Identification of sites phosphorylated by the vaccinia virus B1R kinase in viral protein H5R

Vaccinia virus protein H5R was phosphorylated by the B1R protein kinase in vitro, digested with V8 protease, and phosphopeptides separated by HPLC. The N-terminal sequence of one radioactively labelled phosphopeptide was determined and found to correspond to residues 81-87 of the protein, with Thr-84 and Thr-85 being phosphorylated. A synthetic peptide based on this region of the protein was shown to be a substrate for the B1R protein kinase, and the extent of phosphorylation was substantially decreased if either Thr residue was replaced by an Ala.We have identified the first phosphorylation site for the vaccinia virus B1R protein kinase. This gives important information about the substrate-specificity of the enzyme, which differs from that of other known protein kinases. It remains to be seen whether the same site is phosphorylated in vivo.Vaccinia virus is a large DNA virus that replicates in the host cell cytoplasm in granular sites called virosomes [1]. It encodes at least two protein kinases belonging to the cellular family of serine/threonine protein kinases, the products of the B1R [2,3] and F10L genes [4]. The F10L kinase is encapsidated in the virion and plays an essential role in virion morphogenesis [5,6]. The B1R protein kinase is expressed early in infection, is found in the virosomes, and is also packaged into virions [7]. It appears to be an essential viral protein, and temperature-sensitive mutations that map to the B1R gene produce virus that cannot replicate its DNA at the restrictive temperature [2,8]. The B1R kinase does not appear to have a broad substrate specificity, and, although it has some activity against the acidic protein, casein, this is a poor substrate compared with the enzyme's known physiological substrates. Three proteins which become phosphorylated during infection of cells with vaccinia virus have been shown to be substrates of the B1R protein kinase in vitro. Two of these are the ribosomal proteins Sa and S2 [9,10] and the third