Mutagenesis of the L protein encoded by Bunyamwera virus and production of monospecific antibodies

Abstract

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.

Nucleotide sequence of the gene encoding the viral polymerase of avian pneumovirus

“…Alteration of the GDN sequence to GDD (the core polymerase motif of most positive-strand RNA viruses) or SDD (the core polymerase motif of segmented negative-strand RNA viruses) also resulted in complete abrogation of polymerase activity. These findings are similar to those observed for Bunyamweravirus (Jin & Elliott, 1992) and vesicular stomatitis virus (VSV; Sleat & Bannerjee, 1993) L proteins. However, mutation of the VSV GDN sequence to GDD did not abrogate polymerase activity, but reduced it to 27% of the wild-type polymerase activity.…”

A Single Amino Acid Change in the L-Polymerase Protein of Vesicular Stomatitis Virus Completely Abolishes Viral mRNA Cap Methylation

“…Domain II has a highly charged putative RNA-binding motif, and domain III contains a potential GDNQ phosphodiester bond-forming motif (33,43,45). Consistent with the predicted roles of domains II and III in viral RNA synthesis, most (domain II) or all (domain III) mutations within these motifs abolished RNA synthesis in several viruses (32,53,60,62,63). Although domains I, IV, and V are conserved among NNS viruses, no sequence elements were found which would indicate specific functions of these domains.…”

“…Therefore, the effects of the L protein mutations on viral mRNA cap methylation were studied using detergent-activated purified viruses naturally carrying active virion-bound polymerase (3). wt, the original hr1, and the recombinant viruses were grown under permissive conditions (BHK cells, 34°C), purified, and first tested for their ability to synthesize mRNA in vitro, using a reaction mixture with or without AdoMet and containing [␣- 32 a The numbers represent the average of two to three experiments where the variation was less than 15% (as in Fig. 1 and 2), measuring the percentage of in vitro transcription (% Tnx) of each L compared to wt L (as 100%).…”

Functional L polymerase of La Crosse virus allows in vivo reconstitution of recombinant nucleocapsids

“…However, it is presently unknown whether these four amino acid positions are critical for polymerase activity, since, to our knowledge, the L sequence published previously was not tested for function. Furthermore, none of the changes were situated within the conserved RNA-dependent RNA polymerase motifs identified previously (Jin & Elliott, 1992;Poch et al, 1989). The new LACV L sequence has been submitted to GenBank (accession no.…”