ROLE OF CELLULAR CASEIN KINASE-II IN THE FUNCTION OF THE PHOSPHOPROTEIN-(P) SUBUNIT OF RNA-POLYMERASE OF VESICULAR STOMATITIS-VIRUS

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in vira l transcription, Recent in vitro studies have demonstrated that CKII c onverts the inactive unphosphorylated form of P (P0) to an active phos phorylated form P1, after phosphorylation at two serine residues, Ser- 59 and Ser-61. To gain insight into the role of CKII-mediated phosphor ylation in the structure and function of the P protein, we have carrie d out circular dichroism (CD) and biochemical analyses of both PO and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a sec ondary structure similar to that of P0, P1, at a protein concentration of 50 mu g/ml, elutes from a gel filtration column apparently as a di mer, whereas both P0 and the double serine mutant elute as a monomer a t the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine r equired a high concentration of CKII for optimal phosphorylation. We d emonstrate here that phosphorylation of either Ser-59 or Ser-61 is nec essary and sufficient to transactivate L polymerase although alteratio n of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficient ly than P0 and the formation of P1 is a prerequisite for the subsequen t phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcri ption reconstitution assay in the presence of wild-type P protein.