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

Abstract

A subset (gamma(2)) of late herpes simplex virus 1 genes depends on viral DNA synthesis for its expression. For optimal expression, a small number of these genes, exemplified by U(S)11, also requires two viral proteins, the alpha protein infected cell protein (ICP) 22 and the protein kinase U(L)13. Earlier we showed that U(L)13 and ICP22 mediate the stabilization of cdc2 and the replacement of its cellular partner, cyclin B, with the viral DNA polymerase processivity factor U(L)42. Here we report that cdc2 and its new partner, U(L)42, bind a phosphorylated form of topoisomerase II alpha. The posttranslational modification of topoisomerase II alpha and its interaction with cdc2-U(L)42 proteins depend on ICP22 in infected cells. Although topoisomerase II is required for viral DNA synthesis, ICP22 is not, indicating a second function for topoisomerase II alpha. The intricate manner in which the virus recruits topoisomerase II alpha for post-DNA synthesis expression of viral genes suggests that topoisomerase II alpha also is required for untangling concatemeric DNA progeny for optimal transcription of late genes.

Figure 1

Topoisomerase II is active and modified in HSV-1-infected cells. (A) Topoisomerase II activity assay. Assay controls are shown in the first three lanes: catenated kDNA (Cat), decatenated kDNA (Decat), and linearized kDNA (Linear). Nuclear fractions (2 μg) of mock-infected, nocodazole-arrested, and HSV-1-infected HEp-2 cell lysates were assayed with catenated kDNA. (B and C) Topoisomerase IIα immunoblot of lysates from cycling HEp-2 cells (B) and contact-inhibited pHFF (C). Cells were mock-infected or infected with wild-type HSV-1 or the HSV-1 mutant R325. Also, HEp-2 cells were treated with nocodazole. (D) Two-dimensional gel electrophoresis of lysates of mock- or HSV-1-infected HEp-2 cells immunoblotted for topoisomerase IIα.

Figure 2

Interaction among U_L42, topoisomerase IIα, and cdc2. (A) HEp-2 cells were transfected with plasmids encoding U_L42, cdc2-dn-HA, and empty vector pcDNA3.1. Nuclear fractions were assayed for topoisomerase II activity after 36 h. Immunoblots of the transfected lysates were done for U_L42 and HA to verify protein expression. The decatenated nicked, open circular kDNA (●) was quantified. (B) The activity of lysates of cells transfected with the vector only was assigned a value of 1. (C) Cells transfected with U_L42 with and without the cdc2-dn construct were immunoprecipitated with U_L42 antibody and immunoblotted for topoisomerase IIα. (D) GST pull-downs with lysates from asynchronous or nocodazole-arrested HEp-2 cells. GST fusion proteins included full-length U_L42 (aa 1–488), U_L42 amino-terminal half (N′, aa 1–244), and U_L42 carboxyl-terminal half (C′, aa 226–488). Pull-downs were immunoblotted for topoisomerase IIα. Aliquots of the whole cell lysates are shown on the left. (E) Nocodazole-arrested HEp-2 cell lysates were treated with alkaline phosphatase in the presence or absence of phosphatase inhibitors and then reacted with GST fusion protein encoding the full-length U_L42 protein. Samples were immunoblotted for topoisomerase IIα. Aliquots of the whole cell lysates are shown in the lower row.

Figure 3

U_L42 interacts with topoisomerase IIα in an ICP22-dependent manner in HSV-1-infected cells. RSC were mock-infected or infected with wild-type or R325 mutant HSV-1. R325 lacks the coding region for the carboxyl-terminal region of ICP22. Cell lysates were immunoprecipitated with antibody to U_L42 and immunoblotted for U_L42 and topoisomerase IIα 12 h after infection. Whole cell lysates are shown in lanes 1–3.

Figure 4

A model of the role of topoisomerase II in viral replication. (A) Schematic representation of the steps involved in the synthesis of the subset of γ₂ proteins exemplified by the U_S11 protein. The α regulatory protein ICP22 and the U_L13 protein kinase mediate the stabilization of cdc2 concurrently with the degradation of cyclin B. Cdc2 binds the viral protein U_L42, and this complex recruits topoisomerase IIα. (B) The role of topoisomerase II in the viral life cycle. Shown is a schematic representation of viral DNA synthesis represented as a rolling circle and transcription of late genes off progeny DNA. Viral DNA synthesis requires topoisomerase II activity inasmuch as enzyme inhibitors effectively block this step in viral replication. The schematic diagram suggests a second role for topoisomerase II in the transcription of late genes from the progeny DNA. The topoisomerase IIα–U_L42 complex associates with U_L42 in a cdc2-dependent manner to enable efficient transcription of late viral genes from vast tangles of progeny viral DNA.