Determinants of serotype specificity in transcription of vesicular stomatitis virus synthetic nucleocapsids.
Transcription of the nucleocapsid template of vesicular stomatitis virus (VSV) is serotype specific, since the viral RNA polymerase of the VSV Indiana (Ind) serotype transcribes the Ind nucleocapsid but not the nucleocapsid of the VSV New Jersey (NJ) serotype and, similarly, the NJ RNA polymerase transcribes only the NJ nucleocapsid. We have prepared synthetic nucleocapsid templates from various combinations of purified leader gene RNA and N protein of these two VSV serotypes for analysis of serotype specificity in vitro. In agreement with previous observations, in vitro transcription of synthetic homologous nucleocapsids with the 3' terminal leader RNA gene of either the (+) or (-) strand sense and the N protein of the same serotype is also serotype specific. We show with chimeric nucleocapsids, where the RNA and N protein are of different serotypes, that both components of the template are important for specificity, although the specifics depend on the serotype from which the RNA polymerase is derived. The Ind RNA polymerase will transcribe only the homologous nucleocapsids where both the RNA and N protein are of the Ind serotype, and not the chimeric nucleocapsids. In contrast, the NJ RNA polymerase is active not only on the homologous synthetic nucleocapsid, but also gives significant levels of transcription as long as one of the nucleocapsid components (N protein or RNA) is of the NJ serotype. The divergent RNA sequence of the distal portion of the leader gene (nt22 to 50/51) seems to be a major determinant for specificity, since transcription of synthetic nucleocapsids containing just the conserved proximal 1-22 nucleotides is significantly less serotype-restricted. Restriction appears to occur at the level of elongation of the product rather than initiation of RNA synthesis, since synthesis of template-length RNA, but not reiterated small initiation products, is inhibited. In addition, the serotype of the P protein subunit of the RNA polymerase also contributes to the serotype specificity of transcription, since the NJ P protein can bind equally to NJ and Ind nucleocapsids, while Ind P protein binds preferentially to Ind nucleocapsids.
Smallwood
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S
Department of Immunology and Medical Microbiology, College of Medicine, University of Florida, Gainesville 32610-0266.
Richards-Sumners
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Moyer
S A
SA
D000818:Animals; D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D008969:Molecular Sequence Data; D010750:Phosphoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins / Q000235:genetics; D012703:Serotyping; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000145:classification / Q000235:genetics*; D018116:Vesiculovirus; D015678:Viral Structural Proteins / Q000145:classification / Q000378:metabolism
C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; D012321:DNA-Directed RNA Polymerases