Identification of a unique guanine-7-methyltransferase in Semliki Forest virus (SFV) infected cell extracts.
The methylation of the 5' terminal guanosine residue of the cap structure of Semliki Forest virus (SFV) mRNAs has been shown to occur in vitro concomitantly with their synthesis (R. K. Cross and P. J. Gomatos, Virology, 114, 542-554, 1981). The enzyme responsible for this methylation, a guanine-7-methyltransferase, is associated with the SFV replication complex which contains both the virus-specified polymerase and RNA template in a mitochondrial pellet fraction, P-15, from infected cell lysates. In the present report, evidence has been obtained demonstrating that a virus-specified function is required for this methylating activity. First, the methyltransferase enzyme in these infected P-15 extracts has been found to differ in substrate specificity from that of the BHK host cell enzyme. This enzyme was able to catalyze the methylation of GTP to m7GTP in vitro whereas the cellular enzyme could not methylate GTP. The incorporation of a methyl group onto GTP occurred linearly for at least 2 hr at 30 degrees under conditions of neutral pH and added GTP substrate. Second, a study of the kinetics of appearance of this activity, has demonstrated that the capacity to methylate GTP did not appear until 1 hr after infection and reached maximal levels by about 3 hr. Third, de novo protein synthesis was required. Addition of the protein synthesis inhibitor, cycloheximide, prevented the appearance and subsequent increase in the methylating activity. However, once formed the methyltransferase was found to be stable for at least 3 hr. These results suggest that an early viral function, perhaps a nonstructural polypeptide is required for this novel guanine-7-methyltransferase activity in SFV infected cell extracts.
D000818:Animals; D002457:Cell Extracts; D002460:Cell Line; D002467:Cell Nucleus / Q000201:enzymology; D006224:Cricetinae; D003513:Cycloheximide / Q000494:pharmacology; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008745:Methylation; D008780:Methyltransferases / Q000378:metabolism*; D008928:Mitochondria / Q000201:enzymology; D012314:RNA Cap Analogs / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology*; D013379:Substrate Specificity; D014158:Transcription, Genetic
D002457:Cell Extracts; D012314:RNA Cap Analogs; C017516:7-methylguanosine triphosphate; D006160:Guanosine Triphosphate; D003513:Cycloheximide; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D012321:DNA-Directed RNA Polymerases