Mutagenesis of the sindbis virus nsP1 protein: Effects on methyltransferase activity and viral infectivity

It has been suggested that four amino acids which are absolutely conserved in the nsP1 nonstructural proteins encoded by togaviruses and in the homologous proteins encoded by plant viruses in the Sindbis virus (SV) superfamiiy may constitute a ''methyltransferase motif.'' In the Sindbis virus nsP1 protein (540 amino acids) these four amino acids are represented by His(39), Arg(91), Asp(94), and Tyr(249). Earlier, in assays of methyltransferase (MTase) activity generated in SV-iniected cells, we had shown that amino acid changes at positions B7 and 88 of SV nsP1 resulted in a 10-fold lower K-m for S-adenosyl methionine, the methyl donor in MTase reactions. Using site-directed mutagenesis we now report the expression of nsP1 in Escherichia coli, and in the infectious clone of Sindbis virus, Toto/1101, in which His(39), Arg(91), Asp(94), and Tyr(249) were changed one at a time to Ala. We also expressed nsP1 with C-terminal deletions of varying size, as well as with internal deletions in the C-terminal portion of the protein, in E. coli. Changing His(39), Arg(91), Asp(94), or Tyr(249) to Ala led to a loss of both MTase activity and viral infectivity; however, changing Ile(389) to Vat, a conservative change in the carboxy-terminal half of nsP1, had no effect on either MTase activity or viral infectivity. With respect to the deleted forms of nsP1, a carboxy-terminal deletion of 48 amino acids was still compatible with MTase activity in vitro. However, larger deletions including those in which the amino acids between positions 442 and 492 were deleted abolished MTase activity. (C) 1995 Academic Press, Inc.