The interaction of herpes simplex virus 1 regulatory protein ICP22 with the cdc25C phosphatase is enabled in vitro by viral protein kinases US3 and UL13

Earlier studies have shown that ICP22 and the U(L)13 protein kinase but not the U(S)3 kinase are required for optimal expression of a subset of late (gamma(2)) genes exemplified by U(L)38, U(L)41, and U(S)11. In primate cells, ICP22 mediates the disappearance of inactive isoforms of cdc2 and degradation of cyclins A and B1. Active cdc2 acquires a new partner, the viral DNA synthesis processivity factor U(L)42. The cdc2-U(L)42 complex recruits and phosphorylates topoisomerase IIalpha for efficient expression of the gamma(2) genes listed above. In uninfected cells, the cdc25C phosphatase activates cdc2 by removing two inhibitory phosphates. The accompanying report shows that in the absence of cdc25C, the rate of degradation of cyclin B1 is similar to that occurring in infected wild-type mouse embryo fibroblast cells but the levels of cdc2 increase, and the accumulation of a subset of late proteins and virus yields are reduced. This report links ICP22 with cdc25C. We show that in infected cells, ICP22 and U(S)3 protein kinase mediate the phosphorylation of cdc25C at its C-terminal domain. In in vitro assays with purified components, both U(L)13 and U(S)3 viral kinases phosphorylate cdc25C and ICP22. cdc25C also interacts with cdc2. However, in infected cells, the ability of cdc25C to activate cdc2 by dephosphorylation of the inactive cdc2 protein is reduced. Coupled with the phosphorylation of cdc25C by the U(S)3 kinase, the results raise the possibility that herpes simplex virus 1 diverts cdc25C to perform functions other than those performed in uninfected cells.     

Posttranslational processing of infected cell protein 22 mediated by viral protein kinases is sensitive to amino acid substitutions at distant sites and can be cell-type specific

Infected cell protein 22 (ICP22) is posttranslationally phosphorylated by the viral kinases encoded by U(S)3 and U(L)13 and nucleotidylylated by casein kinase II. In rabbit and rodent cells and in primary human fibroblasts infected with mutants from which the alpha 22 gene encoding ICP22 had been deleted, a subset of late (gamma(2)) gene products exemplified by U(L)38 and U(S)11 proteins are expressed at a reduced level, as measured by the accumulation of both mRNA and protein. The same phenotype was observed in cells infected with mutants lacking the U(L)13 gene. The focus of this report is on three serine- and threonine-rich domains of ICP22. Two of these domains are homologs located between residues 38 to 66 and 300 to 328. The third domain is near the carboxyl terminus and contains the sequence T374SS. The results were as follows. (i) Alanine substitutions in the amino-terminal homolog precluded the posttranslational processing of ICP22 in rabbit skin cells and in Vero cells but had no effect on the accumulation of either U(S)11 or U(L)38 protein. (ii) Alanine substitutions in the carboxyl-terminal homolog had no effect on posttranslational processing of ICP22 accumulating in Vero cells but precluded full processing of ICP22 accumulating in rabbit skin cells. The effect on accumulation of U(L)38 and U(S)11 proteins was insignificant in Vero cells and minimal in rabbit skin cells. (iii) Substitutions of alanine for the threonine and serines in the third domain precluded full processing of ICP22 and caused a reduction of accumulation of U(S)11 and U(L)38 proteins. These results indicate the following. (i) The posttranslational processing of ICP22 is sensitive to mutations within the domains of ICP22 tested and is cell-type dependent. (ii) Posttranslational processing of ICP22 is not required for accumulation of U(L)38 and U(S)11 proteins to the same level as that seen in cells infected with the wild-type virus. (iii) The T374SS sequence shared by ICP22 and the U(S)1.5 proteins is essential for the accumulation of a subset of gamma(2) proteins exemplified by U(S)11 and U(L)38 and is the first step in mapping of the sequences necessary for optimal accumulation of U(S)11 and U(L)38 proteins.     

