We reported previously that the posttranslational processing associated with phosphorylation of the herpes simplex virus 1 infected-cell protein 22 (ICP22), a regulatory protein, is encoded by UL13, a gene encoding a structural protein of the virion. We now report the following. (i) In cells infected with a mutant lacking UL13 (delta UL13), restricted infected cells accumulate reduced levels of the regulatory protein ICP0 and several late viral proteins. Identical reductions have been observed in the same cell lines infected with a mutant from which the alpha 22 gene, encoding ICP22, had been deleted (delta alpha 22). We conclude that the UL13-mediated processing of ICP22 is essential for its gene-regulatory function. (ii) The reduced accumulations of specific viral protein in cells infected with either delta UL13 or delta alpha 22 viruses correlate with reduced levels of specific mRNAs for both ICP0 and the affected late genes. (iii) ICP22 is not modified by the UL13 protein introduced into cells during infection. (iv) ICP22 is also modified by the protein kinase encoded by US3, but this modification is different from that of the UL13 protein kinase. These results predict that UL13 encodes a protein kinase or phosphotransferase which is expressed late in the replicative life cycle and which directly or indirectly phosphorylates ICP22. This modification is essential for stabilization or increased transcription of a specific subset of viral RNAs and, ultimately, for the accumulation of corresponding viral proteins.

The herpes simplex virus 1 genome was shown to encode two genes, US3 and UL13, exhibiting amino acid sequence motifs common to protein kinases. Elsewhere this laboratory reported that the prominent substrate of the US3 protein kinase is the product of the UL34 gene, an essential nonglycosylated membrane protein. In the absence of the US3 kinase, the UL34 protein remains unphosphorylated but forms a complex with four proteins that become phosphorylated uniquely when UL34 is not. To investigate the role of UL13 protein in this process, recombinant viruses lacking UL13 or both UL13 and US3 were constructed. We report that UL13 is dispensable for viral replication in cell culture and is not involved in the processing of UL34 or of associated phosphoproteins. UL13 is, however, responsible for the posttranslational processing associated with phosphorylation of infected-cell protein 22, the product of the alpha 22 gene. This gene was previously reported to play a regulatory role in selected cell lines. UL13 appears to be either a protein kinase or a phosphotransferase and its major substrate is the alpha 22 protein.

Previous studies (F. C. Purves, D. Spector, and B. Roizman, J. Virol. 65:5757-5764, 1991) have shown that the protein kinase encoded by the U(S)3 gene mediates posttranslational modification of a viral phosphoprotein with an apparent M(r) of 30,000 encoded by the UL34 gene. Here we report the following. (i) UL34 protein is not phosphorylated in cells infected with recombinant viruses deleted in the U(S)3 gene. (ii) Several new phosphoproteins (apparent M(r)s, 25,000 to 35,000) are present in cells infected with recombinant viruses deleted in the U(S)3 gene or with viruses carrying a mutation in the UL34 gene that precluded phosphorylation of the UL34 gene product by the U(S)3 protein kinase, but not in cells infected under conditions which permit phosphorylation of the UL34 protein. These proteins are genetically unrelated to the product of the UL34 gene. (iii) Polyclonal rabbit anti-UL34 protein serum precipitated not only the UL34 protein but also the other (25,000- to 35,000-M(r)) phosphoproteins from lysates of cells infected with U(S)3- virus. (iv) The UL34 gene product is a membrane protein inasmuch as the polyclonal anti-UL34 serum reacted with surfaces of intact, unfixed, infected cells and the antigen-antibody complex formed in this reaction contained the UL34 protein. (v) Small amounts of the UL34 protein were present in virions of infected cells. We conclude that the UL34 gene product is a membrane protein exclusively phosphorylated by the U(S)3 protein kinase which can either directly or indirectly form complexes with several other phosphoproteins. Experiments done thus far suggest that these phosphoproteins are present only under conditions in which the UL34 protein is not phosphorylated.

