Alteration of the 5′ terminal caps of the mRNAs of vesicular stomatitis virus by cycloleucine in vivo

Abstract

Cycloleucine, a methionine analog, which inhibits methylation in animal cells also inhibits the reproduction of vesicular stomatitis virus (VSV) in infected cells. The replication of the VSV genome RNA is abolished and the steady state levels of viral mRNAs are greatly reduced. These effects of cycloleucine in vivo appear to be due to the role of the drug as an inhibitor of S-adenosylmethionine formation, since RNA synthesis and methylation in vitro with purified virus in the presence of added S-adenosylmethionine are unaffected by the drug. Pulse label experiments show that VSV mRNAs are synthesized in infected cells at nearly normal rates in the presence of cycloleucine, however, the majority of the transcripts appear to be unstable and are rapidly degraded. The limited amounts of VSV mRNAs which do accumulate during a 2 hr label in vivo contain the undermethylated 5′-terminal caps GpppA and 7mGpppA. No GpppA m capped or pppA initiated RNAs were detected. These last observations suggest that the pathway of viral RNA methylation within the infected cell occurs in the order GpppA → 7mGpppA → 7mGpppA m, instead of the reverse order which was proposed based on transcription studies in vitro. [ Testa, D., and Banerjee, A. K. (1977). J. Virol. 24, 786–793]. At least some of the undermethylated mRNAs are functional since there is a low level of synthesis in vivo of the virus proteins G, N, NS, and M.