The products of the herpes simplex virus type 1 immediate-early US1/US1.5 genes downregulate levels of S-phase-specific cyclins and facilitate virus replication in S-phase Vero cells

Herpes simplex virus type 1 ICP22-/U(S)1.5- mutants initiate viral gene expression in all cells; however, in most cell types, the replication process stalls due to an inability to express gamma2 late proteins. Although the function of ICP22/U(S)1.5 has not been established, it has been suggested that these proteins activate, induce, or repress the activity of cellular proteins during infection. In this study, we hypothesized that cell cycle-associated proteins are targets of ICP22/U(S)1.5. For this purpose, we first isolated and characterized an ICP22-/U(S)1.5- mutant virus, 22/n199. Like other ICP22-/U(S)1.5- mutants, 22/n199 replicates in a cell-type-specific manner and fails to induce efficient gamma2 late gene expression in restrictive cells. Although synchronization of restrictive human embryonic lung cells in each phase of the cell cycle did not overcome the growth restrictions of 22/n199, synchronization of permissive Vero cells in S phase rendered them less able to support 22/n199 plaque formation and replication. Consistent with this finding, expression of cellular S-phase cyclins was altered in an ICP22/U(S)1.5-dependent manner specifically when S-phase Vero cells were infected. Collectively, these observations support the notion that ICP22/U(S)1.5 deregulates the cell cycle upon infection of S-phase permissive cells by altering expression of key cell cycle regulatory proteins either directly or indirectly.     

Small dense nuclear bodies are the site of localization of herpes simplex virus 1 U(L)3 and U(L)4 proteins and of ICP22 only when the latter protein is present

The herpes simplex virus 1 U(L)3 and U(L)4 open reading frames are expressed late in infection and are not essential for viral replication in cultured cells in vitro. An earlier report showed that the U(L)4 protein colocalizes with the products of the alpha22/U(S)1.5 genes in small nuclear dense bodies. Here we report that the U(L)3 protein also colocalized in these small nuclear dense bodies and the localization of U(L)3 and U(L)4 proteins in these bodies required the presence of alpha22/U(S)1.5 genes. In cells infected with a mutant lacking intact alpha22/U(S)1.5 genes, U(L)3 was diffused throughout the nucleus even though the overall accumulation of the gamma2 U(L)3 protein was decreased. The results suggest that ICP22 acts both as a regulator of U(L)3 accumulation and as the structural component and anchor of these small dense nuclear bodies.     

Identification of a herpes simplex virus function that represses late gene expression from parental viral genomes.

The expression of herpes simplex virus gamma 2 (late) genes is inhibited before the onset of viral DNA replication. We report that the block in the expression of certain gamma 2 genes is relieved, at least in part, by defects in the beta ICP8 protein. We have examined the expression of the gamma 2 gene encoding glycoprotein C (gC) in cells infected with a temperature-sensitive ICP8 mutant. Under conditions in which viral DNA replication is inhibited, cells infected with the ICP8 mutant overproduce the gC family of mRNAs relative to the level observed in cells infected with a wild-type virus. The gC mRNA synthesized in cells infected with the ICP8 mutant virus is correctly initiated and spliced and is translated with the same relative efficiency as in cells infected with a replicating wild-type virus. These results suggest that ICP8 is involved in the negative regulation of gamma 2 genes expressed from parental viral genomes. The level of gC expression was greatest in cells infected with a replicating wild-type virus. These data suggest that DNA replication and genome amplification are not absolute requirements for gamma 2 gene expression but may facilitate full-level expression of these genes.     

