The latent form of the dimeric transcription factor NF-kappa B is sequestered in the cytoplasm by proteins containing ankyrin repeats, such as 1 kappa B alpha and beta, or by the p105 precursor form of the NF-kappa B p50 subunit. Tumor necrosis factor alpha or virus infection can cause targeted destruction of 1 kappa B and nuclear translocation of NF-kappa B. Following translocation, NF-kappa B mediates immune, inflammatory, or anti-apoptotic responses. Here we present evidence that beginning at around 6 h postinfection, herpes simplex virus (HSV) induces a persistent translocation of NF-kappa B into the nucleus of C33 cells, coincident with loss of both 1 kappa B alpha and 1 kappa B beta. Translocation failed to occur when infecting virus was preincubated with neutralizing antibody to viral envelope glycoproteins gD or gH, thus preventing entry, or when cells infected with viruses expressing mutated forms of immediate-early regulatory proteins lCP4 or lCP27. Surprisingly, no increase in the trans-activation function of NF-kappa B, as assayed by transient expression of CAT, was detected following HSV infection. The significance of NF-kappa B nuclear translocation for virus replication was demonstrated by an 80-90% reduction in virus yield following infection of C33 cells expressing a constitutive repressor form of 1 kappa B alpha. Models that reconcile nuclear translocation of NF-kappa B with the inability to detect NF-kappa B-dependent gene expression are discussed.

Herpes simplex virus type 1 (HSV-1) infection disrupted cell cycle regulation in at least two ways. First, infection of quiescent human embryonic lung cells simultaneously with readdition of serum caused inhibition of cyclin D/cyclin-dependent kinase (CDK) 4,6-specific and cyclin E/CDK2-specific phosphorylation of the retinoblastoma protein pRb. The inhibition of cyclin D/CDK4,6 kinase activity corresponded to a loss of cyclin D1 protein and a failure of CDK4 and CDK6 to translocate to the nucleus. Failure to detect cyclin E/CDK2 kinase activity was accompanied by a loss of cyclin E protein and a failure of CDK2 to translocate to the nucleus. Levels of pocket protein p130 persisted, whereas p107 did not accumulate. As a result of these effects on cyclin kinase, G(0)-infected cells failed to reenter the cell cycle. The second type of HSV-induced cell cycle dysregulation was observed in asynchronously dividing cell cultures. A rapid inhibition of preexisting cyclin E/CDK2 and cyclin A/CDK2 activities was observed in human embryonic lung cells, as well as two other human cell lines: C33 and U2OS. HSV-1 immediate-early gene expression was necessary for the inhibition of CDK2 kinase activity. Cyclin and CDK subunit protein levels, intracellular localization, and complex stability were unaffected by infection. In addition, levels of cyclin-dependent kinase inhibitors, p27 and p21, were not affected by HSV-1. Previous experiments demonstrated that in asynchronous infected cells, hypophosphorylated pRb and pocket protein-E2F complexes accumulated, and cellular DNA synthesis was rapidly inhibited. Coupled with the present results, this indicates that HSV-1 has evolved mechanisms for preventing cells in G(1) from proceeding through the restriction point and for cells in S from completing a round of DNA replication.

