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 stoma titis virus must undergo phosphorylation-dependent multimerization to become transcriptionally active. Phosphorylation at S-60 and/or T-62 b y 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 trans criptional components of vesicular stomatitis virus (L protein and the N-RNA template) has been characterized by using P immobilized on bead s through its poly(His) tag to facilitate recovery of bound complexes. Multimerization of P was absolutely required for binding to both L an d template, Multimeric P combined with the polymerase enzyme (L) in a stoichiometric 1:1 complex, which bound to the N-RNA template much mor e 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 transcript ional activity, In contrast, substitution of these residues with D res idues had no effect on template binding or activity. However, substitu tion at these sites by either D or A largely abolished phosphorylation by L-associated kinases, thus identifying S-227 and S-233 as the majo r sites targeted by these kinases and confirming that phosphorylation of P protein by L-associated kinases is without transcriptional effect .