Herpes Simplex Virus Immediate-early Protein Research Articles

Binding sites for the herpes simplex virus immediate-early protein ICP4 impose an increased dependence on viral DNA replication on simple model promoters located in the viral genome.

We examined the ability of binding sites for the herpes simplex virus immediate-early protein ICP4 to alter the regulation of closely linked promoters by placing strong ICP4 binding sites upstream or downstream of simple TATA promoters in the intact viral genome. We found that binding sites strongly reduced the levels of expression at early times postinfection and that this effect was partially overcome after the onset of viral DNA replication. These data confirm that DNA-bound ICP4 can inhibit the activity of a closely linked promoter and raise the possibility that ICP4 binding sites contribute to temporal regulation during infection.

Multimerization of ICP0, a herpes simplex virus immediate-early protein.

ICP0, a herpes simplex virus immediate-early gene product, is a highly phosphorylated nuclear protein that is a potent activator of virus and host genes. Using biochemical and genetic assays employing plasmids encoding mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we mutant forms of ICP0 and a recombinant adenovirus that expresses ICP0, we provide evidence that the protein multimerizes. Some mutant forms of ICP0 were transdominant and interfered with activation of a target reporter gene or with complementation of an ICP0-minus virus.

The herpes simplex virus immediate-early protein ICP27 stimulates the transcription of cellular Alu repeated sequences by increasing the activity of transcription factor TFIIIC.

Infection with herpes simplex virus (HSV) results in an increase in the transcription of the endogenous Alu repeated sequence by RNA polymerase III. This effect is also observed in uninfected cells stably transformed with a plasmid expressing the HSV immediate-early protein ICP27 or in cells transfected with the gene encoding this protein. Both uninfected cells expressing ICP27 and cells infected with virus producing functional ICP27 display increased activity of the cellular transcription factor TFIIIC when compared with untreated cells. This increase is not observed, however, in cells infected with a mutant strain of virus which does not produce ICP27. Hence ICP27 induces elevated Alu transcription by activating transcription factor TFIIIC, which is the limiting factor for such transcription. This is the first report of increased activity of a cellular transcription factor during HSV infection, when most cellular gene activity is inhibited.

Promoter-independent activation of heterologous virus gene expression by the herpes simplex virus immediate-early protein ICP27

The herpes simplex virus (HSV) immediate-early protein ICP27 has been postulated to play an auxillary role in HSV gene expression, augmenting or inhibiting the activation of different viral promoters by the other immediate-early proteins ICPO and ICP4. Here we show that ICP27 alone can up-regulate gene expression of a retroviral vector containing Moloney murine leukemia virus (MoMuLV) regulatory sequences. This is the first report of an effect of ICP27 on gene expression driven by heterologous virus regulatory sequences. The effect does not involve the region of ICP27 which inhibits gene activation of HSV early gene promoters by ICPO and ICP4, but rather is dependent on the same region of ICP27 as is required to augment the activation of HSV late gene promoters by ICPO and ICP4. This indicates that the two activation effects are likely to operate via the same mechanism. Activation by ICP27 is dependent on the 3′ LTR and adjacent region of MoMuLV but is independent of the promoter in the 5′ LTR which can be replaced by a heterologous promoter such as that of SV40 without affecting activation by ICP27. The significance of this effect for an understanding of the mechanism of action of ICP27 and its role in regulating the gene expression of HSV and potentially of other viruses is discussed.

Activation of the cellular transcription factor AP-1 in herpes simplex virus infected cells is dependent on the viral immediate-early protein ICPO.

Lytic infection with herpes simplex virus (HSV) results in the repression of most host cell protein synthesis but produces an increased activity of the cellular AP-1 transcription factor. This increase is paralleled by an increase in the transcription rate of the proto-oncogene encoding the AP-1 component, c-Jun resulting in an increase in c-Jun protein in infected cells. The increased AP-1 activity in infected cells is dependent upon the HSV immediate-early protein ICPO. Thus a mutant lacking the gene encoding this protein fails to increase AP-1 activity whilst an ICPO expression plasmid can specifically increase the activity of an AP-1 dependent promoter in co-transfection experiments. The implications of these effects in the interaction of HSV with cultured cells are discussed.