Earlier studies have shown that a herpes simplex virus 1 (HSV-1) open reading frame, US3, encodes a novel protein kinase and have characterized the cognate amino acid sequence which is phosphorylated by this enzyme. This report identifies an apparently essential viral phosphoprotein whose posttranslational processing involves the viral protein kinase. Analyses of viral proteins phosphorylated in the course of productive infection revealed a phosphoprotein whose mobility was viral protein kinase and serotype dependent. Thus, the corresponding HSV-1 and HSV-2 phosphoproteins differ in their electrophoretic mobilities, and the phosphoprotein specified by the HSV-1 mutant deleted in US3 (R7041) differs from that of the corresponding HSV-1 and HSV-2 proteins. Analyses of HSV-1 x HSV-2 recombinants mapped the phosphoprotein between 0.42 and 0.47 map units on the prototype HSV-1 DNA map. Within this region, the UL34 open reading frame was predicted to encode a protein of appropriate molecular weight which would also contain the consensus target site for phosphorylation by the viral protein kinase as previously defined with synthetic peptides. Replacement of the native UL34 gene with a UL34 gene tagged with a 17-amino-acid epitope from the alpha 4 protein identified this gene as encoding the phosphoprotein. Finally, mutagenesis of the predicted phosphorylation site on UL34 in the viral genome, and specifically the substitution of threonine or serine with alanine in the product of the UL34 gene, yielded phosphoproteins whose electrophoretic mobilities could not be differentiated from that of the US3- mutant. We conclude that the posttranslational processing of the UL34 gene product to its wild-type phenotype requires the participation of the viral protein kinase. While the viral protein kinase is not essential for viral replication in cells in culture, the UL34 gene product itself may not be dispensable.

A protein kinase discovered in cells infected with α-herpesviruses has recently been shown to be the product of an evolutionary conserved viral gene. We argue that, oncogenic retroviruses notwithstanding, this is the first authentic eukaryotic viral protein kinase; and consider whether this implies a novel function for protein phosphorylation.

Previous work has shown that a novel protein kinase is induced after infection of cultured cells with herpes simplex virus type 1 (HSV-1). Separately, it has been reported that the protein encoded by HSV-1 gene US3 shows similarity in its amino acid sequence to members of the protein kinase family of eukaryotes. We have investigated the possibility that these two observations are connected by preparing an antiserum to a synthetic oligopeptide corresponding to the carboxy-terminal eight amino acids of the US3 protein. This antiserum reacted on immunoblots with a polypeptide of apparent molecular weight 68,000 from extracts of cells which had been infected with HSV-1. The antiserum also reacted strongly with a 68,000 molecular weight species from a preparation of the novel HSV-1 protein kinase which had been extensively purified and resolved from other protein kinases. In addition, the purified preparation phosphorylated a protein species, also of 68,000 apparent molecular weight, when incubated with [gamma-32P]ATP. These data are consistent with gene US3 encoding the novel protein kinase induced after infection of cells with HSV-1.

Earlier reports have described a novel protein kinase in cells infected with herpes simplex or pseudorabies viruses. These novel enzymes were characterized by their acceptance of protamine as a substrate and by their differential chromatographic behavior in anion-exchange chromatography. We report that this activity was not present in extracts of uninfected cells or of cells infected with a mutant constructed so as to contain a deletion in the US3 open reading frame mapping in the small component of herpes simplex virus 1 DNA. The activity was present in extracts of cells infected with wild-type virus and with a recombinant in which the US3 open reading frame had been rescued. Our results are consistent with the observation reported earlier that the coding sequences predict an amino acid motif common to protein kinases and lead to the conclusion that the US3 open reading frame encodes a virus-specific protein kinase that is not required for virus growth in cells in culture.

The recently described pseudorabies virus protein kinase has been purified from infected hamster fibroblasts by a combination of anion-exchange, hydrophobic-interaction and affinity chromatography. The purification resulted in enzyme with a specific activity in excess of 1,000 nmol phosphate mg-1 min-1 in relatively high yield. Gel electrophoresis of the purified enzyme under denaturing conditions revealed a single stained band at a position of migration corresponding to a Mr 38,000. Incubation of the purified enzyme with [gamma-32P]ATP in the absence of added substrate resulted in incorporation of 32P into this protein band, consistent with the 38-kDa protein being a protein kinase with a capacity for autophosphorylation. The phosphorylated form of the protein has an isoelectric point of approximately 4.9. Gel permeation chromatography of the purified enzyme indicated a native Mr 70,000, suggesting that the protein kinase has a homodimeric structure.

Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with α-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simples virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different.

The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gel-permeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this.

The soluble proteins released by hypotonie lysis of highly purified bovine adrenal chromaffin granules were analysed by one- and two-dimensional electrophoresis, followed by transfer to nitrocellulose and decoration with lectins or specific antibodies. The effects of neuraminidase treatment, and of chemical deglycosylation by trifluoromethanesulphonic acid, were investigated. It was shown that lectins could be used to distinguish the two major series of chromogranins from each other, from dopamine β-hydroxylase and from several minor, unidentified glycoprotein components of the lysate. Antibody decoration revealed a complex series of peptides containing enkephalin sequences, some of which changed their electrophoretic mobility on treatment with trifluoromethanesulphonic acid.