Optimal replication activity of vesicular stomatitis virus RNA polymerase requires phosphorylation of a residue(s) at carboxy-terminal domain II of its accessory subunit, phosphoprotein P

The phosphoprotein, P, of vesicular stomatitis virus (VSV) is a hey subunit of the viral RNA-dependent RNA polymerase complex. The protein is phosphor ylated at multiple sites in two different domains. We recently showed that specific serine and threonine residues within the amino-terminal acidic dom ain I of P protein must be phosphorylated for in vivo transcription activit y, but not for replication activity, of the polymerase complex. To examine the role of phosphorylation of the carboxy-terminal domain II residues of t he P protein in transcription and replication, we have used a panel of muta nt P proteins in which the phosphate acceptor sites (Ser-226, Ser-227, and Ser-233) were altered to alanines either individually or in various combina tions. Analyses of the mutant proteins for their ability to support replica tion of a VSV minigenomic RNA suggest that phosphorylation of either Ser-22 6 or Ser-227 is necessary for optimal replication activity of the protein. The mutant protein (P-226/227) in which both of these residues were altered to alanines was only about 8% active in replication compared to the wild-t ype (wt) protein. Substitution of alanine for Ser-233 did not have any adve rse effect on replication activity of the protein. In contrast, all the mut ant proteins showed activities similar to that of the wt protein in transcr iption. These results indicate that phosphorylation of the carboxy-terminal domain II, residues of P protein are required for optimal replication acti vity but not for transcription activity. Furthermore, substitution of gluta mic acid residues for Ser-226 and Ser-227 resulted in a protein that was on ly 14% active in replication but almost fully active in transcription. Take n together, these results, along with our earlier studies, suggest that pho sphorylation of residues at two different domains in the P protein regulate s its activity in transcription and replication of the VSV genome.