Herpes simplex virus 1 mutant in which the ICP0 HUL-1 E3 ubiquitin ligase site is disrupted stabilizes cdc34 but degrades D-type cyclins and exhibits diminished neurotoxicity

Herpes simplex virus type 1 (HSV-1) infected cell protein 0 (ICP0) is a multifunctional protein that functions as a promiscuous transactivator and promotes the degradation of multiple cellular proteins. In vitro studies indicated that it encodes two physically separated functional E3 ubiquitin ligase domains. One, designated herpesvirus ubiquitin ligase 1 (HUL-1), maps to a region encoded by exon 3 and is contained between residues 543 and 680. Deletion of amino acids 621 to 625 abolishes this activity. The second, designated HUL-2, maps to the RING finger domain present in ICP0 encoded by exon 2. Earlier studies have shown that ICP0 stabilizes cyclins D1 and D3, and several lines of investigation led to the hypothesis that this function of ICP0 is the consequence of degradation of the E2 enzyme cdc34, known to be involved in the proteasome-dependent degradation of D-type cyclins. Consistent with this hypothesis, we have previously shown that cdc34 physically interacts with ICP0 at or near aspartate 199 and at amino acids 621 to 625 and that the former site is required for effective ubiquitylation and degradation of cdc34. Furthermore, the ICP0 HUL-1 domain promotes the polyubiquitination of cdc34 in vitro. If the mechanism by which D-type cyclins are salvaged in wild-type-infected cells is dependent on polyubiquitination and consequent destruction of cdc34, than the mutant virus R6701, which was constructed for these studies and lacks ICP0 residues 621 to 625, should destabilize the D cyclins and preclude the degradation of cdc34. We report that ICP0 residues 621 to 625 are essential for degradation of cdc34 in infected cells and for the ICP0-mediated stabilization of D-type cyclins, that a mutation that specifically disrupted the ring finger domain of the HUL-2 site had no effect on the degradation of cdc34 in infected cells, and that deletion of ICP0 residues 621 to 625 decreased the replicative capacity of the virus in growth-arrested but not in dividing cells and resulted in diminished pathogenicity on intracerebral inoculation of mice. We conclude that the ICP0 HUL-1 domain acts in infected cells to degrade cdc34 and that this function requires the interaction of cdc34 with sequences in exons 2 and 3 but does not involve the HUL-2 RING finger E3 domain.     

US3 Protein Kinase of Herpes Simplex Virus 1 Blocks Caspase 3 Activation Induced by the Products of US1.5 and UL13 Genes and Modulates Expression of Transduced US1.5 Open Reading Frame in a Cell Type-Specific Manner

The coding domain of the herpes simplex virus type 1 (HSV-1) alpha22 gene encodes two proteins, the 420-amino-acid infected-cell protein 22 (ICP22) and U(S)1.5, a protein colinear with the carboxyl-terminal domain of ICP22. In HSV-1-infected cells, ICP22 and U(S)1.5 are extensively modified by the U(L)13 and U(S)3 viral protein kinases. In this report, we show that in contrast to other viral proteins defined by their properties as alpha proteins, U(S)1.5 becomes detectable and accumulated only at late times after infection. Moreover, significantly more U(S)1.5 protein accumulated in cells infected with a mutant lacking the U(L)13 gene than in cells infected with wild-type virus. To define the role of viral protein kinases on the accumulation of U(S)1.5 protein, rabbit skin cells or Vero cells were exposed to recombinant baculoviruses that expressed U(S)1.5, U(L)13, or U(S)3 proteins under a human cytomegalovirus immediate-early promoter. The results were as follows. (i) Accumulation of the U(S)1.5 protein was reduced by concurrent expression of the U(L)13 protein kinase and augmented by concurrent expression of the U(S)3 protein kinase. The magnitude of the reduction or increase in the accumulation of the U(S)1.5 protein was cell type dependent. The effect of U(L)13 kinase appears to be specific inasmuch as it did not affect the accumulation of glycoprotein D in cells doubly infected by recombinant baculoviruses expressing these genes. (ii) The reduction in accumulation of the U(S)1.5 protein was partially due to proteasome-dependent degradation. (iii) Both U(S)1.5 and U(L)13 proteins activated caspase 3, indicative of programmed cell death. (iv) Concurrent expression of the U(S)3 protein kinase blocked activation of caspase 3. The results are concordant with those published elsewhere (J. Munger and B. Roizman, Proc. Natl. Acad. Sci. USA 98:10410-10415, 2001) that the U(S)3 protein kinase can block apoptosis by degradation or posttranslational modification of BAD.     

