Cloning and expression of the vesicular stomatitis virus phosphoprotein gene in Escherichia coli: analysis of phosphorylation status versus transcriptional activity

The phosphoprotein (P, previously known as NS) genes of vesicular stomatitis virus serotypes New Jersey and Indiana have been cloned in the Escherichia coli expression vector pET-3a. Transcription of P genes in these clones initiated from a phage T7 RNA polymerase promoter, whereas translation was driven by the Shine-Dalgarno sequence and the initiator AUG codon of the T7 gene 10 message. The clones were introduced into an appropriate E. coli strain in which T7 RNA polymerase was expressed under the control of the lac promoter. Under optimal conditions of induction with isopropylthiogalactopyranoside, P protein made in these bacterial strains constituted 5 to 20 % of total cellular protein. P protein expressed in bacteria was unphosphorylated and transcriptionally active in an in vitro reconstitution assay with viral L protein and an N-RNA template. However, the P protein was phosphorylated in vitro by the kinase activities associated with L and the N-RNA template. The virion-associated RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV), a prototype rhabdovirus, is composed of a large polypeptide (L) and a phosphoprotein (P). Within the virion, these two proteins are associated with the negative-strand (anti-message sense) genomic RN