Concomitant methylation and synthesis in vitro of Semliki Forest virus (SFV) ss RNAs by a fraction from infected cells.

Abstract

Abstract The 42 S and 26 S ss SFV RNAs synthesized in vitro by the SFV RNA polymerase were also methylated in vitro by an enzyme in the mitochondrial pellet fraction, P-15, which had the activity of a guanine-7-methyltransferase. The 22 S ds RF RNA was not methylated in vitro . The methyl - 3 H radioactivity incorporated in vitro from labeled S -adenosyl- l -methionine was at the 5′-terminus of each of the ss RNAs, and none was detectable at an internal site. The 3 H-labeled cap containing oligonucleotide from the RNase T1 digest of 42 S RNA had a charge of about −6 consistent with its known m 7 GpppApUpGp sequence and that from the 26 S RNA had a charge of about −7 consistent with its known terminus of m 7 GpppApUpUpGp. The in vitro -labeled cap of both ss RNAs was m 7 GpppA. The rates of SFV RNA synthesis and methylation in vitro were linear for at least 40 min. In vitro methylation of the SFV ss RNAs absolutely depended on assay conditions which supported synthesis of ss RNAs. In contrast, transcription of 42 S and 26 S SFV RNAs in vitro was not dependent on methylation. The methylating enzyme in the P-15 fractions distributed during equilibrium centrifugation with membranous fractions of different densities in a manner similar to the distribution of the SFV RNA polymerase. It has not yet been established if this methylating activity was catalyzed by a virus-specific protein or a host protein.