SVLM21, a Sindbis virus mutant resistant to methionine deprivation, encodes an altered methyltransferase.
The replication of Sindbis virus (SVSTD) in cultured Aedes albopictus mosquito cells is sensitive to methionine deprivation. We have suggested from earlier work that this sensitivity is primarily because of a decreased pool of S-adenosyl methionine (ado met) and the resultant failure to methylate the 5' cap of the viral mRNAs. SVLM21, a strain of Sindbis virus derived in our laboratory from SVSTD by serial passage on mosquito cells maintained after infection in low concentrations of methionine, is resistant to methionine starvation. It was proposed that this adaptation to low methionine, and to the resultant low intracellular levels of ado met, reflected the accumulation of mutations which led to the generation of a viral RNA cap methyltransferase with an increased affinity for ado met. We report here kinetic data which distinguished the enzymes coded for by SVSTD and SVLM21. Using guanylylimidodiphosphate (GIDP) as the methyl acceptor, radioactively labeled ado met as the methyl donor, and lysates from infected BHK cells as the enzyme source, we calculated from our results that SVLM21 generated a methyltransferase with a Km for ado met 10-fold lower than that generated by either SVSTD or the related alphavirus, Semliki Forest virus. In addition, we found that BHK cells infected with SVLM21 generated higher levels of methyltransferase activity than did cells infected with SVSTD and that the SVSTD and SVLM21 enzymes differed with respect to their relative activities at elevated temperatures. We conclude from these results that the SVLM21 phenotype is associated with an altered methyltransferase and suggest that this is the basis of the resistance of SVLM21 to methionine deprivation.
Scheidel
L M
LM
Department of Molecular Genetics and Microbiology, University of Medicine and Dentistry of New Jersey, Robert Wood Johnson Medical School, Piscataway 08854-5635.
Durbin
R K
RK
Stollar
V
V
D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002855:Chromatography, Thin Layer; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000378:metabolism*; D008745:Methylation; D009154:Mutation; D012436:S-Adenosylmethionine / Q000378:metabolism; D012845:Sindbis Virus / Q000201:enzymology / Q000235:genetics* / Q000502:physiology; D013696:Temperature; D014779:Virus Replication; D012359:tRNA Methyltransferases / Q000235:genetics* / Q000378:metabolism
D006165:Guanylyl Imidodiphosphate; D012436:S-Adenosylmethionine; D008715:Methionine; D012359:tRNA Methyltransferases