The carboxyl-terminal domain of RNA polymerase II is phosphorylated by a complex containing cdk9 and infected-cell protein 22 of herpes simplex virus 1

Abstract

The infected-cell protein 22 (ICP22), a regulatory protein encoded by the alpha22 gene of herpes simplex virus 1, is required for the optimal expression of a set of late viral proteins that includes the products of the U(S)11, U(L)38, and U(L)41 genes. ICP22 has two activities. Thus, ICP22 and the U(L)13 protein kinase mediate the activation of cdc2 and degradation of its partners, cyclins A and B. cdc2 and its new partner, the DNA polymerase accessory factor (U(L)42), bind topoisomerase IIalpha in an ICP22-dependent manner. In addition, ICP22 and U(L)13 mediate an intermediate phosphorylation of the carboxyl terminus of RNA polymerase II (RNA POL II). Here we report another function of ICP22. Thus, ICP22 physically interacts with cdk9, a constitutively active cyclin-dependent kinase involved in transcriptional regulation. A protein complex containing ICP22 and cdk9 phosphorylates in vitro the carboxyl-terminal domain of RNA POL II in a viral U(S)3 protein kinase-dependent fashion. Finally, the carboxyl-terminal domain of RNA POL II fused to glutathione S-transferase is phosphorylated in reaction mixtures containing complexes pulled down with ICP22 or cdk9 immune precipitated from lysates of wild-type parent virus or deltaU(L)13 but not deltaU(S)3 mutant-infected cells. The experiments described here place ICP22 and cdk9 in a complex with the carboxyl-terminal domain of RNA POL II. At the same time we confirm the requirement of ICP22 and the U(L)13 protein kinase in the posttranslational modification of RNA POL II that alters its electrophoretic mobility, although U(S)3 kinase appears to play a role in a cell-type-dependent fashion.

FIG. 1.

The GST-Cdk9 chimeric protein specifically pulls down two HSV-1 proteins. Panel A: Coomassie brilliant blue-stained GST-chimeric proteins. The molecular weights of the proteins bound to the glutathione beads are of the expected sizes of the GST-cdc2, GST-cdk7, and GST-cdk9 chimeric proteins. Panel B: Autoradiogram of [³⁵S]Met-labeled HEp-2 cell proteins bound to the GST-chimeric proteins. The cells were exposed to the wild-type virus HSV-1(F). After incubation for 6 h, the cells were harvested, lysed, and reacted with GST alone, GST-cdc2, GST-cdk7, or GST-cdk9 as described in Materials and Methods. LMW, low molecular weight; HMW, high molecular weight.

FIG. 2.

GST-cdk9 interacts with ICP22. Lysates of HEp-2 cells mock infected or infected with HSV-1(F) or R325 (ΔICP22) and incubated for 6 h were reacted with GST-cdk9. The bound proteins were solubilized, subjected to electrophoresis on a denaturing gel, transferred to a nitrocellulose sheet, and reacted with anti-ICP22 antibody. The band to the right of the dot comigrates with the ICP22 present in whole-cell lysates (lane 2) and contains ICP22. The heavy band in lanes 4 to 6 is GST. GST does not interact with ICP22 (Fig. 1 and data not shown).

FIG. 3.

The interaction of GST-cdk9 chimeric protein with the 69,000-M_r protein is dependent on ICP22 and the U_S3 protein kinase. Panel A: Autoradiogram of electrophoretically separated lysates of [³⁵S]Met-labeled HEp-2 cell proteins. The cells were harvested and lysed 6 h after mock infection or exposure to HSV-1(F), R325, R7356, or R7041. Panel B: The cell lysates described above were reacted with GST-cdk9. The proteins bound to the GST-cdk9 were solubilized and subjected to electrophoresis on a denaturing gel and autoradiography. The procedures were as described in Materials and Methods.

FIG. 4.

The levels of cdk9 and cyclin T are stable throughout infection. Lysates of cells harvested 3, 6, 9, or 16 h after infection were subjected to electrophoresis in denaturing gels, transferred to a nitrocellulose sheet, and reacted with antibody to cdk9 or cyclin T.

FIG. 5.

ICP22-dependent phosphorylation of RNA POL II CTD requires the US3 protein kinase. (A and D) HEp-2 cells were mock infected or exposed to HSV-1(F), R325 (Δα22), R7356 (ΔU_L13), R7041 (ΔU_S3), or R7353 (ΔU_L13/ΔU_S3). The cells were harvested 18 h after infection, precleared with protein A beads, and immunoprecipitated with either an antibody against cdk9 (panel A) or ICP22 (panel D). This complex was then reacted with a chimeric protein of the C-terminal domain of RNA POL II fused to the GST (GST-CTD) for 30 min at 30°C in the presence of [γ-³²P]ATP. (B) Ponceau S-stained proteins served as a control to ensure that the amounts of proteins in each reaction mixture were identical. (C and E) Quantification of the amounts of radioactivity in each band adjusted for background. The results were normalized with respect to the amounts of radioactivity present in reaction mixtures containing lysates of mock-infected cells. The procedures were as described in the text.

FIG. 6.

U_S3 is not required for the posttranslational generation of the RNA POL IIi form. (A) Vero cells were mock infected or exposed to HSV-1(F), R7356 (ΔU_L13), R7041 (ΔU_S3), or R7353 (ΔU_L13/ΔU_S3). The cells were harvested 18 h after infection and then subjected to electrophoresis in denaturing gels, transferred to a nitrocellulose sheet, and reacted with an antibody to the large subunit of RNA POL II (8WG16; Covance, Princeton, N.J.; catalog no. MM5-126R-500). (B) Rabbit skin cells (RSC) were mock infected or exposed to HSV-1(F), R7356 (ΔU_L13), R7041 (ΔU_S3), or R7353 (ΔU_L13/ΔU_S3). The cells were harvested 18 h after infection and then subjected to electrophoresis in denaturing gels, transferred to a nitrocellulose sheet, and reacted with an antibody to the large subunit of RNA POL II. (C) HEp-2 cells were mock infected or exposed to HSV-1(F), R325 (Δα22-CTD), R7356 (ΔU_L13), R7041 (ΔU_S3), or R7353 (ΔU_L13/ΔU_S3). The cells were harvested 18 h after infection and then subjected to electrophoresis in denaturing gels, transferred to a nitrocellulose sheet, and reacted with an antibody to RNA POL II. a, phosphorylated RNA POL II; b, fast-migrating, nonphosphorylated RNA POL II.