Herpes Simplex Virus Type 1 ICP27-Dependent Activation of NF- B

The ability of herpes simplex virus type 1 (HSV-1) to activate NF-κB has been well documented. Beginning at 3 to 5 h postinfection, HSV-1 induces a robust and persistent nuclear translocation of an NF-κB-dependent (p50/p65 heterodimer) DNA binding activity, as measured by electrophoretic mobility shift assay. Activation requires virus binding and entry, as well as de novo infected-cell protein synthesis, and is accompanied by loss of both IκBα and IκBβ. In this study, we identified loss of IκBα as a marker of NF-κB activation, and infection with mutants with individual immediate-early (IE) regulatory proteins deleted indicated that ICP27 was necessary for IκBα loss. Analysis of both N-terminal and C-terminal mutants of ICP27 identified the region from amino acids 21 to 63 as being necessary for IκBα loss. Additional experiments with mutant viruses with combinations of IE genes deleted revealed that the ICP27-dependent mechanism of NF-κB activation may be augmented by functional ICP4. We also analyzed two additional markers for NF-κB activation, phosphorylation of the p65 subunit on Ser276 and Ser536. Phosphorylation of both serines was induced upon HSV infection and required functional ICP4 and ICP27. Pharmacological inhibitor studies revealed that both IκBα and Ser276 phosphorylation were dependent on Jun N-terminal protein kinase activity, while Ser536 phosphorylation was not affected during inhibitor treatment. These results demonstrate that there are several layers of regulation of NF-κB activation during HSV infection, highlighting the important role that NF-κB may play in infection

Activation of cJUN N-Terminal Kinase by Herpes Simplex Virus Type 1 Enhances Viral Replication

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 ATF2 trans-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

Efficient Replication by Herpes Simplex Virus Type 1 Involves Activation of the I B Kinase-I B-p65 Pathway

Infection by herpes simplex virus type 1 (HSV-1) induces a persistent nuclear translocation of NFκB. To identify upstream effectors of NFκB and their effect on virus replication, we employed mouse embryo fibroblast (MEF)-derived cell lines with deletions of either IKK1 or IKK2, the catalytic subunits of the IκB kinase (IKK) complex. Infected MEFs were assayed for virus yield, loss of IκBα, nuclear translocation of p65, and NFκB DNA-binding activity. Absence of either IKK1 or IKK2 resulted in an 86 to 94% loss of virus yield compared to that of normal MEFs, little or no loss of IκBα, and greatly reduced NFκB nuclear translocation. Consistent with reduced virus yield, accumulation of the late proteins VP16 and gC was severely depressed. Infection of normal MEFs, Hep2, or A549 cells with an adenovirus vector expressing a dominant-negative (DN) IκBα, followed by superinfection with HSV, resulted in a 98% drop in virus yield. These results indicate that the IKK-IκB-p65 pathway activates NFκB after virus infection. Analysis of NFκB activation and virus replication in control and double-stranded RNA-activated protein kinase-null MEFs indicated that this kinase plays no role in the NFκB activation pathway. Finally, in cells where NFκB was blocked because of DNIκB expression, HSV failed to suppress two markers of apoptosis, cell surface Annexin V staining and PARP cleavage. These results support a model in which activation of NFκB promotes efficient replication by HSV, at least in part by suppressing a host innate response to virus infection

Direct BRLF1 binding is required for cooperative BZLF1/BRLF1 activation of the Epstein-Barr virus early promoter, BMRF1

Disruption of Epstein-Barr virus (EBV) latency is mediated through the activation of the viral immediate-early proteins, BZLF1 (Z) and BRLF1 (Ft).I.; (Chevallier-Greco, A.

Structure of replicating herpes simplex virus DNA.

We have investigated the molecular anatomy of the herpes simplex virus replicative intermediates by cleavage with the restriction endonuclease BglII. We find that in populations of multiply infected cells, pulse-labeled replicating herpes simplex virus DNA contains at least two and probably all four sequence isomers. Also, it contains no detectable termini. In pulse-chase experiments, we show that endless replicative intermediates are the precursors to virion DNA and that maturation is a relatively slow process. The results are discussed in terms of their significance to possible models of herpes simplex virus DNA replication

Differential dependence of herpes simplex virus immediate-early gene expression on de novo-infected cell protein synthesis.

The time course of accumulation of herpes simplex virus immediate-early (IE) mRNA and the requirement for infected cell protein synthesis for mRNA transcription and accumulation were compared. Measurements of transcription in nuclear run-on assays, accumulation of cytoplasmic mRNA by Northern (RNA) blot hybridization, and rates of infected cell protein synthesis by pulse-labeling did not indicate differences among the five IE gene, consistent with previous studies. However, as a result of varying the amount of de novo protein synthesis after infection, at least three patterns of maximal expression of the IE genes were revealed. Addition of the protein synthesis inhibitor anisomycin to cells coincident with infection resulted in maximal rates of transcription and accumulation of functional ICP0 mRNA, while 0.5 h of infected cell protein synthesis prior to addition of the drug was required for maximal expression of ICP22/47 and ICP27 mRNAs. Maximal expression of ICP4 mRNA occurred only when 1 h of de novo protein synthesis occurred prior to the addition of the drug. These results are discussed in the context of alternative mechanisms for regulating IE gene expression

Efficient Replication by Herpes Simplex Virus Type 1 Involves Activation of the IκB Kinase-IκB-p65 Pathway

Infection by herpes simplex virus type 1 (HSV-1) induces a persistent nuclear translocation of NFκB. To identify upstream effectors of NFκB and their effect on virus replication, we employed mouse embryo fibroblast (MEF)-derived cell lines with deletions of either IKK1 or IKK2, the catalytic subunits of the IκB kinase (IKK) complex. Infected MEFs were assayed for virus yield, loss of IκBα, nuclear translocation of p65, and NFκB DNA-binding activity. Absence of either IKK1 or IKK2 resulted in an 86 to 94% loss of virus yield compared to that of normal MEFs, little or no loss of IκBα, and greatly reduced NFκB nuclear translocation. Consistent with reduced virus yield, accumulation of the late proteins VP16 and gC was severely depressed. Infection of normal MEFs, Hep2, or A549 cells with an adenovirus vector expressing a dominant-negative (DN) IκBα, followed by superinfection with HSV, resulted in a 98% drop in virus yield. These results indicate that the IKK-IκB-p65 pathway activates NFκB after virus infection. Analysis of NFκB activation and virus replication in control and double-stranded RNA-activated protein kinase-null MEFs indicated that this kinase plays no role in the NFκB activation pathway. Finally, in cells where NFκB was blocked because of DNIκB expression, HSV failed to suppress two markers of apoptosis, cell surface Annexin V staining and PARP cleavage. These results support a model in which activation of NFκB promotes efficient replication by HSV, at least in part by suppressing a host innate response to virus infection