Tyrosine phosphorylation of A17 during vaccinia virus infection: Involvement of the H1 phosphatase and the F10 kinase

Vaccinia virus encodes two protein kinases (B1 and F10) and a dual-specific ity phosphatase (VH1), suggesting that phosphorylation and dephosphorylatio n of substrates on serine/threonine and tyrosine residues are important in regulating diverse aspects of the viral life cycle. Using a recombinant in which expression of the H1 phosphatase can be regulated experimentally (vin dH1), we have previously demonstrated that repression of H1 leads to the ma turation of noninfectious virions that contain several hyperphosphorylated substrates (K. Liu et al., J. Virol. 69:7823-7834). In this report, we demo nstrate that among these is a 25-kDa protein that is phosphorylated on tyro sine residues in H1-deficient virions and can be dephosphorylated by recomb inant H1. We demonstrate that the 25-kDa phosphoprotein represents the prod uct of the A17 gene and that A17 is phosphorylated on serine, threonine, an d tyrosine residues during infection. Detection of phosphotyrosine within A 17 is abrogated when Tyr(203) (but not Tyr(3), Tyr(6), or Tyr(7)) is mutate d to phenylalanine, suggesting strongly that this amino acid is the site of tyrosine phosphorylation. Phosphorylation of A17 fails to occur during non permissive infections performed with temperature-sensitive mutants defectiv e in the F10 kinase. Our data suggest that this enzyme, which was initially characterized as a serine/threonine kinase, might in fact have dual specif icity. This hypothesis is strengthened by the observation that Escherichia call induced to express F10 contain multiple proteins which are recognized by antiphosphotyrosine antiserum. This study presents the first evidence fo r phosphotyrosine signaling during vaccinia virus infection and implicates the F10 kinase and the H1 phosphatase as the dual-specificity enzymes that direct this cycle of reversible phosphorylation.