Transcriptional activity and mutational analysis of recombinant vesicular stomatitis virus RNA polymerase.
The 241-kDa large (L) protein of vesicular stomatitis virus (VSV) is the multifunctional catalytic component of the viral RNA polymerase. A protocol has been developed for the synthesis of recombinant L protein that will support viral mRNA synthesis in vitro. COS cells were transfected with a transient expression vector (pSV-VSL1 [M. Schubert, G. G. Harmison, C. D. Richardson, and E. Meier, Proc. Natl. Acad. Sci. USA 82:7984-7988, 1985]) which contains the simian virus 40 late promoter for the transcription of a cDNA copy of the L protein of the Indiana serotype of VSV. Cytoplasmic extracts of these cells efficiently transcribed VSV mRNAs in vitro in conjunction with N protein-RNA template purified from virus and recombinant phosphoprotein synthesized in Escherichia coli. mRNA synthesis was completely dependent upon addition of both bacterial phosphoprotein and extracts from cells transfected with the L gene. Extracts from mock-transfected cells or from cells transfected with the expression vector alone did not support VSV RNA synthesis. RNA synthesis was proportional to the concentration of cell extract used, with an optimum of 0.2 mg/ml. Rhabdoviruses and paramyxoviruses contain a highly conserved GDNQ motif which was mutated in the transfected L gene. All constructs with mutations within the core GDN abrogated transcriptional activity except for the mutant containing GDD, which retained 25% activity. Conserved amino acid changes outside of the core GDN and changes corresponding to other paromyxovirus and rhabdovirus L proteins retained variable transcriptional activity. These findings provide experimental evidence that the GDN of negative-strand, nonsegmented RNA viruses is a variant of the GDD motif of plus-strand RNA viruses and of the XDD motif of DNA viruses and reverse transcriptases.
Department of Molecular Biology, Cleveland Clinic Foundation, Ohio 44195-5178.
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Sleat
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Banerjee
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D002478:Cells, Cultured; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010252:Paramyxoviridae / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D011994:Recombinant Proteins / Q000235:genetics; D012209:Rhabdoviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014162:Transfection; D014721:Vesicular stomatitis Indiana virus / Q000201:enzymology* / Q000235:genetics
D012333:RNA, Messenger; D012367:RNA, Viral; D011994:Recombinant Proteins; D012321:DNA-Directed RNA Polymerases