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Published November 1990 | public
Journal Article

Phosphorylation of sindbis virus nsP3 in vivo and in vitro


nsP3 is one of four viral nonstructural proteins required for RNA replication of Sindbis virus. In this report, post-translational modifications of nsP3 which occur in both vertebrate and mosquito cell cultures have been examined. In pulse-chase experiments, analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nsP3 was initially observed as a single species (termed nsP3a, ∼ 76 kDa) which was gradually converted to slower mobility forms ranging from 78 kDa (termed nsP3b) to 106 kDa (termed nsP3c). The slower mobility forms, but not nsP3a or the other nonstructural proteins, could be labeled in vivo with [^(34)P]orthophosphate. Treatment of nsP3 immunoprecipitates with calf intestinal alkaline phosphatase converted the slower mobility forms to nsP3a. Phosphoamino acid analysis of nsP3b and nsP3c demonstrated that both contained phosphoserine and phosphothreonine but not phosphotyrosine. nsP34, a polyprotein produced by readthrough of the in-frame opal codon preceding nsP4, was also phosphorylated on serine and threonine residues. nsP3 phosphorylation did not require ongoing RNA synthesis since phosphorylated forms were also observed in the absence of Sindbis-specific RNA synthesis. Furthermore, when immunoprecipitates of nsP3 were incubated with [γ-^(32)P]ATP in the presence of Mg^(2+) or Mn^(2+), a kinase activity which was able to phosphorylate nsP3 on serine and threonine residues in vitro was detected. This kinase activity was inhibited by heparin, was activated by spermidine, and could utilize GTP and ATP as the phosphate donor. These latter properties are similar to those of cellular casein kinase II. Although it is possible that this nsP3-associated kinase is of cellular origin, autophosphorylation of nsP3 has not been excluded.

Additional Information

© 1990 Academic Press, Inc. Received May 29, 1990; accepted July 19, 1990. We thank Dr. J. R. McDonald for expert advice on phosphoamino acid analysis and Drs. H. Huang, S. Schlesinger, M. Schlesinger, and members of our laboratories for helpful suggestions on the manuscript. This work was supported by Public Health Service Grants AI 24134, AI 20612, and AI 10793 from the National Institutes of Health and by a grant from the Pew Memorial Trust. C.M.R. is a Pew Scholar in the Biomedical Sciences.

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