Cooperative binding of multimeric phosphoprotein (P) of vesicular stomatitis virus to polymerase (L) and template: pathways of assembly.
It was previously shown that the phosphoprotein (P) of vesicular stomatitis virus must undergo phosphorylation-dependent multimerization to become transcriptionally active. Phosphorylation at S-60 and/or T-62 by casein kinase II or substitution of these residues by D is required for multimer formation. We now find that substitution of either one of these residues by A prevents phosphorylation by casein kinase II and multimer formation. The binding of multimeric P to the other two transcriptional components of vesicular stomatitis virus (L protein and the N-RNA template) has been characterized by using P immobilized on beads through its poly(His) tag to facilitate recovery of bound complexes. Multimerization of P was absolutely required for binding to both L and template. Multimeric P combined with the polymerase enzyme (L) in a stoichiometric 1:1 complex, which bound to the N-RNA template much more strongly than multimeric P alone. Substitution of S-227 and S-233 by A residues had no effect on multimerization or binding of L to P but prevented binding of both P and L to template and abolished transcriptional activity. In contrast, substitution of these residues with D residues had no effect on template binding or activity. However, substitution at these sites by either D or A largely abolished phosphorylation by L-associated kinases, thus identifying S-227 and S-233 as the major sites targeted by these kinases and confirming that phosphorylation of P protein by L-associated kinases is without transcriptional effect.
D000818:Animals; D002460:Cell Line; D006224:Cricetinae; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007668:Kidney; D007700:Kinetics; D016296:Mutagenesis; D010750:Phosphoproteins; D010766:Phosphorylation; D011485:Protein Binding; D012367:RNA, Viral / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012324:RNA-Dependent RNA Polymerase; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D016324:Sequence Tagged Sites; D013698:Templates, Genetic; D014158:Transcription, Genetic; D014721:Vesicular stomatitis Indiana virus / Q000378:metabolism*; D014764:Viral Proteins / Q000302:isolation & purification / Q000378:metabolism*; D015678:Viral Structural Proteins / Q000302:isolation & purification / Q000378:metabolism*
C081250:P protein, Vesicular stomatitis virus; D010750:Phosphoproteins; D012367:RNA, Viral; D011994:Recombinant Proteins; D014764:Viral Proteins; D015678:Viral Structural Proteins; C040530:L protein, vesicular stomatitis virus; D012324:RNA-Dependent RNA Polymerase; D012321:DNA-Directed RNA Polymerases