Differential effect of ATP concentration on synthesis of vesicular stomatitis virus leader RNAs and mRNAs

Abstract

Cleavage of the beta-gamma bond of ATP is required for wild-type (wt) vesicular stomatitis virus transcription in vitro. Recent findings have established that a domain-specific phosphorylation of the virus NS protein is necessary for activity. We report here that RNA synthesis catalyzed by purified standard wt virions responded cooperatively to various ATP concentrations, with half-maximal activity at approximately 500 microM. In contrast, mutant polR1 standard virions and wt defective interfering particles both showed conventional Michaelis-Menten kinetic profiles with Km values of approximately 143 and approximately 133 microM, respectively. The former synthesize readthrough products of the leader-N gene junction in addition to plus-strand leader RNA and mRNAs, whereas the latter synthesize only minus-strand leader RNA. The cooperative response of wt virus products, however, was specific to mRNAs; the small fraction of the total products corresponding to plus-strand leader approximated Michaelis-Menten behavior. Since the unique phenotype of the polR mutants correlates with the synthesis of replicationlike products in vitro, the affected ATP-requiring function most likely regulates both transcription and replication. We suggest that this mutated function involves phosphorylation of viral proteins.