Altered ATP utilization by the poIR mutants of vesicular stomatitis virus maps to the N-RNA template.
In a continuation of previous efforts to study the modified ATP requirements for RNA synthesis by poIR mutants of vesicular stomatitis virus (VSV), we have used a novel reconstitution assay to show that it is the template moiety of the mutants, not the polymerase proteins, which governs both the increased utilization of the ATP analog, beta, gamma-imido ATP (AMP-PMP), and the loss of a positive cooperativity-like response to varying ATP concentrations. Assays utilized uv-irradiated virus as a source of polymerase proteins and purified N-RNA as templates. Homologous and heterologous transcriptase reactions were carried out with wild-type (wt) virus and each of the two independently isolated poIR mutants. We show that in the presence of wt N-RNA template, substitution of AMP-PNP for ATP resulted in only approximately 5% of control RNA synthesis regardless of which source of polymerase was used. Furthermore, all reactions containing wt N-RNA template responded to varying ATP concentrations with a concave, upward-shaped Lineweaver-Burke plot generally indicative of positive cooperativity effects. In contrast, all reactions which utilized N-RNA templates from the poIR mutants showed an increased utilization of AMP-PNP (greater than 20%) and a more characteristic Michaelis-Menten response to changing ATP concentrations. These findings strongly support the notion that the template-associated nucleocapsid protein modulates the utilization of an ATP site which is directly or indirectly involved in VSV RNA synthesis.
Helfman
W B
WB
Department of Biology, San Diego State University, California 92182.
Perrault
J
J
D000255:Adenosine Triphosphate / Q000378:metabolism*; D009154:Mutation; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D014779:Virus Replication
D012333:RNA, Messenger; D012367:RNA, Viral; D000255:Adenosine Triphosphate