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.