cdc2 Cyclin-Dependent Kinase Binds and Phosphorylates Herpes Simplex Virus 1 U _L 42 DNA Synthesis Processivity Factor

Abstract

Earlier studies have shown that cdc2 kinase is activated during herpes simplex virus 1 infection and that its activity is enhanced late in infection even though the levels of cyclin A and B are decreased below levels of detection. Furthermore, activation of cdc2 requires the presence of infected cell protein no. 22 and the U L 13 protein kinase, the same gene products required for optimal expression of a subset of late genes exemplified by U S 11, U L 38, and U L 41. The possibility that the activation of cdc2 and expression of this subset may be connected emerged from the observation that dominant negative cdc2 specifically blocked the expression of U S 11 protein in cells infected and expressing dominant negative cdc2. Here we report that in the course of searching for a putative cognate partner for cdc2 that may have replaced cyclins A and B, we noted that the DNA polymerase processivity factor encoded by the U L 42 gene contains a degenerate cyclin box and has been reported to be structurally related to proliferating cell nuclear antigen, which also binds cdk2. Consistent with this finding, we report that (i) U L 42 is able to physically interact with cdc2 at both the amino-terminal and carboxyl-terminal domains, (ii) the carboxyl-terminal domain of U L 42 can be phosphorylated by cdc2, (iii) immunoprecipitates obtained with anti U L 42 antibody contained a roscovitine-sensitive kinase activity, (iv) kinase activity associated with U L 42 could be immunodepleted by antibody to cdc2, and (v) U L 42 transfected into cells associates with a nocodazole-enhanced kinase. We conclude that U L 42 can associate with cdc2 and that the kinase activity has the characteristic traits of cdc2 kinase.In this report we show that the mitotic cyclin-dependent kinase interacts with the herpes simplex virus 1 (HSV-1) protein product of the U L 42 open reading frame. The viral protein functions as a DNA polymerase-associated processivity factor. We have identified this protein as a potential virally encoded partner for cdc2 on the basis of its cyclin-like characteristics, and in this report we show that it interacts physically with cdc2. Relevant to this report are the following.(i) The roots of this investigation rest on the observation that infected-cell protein 0 (ICP0), a promiscuous transactivator encoded by the ␣0 gene, binds to and stabilizes cyclin D3 (13, 31). In the ensuing investigation it became apparent that the stabilization of cyclin D3 was not associated with the transition from G 1 to S phase of the cell cycle. Specifically, cdk2 was inactive and members of the E2F family of proteins required for transcriptional activation of the S phase genes were either sequestered in the cytoplasm or rendered inactive (2,8,23,32). It also became apparent that while HSV stabilized cyclin D3, at least two other herpesviruses, herpessaimiri virus and human herpesvirus 8, encoded cyclin D homologs (17, 21). The obvious conclusion was that herpesviruses require D cyclins although, at least in the case of HSV, for other purposes tha...

Roscovitine Inhibits Activation of Promoters in Herpes Simplex Virus Type 1 Genomes Independently of Promoter-Specific Factors

“…Rosco is highly selective in that among 68 proteins tested, it inhibited only CDK1, CDK2, CDK5, and CDK7 with high potency and DYRK1a, ERK1 and ERK2 with lower potency (recently reviewed in reference 70). Based on independent evidence, two of the Rosco-sensitive CDKs, CDK1 and CDK2, have been postulated to be required for HSV-1 transcription (2,3,71,73), both these CDKs are also inhibited by IFN-␣. Since the human genome encodes 518 potential protein kinases (47) and numerous other ATP-binding proteins, however, it is impossible to conclude from the experiments described in this paper which specific kinases are required for activation of HSV-1 transcription and inhibited by Rosco.…”

Ionizing Radiation Activates Late Herpes Simplex Virus 1 Promoters via the p38 Pathway in Tumors Treated with Oncolytic Viruses

“…In recent studies, it has been shown that the expression of glycoprotein C and U S 11 is enhanced by the ICP22-mediated interaction of the viral DNA processivity factor U L 42 (a viral functional homologue of proliferating cell nuclear antigen), cyclin-dependent kinase cdc2, and topoisomerase IIa (34)(35)(36). One hypothesis that could explain the mechanism of activation of late promoters and the role of ionizing radiation in inducing these promoters is that these are activated by modifications in the structure of the DNA, either as a result of nicks, gaps, or other perturbations in the DNA structure.…”

Herpes simplex virus 1 activates cdc2 to recruit topoisomerase IIα for post-DNA synthesis expression of late genes

“…The construction of a U L 42-expressing plasmid in the multiple coding site of pcDNA3.1(ϩ) has been described elsewhere (7). A plasmid encoding HA-tagged dn cdc2 (cdc2-dn-HA) kinase was provided by S. van den Heuvel (Massachusetts General Hospital Cancer Center, Charlestown, MA) (22).…”

“…Immunoprecipitation with antibody to U L 42 was done as described (7). The electrophoretically separated proteins in the precipitate were reacted with antibodies to topoisomerase II␣ and U L 42.…”