence; D002042:Bunyamwera virus / Q000235:genetics / Q000276:immunology*; D006224:Cricetinae; D005784:Gene Amplification; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D012367:RNA, Viral / Q000096:biosynthesis; D012324:RNA-Dependent RNA Polymerase / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis*; D017385:Sequence Homology; D014162:Transfection; D014764:Viral Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology*
D000914:Antibodies, Viral; C069882:L protein, Bunyamwera virus; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D012324:RNA-Dependent RNA Polymerase