ABSTRACT Sinefungin (SIN), a natural S -adenosyl- l -methionine analog produced by Streptomyces griseolus , is a potent inhibitor of methyltransferases. We evaluated the effect of SIN on replication of vesicular stomatitis virus (VSV), a prototype of the nonsegmented negative-strand RNA viruses. The 241-kDa large polymerase (L) protein of VSV methylates viral mRNA cap structures at the guanine-N-7 (G-N-7) and ribose-2′-O (2′-O) positions. By performing transcription reactions in vitro, we show that both methylations are inhibited by SIN and that methylation was more sensitive at the G-N-7 than at 2′-O position. We further show that SIN inhibited growth of VSV in cell culture, reducing viral yield by 50-fold and diminishing plaque size. We isolated eight mutants that were resistant to SIN as judged by their growth characteristics. The SIN-resistant (SIN R ) viruses contained mutations in the L gene, the promoter for L gene expression provided by the conserved sequence elements of the G - L gene junction and the M gene. Five mutations resulted in amino acid substitutions to conserved regions II/III and VI of the L protein. For each mutant, we examined viral gene expression in cells and cap methylation in vitro. SIN R mutants upregulated RNA synthesis in the presence of SIN, which may be responsible for their resistance. We also found that some SIN R viruses with L gene mutations were defective in cap methylation in vitro, yet their methylases were less sensitive to SIN inhibition than those of the wild-type parent. These studies show that the VSV methylases are inhibited by SIN, and they define new regions of L protein that affect cap methylation. These studies also provide experimental evidence that inhibition of cap methylases is a potential strategy for development of antiviral therapeutics against nonsegmented negative-strand RNA viruses.