Role of cellular phosphatase cdc25C in herpes simplex virus 1 replication

Abstract

Earlier studies have shown that in herpes simplex virus 1-infected cells, ICP22 upregulates the accumulation of a subset of gamma(2) proteins exemplified by the products of the U(L)38, U(L)41, and U(S)11 genes. The ICP22-dependent process involves degradation of cyclins A and B1, the stabilization and activation of cdc2, physical interaction of activated cdc2 with the U(L)42 DNA synthesis processivity factor, and recruitment and phosphorylation of topoisomerase IIalpha by the cdc2/U(L)42 complex. Activation of cdc2, the first step in the process, is a key function of the mitotic phosphatase cdc25C. To define the role of cdc25C, we probed some features of the ICP22-dependent pathway of upregulation of gamma(2) genes in cdc25C(-/-) cells and in cdc25C(+/+) cells derived from sibling mice. We report that cyclin B1 turned over in cdc25C(+/+) or cdc25C(-/-) cells at the same rate, that cdc2 increased in amount, and that U(S)11 and U(L)38 proteins and infectious virus accumulated in smaller amounts than in wild-type infected cells. The reduction in U(L)38 protein accumulation and virus was greater in cdc25C(-/-) cells infected with virus lacking ICP22 than in cells infected with wild-type virus. We conclude that cdc25C phosphatase plays a role in viral replication and that this role extends beyond its function of activating cdc2 for initiation of the ICP22-dependent cascade for upregulation of gamma(2) gene expression.

FIG. 1.

The metabolism of cdc2 and cyclin B1 proteins in MEFs infected with wild-type HSV-1(F). cdc25C^+/+ or cdc25C^−/− MEF cells were mock or HSV-1(F) infected for the indicated amount of time, and cell lysates were electrophoretically separated and immunoblotted. The blots were scanned with the aid of a Molecular Dynamics PhosphorImager and normalized with respect to the amounts detected in corresponding mock-infected cells. (A) Immunoblot and results of scanning of cyclin B1. (B) Immunoblot and results of scanning of cdc2.

FIG. 2.

cdc25C is not required for increase in cdc2 kinase activity after HSV-1(F) infection. cdc25C^+/+ or cdc25C^−/− MEF cells were mock or HSV-1(F) infected for 16 h. cdc2 kinase complex immunoprecipitated using antibody against cyclin B1 (lanes 1 to 4) or against cdc2 (lanes 5 to 8) was incubated with purified histone H1 in a [γ-³²P]ATP-labeled kinase assay. (A) Light exposure of autoradiogram. (B) Dark exposure of same autoradiogram.

FIG. 3.

cdc25C is not required for modification of TopoIIα to negatively charged forms. cdc25C^+/+ and cdc25C^−/− MEF cells were mock infected or infected with 10 PFU/cell HSV-1(F) for 12 h. Cell lysates were separated electrophoretically for linear analysis or separated by charge followed by electrophoresis for two-dimensional analysis. (A) Linear separation, immunoblot for topoisomerase IIα. (B) Two-dimensional separation, immunoblot for topoisomerase IIα. Linear migration is marked with an arrowhead; charge distribution is indicated with a line.

FIG. 4.

cdc25C is required for maximal production of HSV-1(F) DNA. Total viral and cellular DNA was purified from Vero and MEF cells 20 h after mock or HSV-1(F) infection. DNA was digested with BamHI restriction endonuclease, electrophoretically separated in a 0.7% agarose gel, transferred to a membrane, and labeled with a probe specific for the genomic junction region designated BamHI fragment S. Digested genomic concatemers and circles migrate more slowly and are identified as BamHI SP. Digested genomic terminal fragments migrate more quickly and are identified as BamHI S.

FIG. 5.

Accumulation of a subset of γ₂ proteins is depends on cdc25C and functional ICP22. cdc25C^+/+ and cdc25C^−/− MEF cells were mock infected or infected with HSV-1(F) (A and B) or R325 (B) and harvested at the times indicated. Electrophoretically separated proteins were immunoblotted for ICP4 (A), ICP0 (A and B), ICP22 (A), U_L38 (A and B), U_S11 (A and B), or actin (B).

FIG. 6.

cdc25C and α22 are each important for virus growth at a low multiplicity of infection. cdc25C^+/+ and cdc25C^−/− MEF cells were infected with 0.01 PFU of R7356 (ΔU_L13), R7802 (Δα22), R325 (α22ΔCTD), or wild-type HSV-1(F) per cell. At 24 h after infection, the cells were harvested, the virus was extracted by repeated freeze thawing, and titers were determined on Vero cells.