Cooperativity among herpes simplex virus type 1 immediate-early regulatory proteins: ICP4 and ICP27 affect the intracellular localization of ICP0.

The results of transient expression assays and studies of viral mutants have shown that three of the five immediate-early proteins of herpes simplex virus type 1 (HSV-1) perform regulatory functions, individually and cooperatively. As part of efforts designed to explore the molecular basis for the functional cooperativity among ICP0, ICP4, and ICP27 in the regulation of HSV gene expression, we have examined the intracellular localization of ICP0 in cells infected with ICP4 and ICP27 null mutant viruses by indirect immunofluorescence. Although ICP0 was localized predominantly to the nuclei of wild-type virus-infected cells, it was found exclusively in the nuclei of ICP27 mutant-infected cells and in both the cytoplasm and nuclei of ICP4 mutant-infected cells, the cytoplasmic component being especially strong. These observations indicate that both ICP4 and ICP27 can affect the intracellular localization of ICP0. Transient expression assays with plasmids that express wild-type and mutant forms of ICP0, ICP4, and ICP27 confirmed that ICP4 promotes and that ICP27 inhibits the nuclear localization of ICP0. These results confirm the observations made for mutant virus-infected cells and indicate that the localization pattern seen in infected cells can be established by these three immediate-early proteins exclusive of other viral proteins. The C-terminal half of ICP27 was shown to be required to achieve its inhibitory effect on the nuclear localization of ICP0. The region of ICP0 responsive to ICP27 was mapped to the C terminus of the molecule between amino acid residues 720 and 769. In addition, the concentration of ICP27 was shown to have a significant effect on the intracellular localization of ICP0. Because the major regulatory activities of ICP0, ICP4, and ICP27 are expressed in the nucleus, the ability of these three proteins collectively to determine their own localization patterns within cells adds a new dimension to the complex process of viral gene regulation in HSV.     

Herpes simplex virus ICP27 activation of stress kinases JNK and p38

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 and p38 activation required viral gene expression. Cycloheximide reversal or phosphonoacetic acid treatment of wild-type virus-infected cells as well as infection with the ICP4 mutant vi13 indicated that only the immediate-early class of viral proteins were required for SAPK activation. Infection with ICP4, ICP27, or ICP0 mutant viruses indicated that only ICP27 was necessary. Additionally, we determined that in the context of virus infection ICP27 was sufficient for SAPK activation and activation of the p38 targets Mnk1 and MK2 by infecting with mutants deleted for various combinations of immediate-early proteins. Specifically, the d100 (0-/4-) and d103 (4-/22-/47-) mutants activated p38 and JNK, while the d106 (4-/22-/27-/47-) and d107 (4-/27-) mutants did not. Finally, infections with a series of ICP27 mutants demonstrated that the functional domain of ICP27 required for activation was located in the region encompassing amino acids 20 to 65 near the N terminus of the protein and that the C-terminal transactivation activity of ICP27 was not necessary.     

Prolonged gene expression and cell survival after infection by a herpes simplex virus mutant defective in the immediate-early genes encoding ICP4, ICP27, and ICP22.

Very early in infection, herpes simplex virus (HSV) expresses four immediate-early (IE) regulatory proteins, ICP4, ICP0, ICP22, and ICP27. The systematic inactivation of sets of the IE proteins in cis, and the subsequent phenotypic analysis of the resulting mutants, should provide insights into how these proteins function in the HSV life cycle and also into the specific macromolecular events that are altered or perturbed in cells infected with virus strains blocked very early in infection. This approach may also provide a rational basis to assess the efficacy and safety of HSV mutants for use in gene transfer experiments. In this study, we generated and examined the phenotype of an HSV mutant simultaneously mutated in the ICP4, ICP27, and ICP22 genes of HSV. Unlike mutants deficient in ICP4 (d120), ICP4 and ICP27 (d92), and ICP4 and ICP22 (d96), mutants defective in ICP4, ICP27, and ICP22 (d95) were visually much less toxic to Vero and human embryonic lung cells. Cells infected with d95 at a multiplicity of infection of 10 PFU per cell retained a relatively normal morphology and expressed genes from the viral and cellular genomes for at least 3 days postinfection. The other mutant backgrounds were too toxic to allow examination of gene expression past 1 day postinfection. However, when cell survival was measured by the capacity of the infected cells to form colonies, d95 inhibited colony formation similarly to d92. This apparent paradox was reconciled by the observation that host cell DNA synthesis was inhibited in cells infected with d120, d92, d96, and d95. In addition, all of the mutants exhibited pronounced and distinctive alterations in nuclear morphology, as determined by electron microscopy. The appearance of d95-infected cells deviated from that of uninfected cells in that large circular structures formed in the nucleus. d95-infected cells abundantly expressed ICP0, which accumulated in fine punctate structures in the nucleus at early times postinfection and coalesced or grew to the large circular objects that were revealed by electron microscopy. Therefore, while the abundant accumulation of ICPO in the absence of ICP4, ICP22, and ICP27 may allow for prolonged gene expression, cell survival is impaired, in part, as a result of the inhibition of cellular DNA synthesis.     