The disappearance of cyclins A and B and the increase in activity of the G(2)/M-phase cellular kinase cdc2 in herpes simplex virus 1-infected cells require expression of the alpha22/U(S)1.5 and U(L)13 viral genes

In uninfected cells the G₂/M transition is regulated by cyclin kinase complex containing cdc2 and, initially, cyclin A, followed by cyclin B. cdc2 is downregulated through phosphorylation by wee-1 and myt-1 and upregulated by cdc-25C phosphatase. We have examined the accumulation and activities of these proteins in cells infected with wild type and mutants of herpes simplex virus 1. The results were as follows. (i) Cyclin A and B levels were reduced beginning 4 h after infection and were undetectable at 12 to 16 h after infection. (ii) cdc2 protein also decreased in amount but was detectable at all times after infection. In addition, a fraction of cdc2 protein from infected cells exhibited altered electrophoretic mobility in denaturing gels. (iii) The levels of cdk7 or myt-1 proteins remained relatively constant throughout infection, whereas the level of wee-1 was significantly decreased. (iv) cdc-25C formed novel bands characterized by slower electrophoretic mobility that disappeared after treatment with phosphatase. In addition, one phosphatase-sensitive band reacted with MPM-2 antibody that recognizes a phosphoepitope phosphorylated exclusively in M phase. (v) cdc2 accumulating in infected cells exhibited kinase activity. The activity of cdc2 was higher in infected cell lysates than those of corresponding proteins present in lysates of mock-infected cells even though cyclins A and B were not detectable in lysates of infected cells. (vi) The decrease in the levels of cyclins A and B, the increase in activity of cdc2, and the hyperphosphorylation of cdc-25C were mediated by U_L13 and α22/U_S1.5 gene products. In light of its normal functions, the activated cdc2 kinase may play a role in the changes in the morphology of the infected cell. These results are consistent with the accruing evidence that herpes simplex virus scavenges the cell for useful cell cycle proteins and subverts them for its own use.     

UL31 of herpes simplex virus 1 is necessary for optimal NF-kappaB activation and expression of viral gene products

Previous results suggested that the U(L)31 gene of herpes simplex virus 1 (HSV-1) is required for envelopment of nucleocapsids at the inner nuclear membrane and optimal viral DNA synthesis and DNA packaging. In the current study, viral gene expression and NF-κB and c-Jun N-terminal kinase (JNK) activation of a herpes simplex virus mutant lacking the U(L)31 gene, designated ΔU(L)31, and its genetic repair construct, designated ΔU(L)31-R, were studied in various cell lines. In Hep2 and Vero cells infected with ΔU(L)31, expression of the immediate-early protein ICP4, early protein ICP8, and late protein glycoprotein C (gC) were delayed significantly. In Hep2 cells, expression of these proteins failed to reach levels seen in cells infected with ΔU(L)31-R or wild-type HSV-1(F) even after 18 h. The defect in protein accumulation correlated with poor or no activation of NF-κB and JNK upon infection with ΔU(L)31 compared to wild-type virus infection. The protein expression defects of the U(L)31 deletion mutant were not explainable by a failure to enter nonpermissive cells and were not complemented in an ICP27-expressing cell line. These data suggest that pU(L)31 facilitates initiation of infection and/or accelerates the onset of viral gene expression in a manner that correlates with NF-κB activation and is independent of the transactivator ICP27. The effects on very early events in expression are surprising in light of the fact that U(L)31 is designated a late gene and pU(L)31 is not a virion component. We show herein that while most pUL31 is expressed late in infection, low levels of pU(L)31 are detectable as early as 2 h postinfection, consistent with an early role in HSV-1 infection.     

