Molecular analysis of the inhibitory effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro polymerase reaction

The effect of phosphorylated ribavirin on the vesicular stomatitis virus in vitro transcription reaction was examined. Analysis of the kinetics observed when the concentrations of nucleoside triphosphates were varied was performed with vesicular stomatitis virus wild-type standard virions. Double-reciprocal and Eadie-Hofstee plots showed competitive inhibition with all natural nucleoside triphosphates when both ribavirin diphosphate (RDP) and ribavirin triphosphate (RTP) were used. The K(m) values for ATP obtained for the wild-type polymerase were similar to those reported previously. To further characterize the observed inhibition kinetics, in vitro transcription products synthesized in the presence or absence of RDP and RTP were purified by CsCl centrifugation and were primer extended with oligonucleotides specific for either positive-sense leader or nucleocapsid mRNA transcripts. The ratios of leader to nucleocapsid mRNA were measured from primer-extended in vitro transcription products. It was found that the addition of RDP or RTP did not significantly change the in vitro ratio, suggesting that the polymerase is blocked before it enters the 3' end of the template.