We previously reported that herpes simplex virus type 1 (HSV-1) can activate the stress-activated protein kinases (SAPKs) p38 and JNK. In the present study, we undertook a comprehensive and comparative analysis of the requirements for viral protein synthesis in the activation of JNK and p38. Infection with the UL36 mutant tsB7 or with UV-irradiated virus indicated that both JNK The stress-activated protein kinases (SAPKs) p38 and JNK are part of a larger family of serine/threonine terminal kinases termed mitogen-activated protein kinases (MAPK), which includes ERK1 and -2. SAPK pathways are normally activated by UV irradiation, anoxia, and engagement of proinflammatory cytokines or Fas ligand by their cognate receptors. Following ligand binding to a cognate receptor, signaling is initiated through receptor-associated kinases to a MAPKKK (MAPK kinase kinase). MAPKKKs capable of initiating the p38 or JNK pathway include MEKK1, -2, -3, and -4, ASK-1, TAK-1, and TAO. These in turn activate dual-specificity MAPK kinase kinases (MAPKKs) MKK3/6 and MKK4/7, which directly bind and phosphorylate p38 and JNK, respectively, on both tyrosine and threonine, resulting in their activation (for a review, see reference 78).All three subfamilies of human herpesviruses activate one or more of the MAPKs during infection. The betaherpesvirus cytomegalovirus activates both p38 and ERK by a mechanism dependent on viral gene expression (14,39,76) and the upstream kinases MKK3/6 and MKK1/2, respectively, (39, 40). ERK activates the early gene UL112-113 promoter (76) and phosphorylates immediate-early 86 and 72 proteins to alter their transactivation activity (40), while p38 phosphorylates retinoblastoma protein and HSP27 (39). ERK and p38 activation are required for cytomegalovirus DNA replication (16). The gammaherpesvirus Kaposi's sarcoma-associated herpesvirus G protein-coupled receptor activates ERK and p38, causing increased vascular endothelial growth factor expression via phosphorylation of hypoxia-inducible factor 1␣ (86). The gammaherpesvirus Epstein-Barr virus activates p38 and JNK (1, 21). The immediate-early proteins BZLF1 and BRLF1 induce increased phosphorylation of p38 and JNK and activation of the ATF-2 transcription factor (1). BZLF1 expression during BRLF1-driven virus reactivation requires p38 activity (1). Among the alphaherpesviruses, HSV-2 has been reported to activate ERK through ICP10-PK binding to RAS-GAP, leading to further expression of ICP10 and prevention of apoptosis (68).Two previous reports documented the activation of the p38 and JNK (SAPK) pathways by HSV-1. Increased JNK and p38 kinase activity was observed as early as 3 hours postinfection and remained active throughout the course of infection (56,97). SAPKs were activated downstream of Ras, as infection with a dominant-negative form of Ras did not affect activation of the transcription factors ATF-2 and c-Jun, downstream targets of p38 and JNK, respectively (56). Furthermore, activation was specific for SAPKs, as ERK was not activated during infect...

Signal transduction pathways convey signals generated at the cell surface into the cell nucleus in order to initiate a program of gene expression that is characteristic for particular stimuli. Here we present evidence that infection by herpes simplex virus type 1 activated the two terminal kinases, cJUN N-terminal kinase (JNK) and p38, of stress-activated signal transduction kinase cascades. By using a solid-phase kinase assay, a phospho-specific antibody, and extracts prepared from a variety of infected cell types, we determined that activation of both kinases began 3 to 4 h postinfection (p.i.) and remained elevated out to 14 h p.i. Through the use of UV-irradiated or antibody-neutralized wild-type virus and the temperature-sensitive mutant tsB7, the high level of JNK activation was shown to be dependent on viral gene expression. Activation of JNK following infection by vi13, an ICP4 mutant virus that does not express early or late genes, suggested that only virus entry and immediate-early gene expression were necessary for JNK activation. The activation of JNK and p38 correlated with increased chloramphenicol acetyltransferase (CAT) activity in reporter assays dependent upon the activity of cJUN and ATF2trans-activation domains. Increased CAT activity dependent on TRE and CRE promoter sites was also observed in response to herpes simplex virus infection. The activities of ERK and ERK-dependent transcription factors were unchanged or depressed following infection, showing that activation of JNK and p38 was a specific event. Finally, the activation of JNK was important for the efficiency of viral replication. The yield of virus in NIH 3T3 cells stably expressing JIP-1, an inhibitor of JNK translocation to the nucleus, was reduced 70% compared to that of control cells, in single-step growth experiments.

We have determined that HSV causes rapid and large increases in cell-cycle-regulated free E2F and S-phase p107/E2F DNA binding activities in asynchronous cultures of C33A cells. Induction occurred by 4 hr postinfection and coincided with the appearance of viral encoded immediate-early and delayed-early proteins, i.e., when viral DNA replication normally commences. No increase in E2F activities occurred when cells were infected with viruses expressing mutant regulatory proteins ICP4 or ICP27, or mutant replication proteins ICP8, pol or helicase, or when cells were infected with wild-type virus in the presence of inhibitors of DNA synthesis. In contrast, ICP8 mutant-infected cells contained elevated amounts of NF kappa B activity equivalent to WT virus, no induction of Sp1 relative to WT virus, and reduced ATF/CREB activity relative to WT virus. Results of transient expression assays with E2F-responsive reporters indicated that the net effect of induction of both active (free E2F) and repressive (p107/E2F) complexes was a decrease in AdE2 promoter activity and an increase in c-myc promoter activity. Taken together these results suggest that HSV can cause unscheduled changes in the amount and functional status of a cell-cycle-regulated transcription factor. These results are discussed in light of possible roles for viral-induced alterations in E2F, especially as related to imposing or overriding cell-cycle checkpoints.