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J. Virol., 12 1995, 7718-7723, Vol 69, No. 12
Copyright © 1995, American Society for Microbiology

Cooperative binding of multimeric phosphoprotein (P) of vesicular stomatitis virus to polymerase (L) and template: pathways of assembly

Y Gao and J Lenard
Department of Physiology and Biophysics, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway 08854-5635, USA.

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.

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