Role of reactive oxygen species and MAPKs in vanadate-induced G(2)/M phase arrest

Cell growth arrest is an important mechanism in maintaining genomic stability and integrity in response to environmental stress. Using the human lung alveolar epithelial cancer cell line A549, we investigated the role of reactive oxygen species (ROS), extracellular signal-regulated protein kinase (ERK), and p38 protein kinase in vanadate-induced cell growth arrest. Exposure of cells to vanadate led to cell growth arrest at the G(2)/M phase and caused upregulation of p21 and phospho-cdc2 and degradation of cdc25C in a time- and dose-dependent manner. Vanadate stimulated mitogen-activated protein kinases (MAPKs) family members, as determined by the phosphorylation of ERK and p38. PD98059, an inhibitor of ERK, and SB202190, an inhibitor of p38, inhibited vanadate-induced cell growth arrest, upregulation of p21 and cdc2, and degradation of cdc25C. In addition to hydroxyl radical ((*)OH) formation, cellular reduction of vanadate generated superoxide radical (O(2)(*)(-)) and hydrogen peroxide (H(2)O(2)), as determined by confocal microscopy using specific dyes. Generation of O(2)(*)(-) and H(2)O(2) was inhibited by specific antioxidant enzymes, superoxide dismutase (SOD) and catalase, respectively. ROS activate ERK and p38, which in turn upregulate p21 and cdc2 and cause degradation of cdc25C, leading to cell growth arrest at the G(2)/M phase. Specific ROS affect different MAPK family members and cell growth regulatory proteins with different potencies.     

Induction and prevention of apoptosis in human HEp-2 cells by herpes simplex virus type 1

Cultured human epithelial cells infected with an ICP27 deletion strain of herpes simplex virus type 1 (HSV-1) show characteristic features of apoptotic cells including cell shrinkage, nuclear condensation, and DNA fragmentation. These cells do not show such apoptotic features when infected with a wild-type virus unless the infections are performed in the presence of a protein synthesis inhibitor. Thus, both types of virus induce apoptosis, but the ICP27-null virus is unable to prevent this process from killing the cells. In this report, we show that this ICP27-deficient virus induced apoptosis in human HEp-2 cells through a pathway which involved the activation of caspase-3 and the processing of the death substrates DNA fragmentation factor and poly(ADP-ribose) polymerase. The induction of apoptosis by wild-type HSV-1 occurred prior to 6 h postinfection (hpi), and de novo viral protein synthesis was not required to induce the process. The ability of the virus to inhibit apoptosis was shown to be effective between 3 to 6 hpi. Wild-type HSV-1 infection was also able to block the apoptosis induced in cells by the addition of cycloheximide, staurosporine, and sorbitol. While U(S)3- and ICP22-deficient viruses showed a partial prevention of apoptosis, deletion of either the U(L)13 or vhs gene products did not affect the ability of HSV-1 to prevent apoptosis in infected cells. Finally, we demonstrate that in UV-inactivated viruses, viral binding and entry were not sufficient to induce apoptosis. Taken together, these results suggest that either gene expression or another RNA metabolic event likely plays a role in the induction of apoptosis in HSV-1-infected human cells.     

A novel cellular protein, p60, interacting with both herpes simplex virus 1 regulatory proteins ICP22 and ICP0 is modified in a cell-type-specific manner and Is recruited to the nucleus after infection

