The role of cdc2 in the expression of herpes simplex virus genes

Abstract

Earlier reports have shown that cdc2 kinase is activated in cells infected with herpes simplex virus 1 and that the activation is mediated principally by two viral proteins, the infected cell protein 22 (ICP22) and the protein kinase encoded by U(L)13. The same proteins are required for optimal expression of a subset of late (gamma(2)) genes exemplified by U(S)11. In this study, we used a dominant-negative cdc2 protein to determine the role of cdc2 in viral gene expression. We report the following. (i) The cdc2 dominant-negative protein had no effect in the synthesis and accumulation of at least two alpha-regulatory proteins (ICP4 and ICP0), two beta-proteins (ribonucleotide reductase major subunit and single-stranded DNA-binding protein), and two gamma(1)-proteins (glycoprotein D and viral protease). U(S)11, a gamma(2)-protein, accumulated only in cells in which cdc2 dominant-negative protein could not be detected or was made in very small amounts. (ii) The sequence of amino acids predicted to be phosphorylated by cdc2 is present in at least 27 viral proteins inclusive of the regulatory proteins ICP4, ICP0, and ICP22. In in vitro assays, we demonstrated that cdc2 specifically phosphorylated a polypeptide consisting of the second exon of ICP0 but not a polypeptide containing the sequence of the third exon as would be predicted from the sequence analysis. We conclude that cdc2 is required for optimal expression of a subset of gamma(2)-proteins whose expression is also regulated by the viral proteins (ICP22 and U(L)13) that mediate the activation of cdc2 kinase.

Figure 1

(A) Immunoblot of electrophoretically separated lysates of HeLa cells reacted with antibody to HA tag. HeLa cells were mock transfected or transfected with cytomegalovirus expression plasmids carrying HA-tagged dominant-negative cdc2 (pCMVcdc2-dn). The cells were harvested 36 h after transfection, solubilized, electrophoretically separated on 10% bisacrylamide gels, and probed with antibody to HA. (B) Autoradiographic image of histone H1 phosphorylated in vitro by immunoprecipitated cdc2. HeLa cells were infected with HSV-1(F) 36 h after transfection with pCMVcdc2-dn and incubated for an additional 14 h at 37°C. Histone H1 was mixed with immune-precipitated Cdc2 from harvested, lysed cells. The reaction mixtures were separated on denaturing gels as described in Materials and Methods.

Figure 2

Accumulation of viral proteins belonging to different kinetic classes in cells expressing cdc2-dn protein. HEp-2 cells were infected with HSV-1(F) 36 h after transfection with plasmid pCMVcdc2-dn. The cells were fixed 12 h after infection and reacted with anti-HA tag (FITC) or an antibody against a viral protein (Texas red). The slides were examined in a Zeiss confocal microscope, and images were captured with software provided by the manufacturer. (A) Monoclonal antibody against ICP4. (B) Monoclonal antibody against ICP0. (C) Monoclonal antibody against ICP6, the major subunit of ribonucleotide reductase. (D) Monoclonal antibody against glycoprotein D. (E) Monoclonal antibody against U_S11 protein. (F) Monoclonal antibody against U_S11 protein. In this image, the signal for green fluorescence was amplified to make apparent a very weak signal for cdc2-dn protein in the cells identified by the arrow and showing the presence of U_S11 protein.

Figure 3

(A) Coomassie blue staining of GST fusion proteins. GST alone, GST-ICP0 20–241, GST-ICP0 543–768, and U_L34 1–240 were purified with the aid of glutathione-agarose beads, electrophoretically separated on 10% bisacrylamide gels, and stained with Coomassie blue. (B) Autoradiographic image of cdc2-mediated phosphorylation of histone H1 or indicated GST fusion proteins. Cdc2 was immunoprecipitated from uninfected HeLa cells with cdc2 antibody, and histone H1 or GST fusion proteins were tested as substrates for cdc2. Reactions were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film.

Figure 4

(A) Autoradiographic image of nocodazole-induced cdc2-mediated phosphorylation of histone H1 or indicated GST fusion proteins. Replicate HeLa cell cultures were mock-treated or treated with nocodazole for 20 h. cdc2 was immunoprecipitated from lysates and reacted with indicated substrates. Reaction mixtures were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film. (B) Autoradiographic image of electrophoretically separated histone H1 or GST-ICP0 20–241 polypeptide reacted with immune-precipitated cdc2 in the presence of various concentrations of roscovitine. The cdc2 kinase was immune-precipitated from HEp-2 cells treated with nocodazole for 20 h and reacted with roscovitine before the addition of complete kinase buffer and substrates as described in Materials and Methods. Reactions were electrophoretically separated on 10% bisacrylamide gels, transferred to nitrocellulose membrane, and exposed to film. (C) Quantification of the amount of radioactivity in substrates phosphorylated by cdc2 in the presence or absence of roscovitine. Radioactivity in each band shown in B was quantified with the aid of Storm 860 PhosphorImager. The radioactivity in each band was normalized with respect to that of untreated (no roscovitine) reaction mixtures (100%). The dashed line represents 50% reduction in phosphorylation.