PHOSPHORYLATED STATES OF VESICULAR STOMATITIS-VIRUS P-PROTEIN IN-VITRO AND IN-VIVO

We have previously shown that the phosphoprotein (P) of vesicular stom atitis virus (VSV), New Jersey serotype (P-NJ) is phosphorylated by ca sein kinase II, within the N-terminal domain I (P1 form), whereas the C-terminal domain II is phosphorylated by a protein kinase activity as sociated with the L protein (P2 form) (D. J. Chattopadhyay and A. K. B anerjee, Cell 49, 407, 1987; A. M. Takacs et al., J. Virol. 66, 5842, 1992). In the present studies, we have mapped the corresponding P1 and P2 phosphorylation sites in the P protein of the well-studied Indiana serotype (PIIND) and compared that with the two previously designated NS1 and NS2 forms present in vivo. The P-IND expressed in Escherichia coli in an unphosphorylated form (PO) was used as substrate for recom binant casein kinase II (CKII). By site-directed mutagenesis, the CKII -mediated phosphorylation sites in the P protein were mapped at S60, T 62, and S64 within the acidic domain I in vitro. In contrast, using BH K cell extract as the source of CKII or expressing P protein in COS ce lls labeled with (32)Pi, th, phosphorylation sites were mapped at S60 and S64 with no phosphorylation at T62 residue, We used a peptide mapp ing technique by which the phosphorylation sites within domain I and d omain II were determined. Using this method we demonstrated that the P 1 and P2 forms are similar, if not identical, to the previously design ated NS1 and NS2 forms, respectively. The domain II phosphorylating ki nase activity, associated with the L protein, is shown to be present a lso in the N-RNA complex, indicating that this activity is of cellular origin. By site-directed mutagenesis, we have shown that S226 and S22 7 are involved in phosphorylation within domain II. We also demonstrat e that the P1 and P2 forms are interconvertible and arise by phosphory lation/dephosphorylation of the phosphate groups in domain II, confirm ing the precursor-product relationship between the two phosphorylated forms of P protein. (C) 1997 Academic Press.