Herpes simplex virus 1 encodes two multifunctional regulatory proteins, infected-cell proteins 22 and 0 (ICP22 and ICP0). ICP0 is a promiscuous transactivator, whereas ICP22 is required in vivo and for efficient replication and expression of a subset of late (gamma2) genes in rodent or rabbit cell lines and in primary human cell strains (restrictive cells) but not in HEp-2 or Vero (permissive) cells. We report the identification in the yeast two-hybrid system of a cellular protein designated p60 that interacts with ICP22. This protein (apparent Mr of 60,000) has not been previously described and has no known motifs. Analyses of p60 revealed the following. (i) p60 bound fast-migrating, underprocessed wild-type ICP22 and ICP22 lacking the carboxyl-terminal 24 amino acids but not ICP22 lacking the carboxyl-terminal 40 amino acids, whereas the previously identified cellular protein p78 (R. Bruni and B. Roizman, J. Virol. 72:8525-8531, 1998) bound all forms of ICP22. The interaction of p60 with only one isoform of ICP22 supports that hypothesis that each isoform of herpes simplex virus proteins performs a specific function that may be different from that of other isoforms. (ii) p60 also bound ICP0; the binding of ICP0 was independent of that of ICP22. (iii) p60 localized in uninfected rabbit skin cells in both nuclei and cytoplasm. In rabbit skin cells infected with wild-type virus, p60 was posttranslationally processed to a higher apparent Mr but was not redistributed. Posttranslational processing required the presence of the genes encoding ICP22 and UL13 protein kinase. (iv) In uninfected HEp-2 cells, p60 localized primarily in nuclei. Soon after infection with wild-type virus, the p60 localized in discrete small nuclear structures with ICP0. Late in infection, both ICP0 and p60 tended to disperse but p60 did not change in apparent Mr. The localization of p60 was independent of ICP22, but p60 tended to be more localized in small nuclear structures and less dispersed in cells infected with mutants lacking the genes encoding the UL13 or US3 protein kinases. The results suggest that posttranslational modification of p60 is mediated either by ICP0 (permissive cells) or by ICP22 and UL13 protein kinase (restrictive rabbit skin cells) and that the restrictive phenotype of rabbit skin cells may be related to the failure to process p60 by mutants lacking the genes encoding UL13 or ICP22.     

14-3-3 family members act coordinately to regulate mitotic progression

The mitosis promoting phosphatase, cdc25C, is a target of both the DNA replication and DNA damage checkpoint pathways. These pathways regulate cdc25C function, in part, by promoting the association of cdc25C with 14-3-3 proteins, which results in the retention of cdc25C in the cytoplasm. To determine which 14-3-3 proteins were required to regulate cdc25C function, we tested the ability of various 14-3-3 family members to form a complex with and negatively regulate cdc25C in human cells. Two 14-3-3 family members, 14-3-3epsilon and 14-3-3gamma specifically formed a complex with cdc25C but not with the 14-3-3 binding defective cdc25C mutant, S216A. In addition, 14-3-3epsilon and 14-3-3gamma inhibited the ability of cdc25C, but not the S216A mutant, to induce premature chromatin condensation (PCC) in U-2OS cells. These results suggested that the reduction in PCC by 14-3-3epsilon and 14-3-3gamma was due to inhibition of cdc25C function. In contrast, 14-3-3sigma was unable to form a complex with cdc25C, but was able to inhibit the ability of both wild type cdc25C and S216A to induce PCC. This suggests that 14-3-3sigma regulates entry into mitosis independently of cdc25C and 14-3-3epsilon and 14-3-3gamma. Thus, specific members of the 14-3-3 family of proteins may act coordinately to maintain the DNA replication checkpoint by regulating the activity of different cell cycle proteins.     

Colocalization of the herpes simplex virus 1 UL4 protein with infected cell protein 22 in small, dense nuclear structures formed prior to onset of DNA synthesis

Herpes simplex virus 1 infected cell protein 22 (ICP22) localizes in small, dense nuclear bodies of primate cells early in infection and in the more diffuse replicative complexes after the onset of DNA synthesis. UL4, a gamma2 protein, localizes in cytoplasm and in the small nuclear structures containing ICP22 but not in replicative complexes. In rabbit skin cells, both ICP22 and UL4 localize in the dense nuclear bodies late in infection. The results suggest that the small nuclear structures perform a function involving both proteins late in infection.