Identification of a set of proteins (C' and C) encoded by the bicistronic P gene of the Indiana serotype of vesicular stomatitis virus and analysis of their effect on transcription by the viral RNA polymerase.
Previous work (C.F. Spiropoulou and S.T. Nichol, 1993, J. Virol. 67, 3103-3110) has demonstrated the existence in cells infected with the New Jersey serotype of vesicular stomatitis virus (VSV) of two small carboxy-coterminal proteins encoded by the P mRNA. These proteins have been named C' and C. We are interested in studying the function of these proteins in the virus life cycle. Toward this end, we have cloned the ORF encoding the potential C' protein of the Indiana serotype as a histidine-tagged fusion protein, purified the expressed protein from Escherichia coli, and used the fusion protein as an immunogen to raise antiserum in a rabbit. We have used this anti-C' protein serum to demonstrate that both of the predicted C' and C proteins are synthesized in cells infected with the Indiana serotype of VSV. In addition we have localized a portion of these proteins to nucleocapsids isolated from infected cells, suggesting that they may play a role in RNA synthesis. Reconstitution of the viral polymerase activity by expressing the L and P protein subunits with or without the C proteins failed to demonstrate any effect of the presence of these latter proteins on reconstituted transcription using purified nucleocapsids as templates. However, we have been able to show a dramatic stimulation of the polymerase activity in purified virions by the addition of purified C' protein to in vitro transcription reactions. Both the level and the fidelity of mRNA synthesis are stimulated by this protein. Evidence for the specificity of this effect comes from the fact that stimulation appears to be serotype specific; C' protein of the Indiana serotype stimulates transcription by purified Indiana serotype virions but has a minimal effect on transcription by purified virions of the New Jersey serotype. We are continuing our studies to determine the mechanism of this stimulation.
Peluso
R W
RW
Department of Microbiology, Mt. Sinai School of Medicine, New York, New York 10029, USA.
Richardson
J C
JC
Talon
J
J
Lock
M
M
D000818:Animals; D000914:Antibodies, Viral; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D005814:Genes, Viral / Q000502:physiology; D006639:Histidine / Q000235:genetics; D016366:Open Reading Frames / Q000235:genetics; D010455:Peptides / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis; D012367:RNA, Viral / Q000096:biosynthesis; D011817:Rabbits; D011993:Recombinant Fusion Proteins; D013095:Spermidine / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects / Q000502:physiology*; D014721:Vesicular stomatitis Indiana virus / Q000276:immunology / Q000378:metabolism*; D018116:Vesiculovirus; D017361:Viral Nonstructural Proteins / Q000032:analysis / Q000096:biosynthesis / Q000494:pharmacology / Q000502:physiology*; D014771:Virion / Q000737:chemistry / Q000378:metabolism
D000914:Antibodies, Viral; D010455:Peptides; D012333:RNA, Messenger; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; D017361:Viral Nonstructural Proteins; C033223:polyhistidine; D006639:Histidine; D012321:DNA-Directed RNA Polymerases; D013095:Spermidine