Expression of Sindbis virus nsP1 and methyltransferase activity in Escherichia coli.
We have constructed two plasmids, pSR5-42 and pSR5-Toto, which under lac control expressed the SVLM21 and the SVToto forms, respectively, of the Sindbis virus nonstructural protein, nsP1. The induced protein, which was the major protein made following induction with IPTG, had an apparent molecular weight of 60,000 and an amino terminal sequence in agreement with that expected for nsP1. Following induction with IPTG, cells carrying pSR5-42 (which contains the SVLM21 gene sequence) generated much higher RNA methyltransferase activity than cells carrying pSR5-Toto (which contains the SVToto gene sequence). This result is in agreement with what is observed when methyltransferase is measured in cells infected with SVLM21 and SVSTD (or SVToto), respectively. These results provide strong evidence that nsP1 has methyltransferase activity in the absence of any other viral nonstructural proteins.
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Department of Molecular Genetics and Microbiology, UMDNJ-Robert Wood Johnson Medical School, Piscataway, New Jersey 08854-5635.
Stollar
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D002213:Capsid / Q000096:biosynthesis / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D015870:Gene Expression; D005814:Genes, Viral; D007700:Kinetics; D008970:Molecular Weight; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D012845:Sindbis Virus / Q000201:enzymology / Q000235:genetics*; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*; D017361:Viral Nonstructural Proteins; D012359:tRNA Methyltransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification
D011994:Recombinant Proteins; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins; D012359:tRNA Methyltransferases