Neurons differentially activate the herpes simplex virus type 1 immediate-early gene ICP0 and ICP27 promoters in transgenic mice

Herpes simplex virus type 1 (HSV-1) immediate-early (IE) proteins are required for the expression of viral early and late proteins. It has been hypothesized that host neuronal proteins regulate expression of HSV-1 IE genes that in turn control viral latency and reactivation. We investigated the ability of neuronal proteins in vivo to activate HSV-1 IE gene promoters (ICP0 and ICP27) and a late gene promoter (gC). Transgenic mice containing IE (ICP0 and ICP27) and late (gC) gene promoters of HSV-1 fused to the Escherichia coli beta-galactosidase coding sequence were generated. Expression of the ICP0 and ICP27 reporter transgenes was present in anatomically distinct subsets of neurons in the absence of viral proteins. The anatomic locations of beta-galactosidase-positive neurons in the brains of ICP0 and ICP27 reporter transgenic mice were similar and included cerebral cortex, lateral septal nucleus, cingulum, hippocampus, thalamus, amygdala, and vestibular nucleus. Trigeminal ganglion neurons were positive for beta-galactosidase in adult ICP0 and ICP27 reporter transgenic mice. The ICP0 reporter transgene was differentially regulated in trigeminal ganglion neurons depending upon age. beta-galactosidase-labeled cells in trigeminal ganglia and cerebral cortex of ICP0 and ICP27 reporter transgenic mice were confirmed as neurons by double labeling with antineurofilament antibody. Nearly all nonneuronal cells in ICP0 and ICP27 reporter transgenic mice and all neuronal and nonneuronal cells in gC reporter transgenic mice were negative for beta-galactosidase labeling in the absence of HSV-1. We conclude that factors in neurons are able to differentially regulate the HSV-1 IE gene promoters (ICP0 and ICP27) in transgenic mice in the absence of viral proteins. These findings are important for understanding the regulation of the latent and reactivated stages of HSV-1 infection in neurons.     

Replication of ICP0-null mutant herpes simplex virus type 1 is restricted by both PML and Sp100

Herpes simplex virus type 1 (HSV-1) mutants that fail to express the viral immediate-early protein ICP0 have a pronounced defect in viral gene expression and plaque formation in limited-passage human fibroblasts. ICP0 is a RING finger E3 ubiquitin ligase that induces the degradation of several cellular proteins. PML, the organizer of cellular nuclear substructures known as PML nuclear bodies or ND10, is one of the most notable proteins that is targeted by ICP0. Depletion of PML from human fibroblasts increases ICP0-null mutant HSV-1 gene expression, but not to wild-type levels. In this study, we report that depletion of Sp100, another major ND10 protein, results in a similar increase in ICP0-null mutant gene expression and that simultaneous depletion of both proteins complements the mutant virus to a greater degree. Although chromatin assembly and modification undoubtedly play major roles in the regulation of HSV-1 infection, we found that inhibition of histone deacetylase activity with trichostatin A was unable to complement the defect of ICP0-null mutant HSV-1 in either normal or PML-depleted human fibroblasts. These data lend further weight to the hypothesis that ND10 play an important role in the regulation of HSV-1 gene expression.