Variable requirements for herpes simplex virus immediate-early proteins in the expression of the adenovirus E2 gene

Regulation of the adenovirus E2 gene by trans-activating proteins encoded by herpes simplex virus was investigated. Coinfection of Vero cells was performed with Ad5 dI3I2 (an E1 deletion mutant) and either wildtype HSV, mutant virus encoding a temperature-sensitive ICP4 protein (tsK), or mutants carrying deletions in the ICP4 (d120) or ICPO (dl × 3.1) gene. As detected by the presence of E2 mRNA, or the product of the E2 gene, 72-kDa DNA binding protein (DBP), functional ICP4 was sufficient for expression of the E2 gene. Regulation of E2 gene expression was at the level of transcription activation as judged by nuclear run-on assay. In contrast to results when Vero cells were coinfected, expression of 72-kDa DBP in CN3 cells, carrying an integrated copy of the E2 gene, required expression of both HSV immediate-early proteins. These results suggest that the DNA-protein organization of the target gene sequence may play a significant role in the ability of viral regulatory proteins to activate expression of heterologous as well as homologous genes.

The herpes simplex virus immediate-early protein, ICP4, is required to potentiate replication of human immunodeficiency virus in CD4+ lymphocytes

The interaction of human immunodeficiency virus (HIV) and herpes simplex virus (HSV) was investigated in an acute whole-virus coinfection system. CD4+ lymphoid CEM cells were infected with HIV-1 and, 24 h later, superinfected with HSV-1 (strain KOS) or HSV mutants possessing defined deletions in genes specifying the immediate-early transcriptional regulatory proteins ICP0, ICP4, or ICP27. Marked potentiation of HIV replication was demonstrated with the KOS strain, the ICP0 mutant, and the ICP27 mutant, but not with the ICP4 mutant, indicating that ICP4 is essential and ICP0 and ICP27 are nonessential for this effect. These studies demonstrate that HSV can be a potent stimulator of HIV replication and gene expression in coinfected CD4+ cells through the activity of the HSV regulatory protein ICP4.

In cell lines constitutively synthesizing a temperature-sensitive ICP4 protein of herpes simplex virus type 1, amount and function of ICP4 are both regulated by temperature

We have established two cell lines that constitutively synthesize a temperature-sensitive form of ICP4, the herpes simplex virus immediate-early protein that activates early and late transcription. ICP4 in both cell lines was confirmed to be functionally temperature sensitive when tested by complementation of an ICP4 deletion mutant virus for expression of viral early and late genes. When grown at the permissive temperature the two cell lines contained approximately 5 and 25%, respectively, of the ICP4 present in control HSV-infected cells. If the cells were grown at the nonpermissive temperature, ICP4 levels were reduced by approximately fourfold; a twofold reduction was observed in control cells synthesizing the wild-type protein. The lower levels of ICP4 at the nonpermissive temperature were the result of two effects: a decrease in mRNA which was similar in cells producing the mutant or wild-type form of ICP4 and a more rapid turnover of the protein which was greater for the mutant than for the wild-type form. Our observations of lower levels of ICP4 in producer cells differ from published reports of overproduction of immediate-early proteins at the nonpermissive temperature in human or hamster cells infected with ICP4 temperature-sensitive mutant viruses. This discrepancy may be related to cell species differences since we observed only a modest twofold overproduction of immediate-early proteins at the nonpermissive temperature in infections of mouse cell lines with an ICP4 temperature-sensitive mutant virus.

Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that ICP 4 is associated with progeny virus DNA.

The localization of ICP 4, ICP 8, DNA polymerase and alkaline exonuclease within herpes simplex virus type 1 (HSV)-infected cells has been examined by immunofluorescence using specific antibodies to these proteins. Cells were simultaneously counterstained with the DNA-binding fluorochrome 4,6-diamidino-2-phenylindole (DAPI) to reveal the intranuclear distribution of DNA. These studies showed that in the absence of virus DNA replication ICP 4, ICP 8 and DNA polymerase were diffusely distributed throughout the nucleus but during virus DNA replication these proteins accumulated at specific foci within the nucleus. Initially these foci were near the nuclear membrane but with continuing virus DNA replication they increased in size until the whole of the nucleus became affected. The increase in size of these foci was coincident with a redistribution of nuclear DNA and margination of chromatin at the nuclear membrane, as revealed by DAPI staining. The number of foci initially present in an infected cell was dependent on the multiplicity of infection. The distribution of ICP 4, ICP 8 and DNA polymerase within the nucleus was altered by treating the cells with DNase. The majority of alkaline exonuclease was diffusely distributed throughout the nucleus during virus DNA replication and did not localize at specific foci within the nucleus. Autoradiographic examination of the incorporation of [3H]thymidine in cells infected with HSV showed that viral DNA replication occurred in restricted areas within the nucleus that were similar, in terms of number, location and size, to the foci where ICP 4, ICP 8 and DNA polymerase accumulated. Furthermore, in cells blocked in mitosis following infection with HSV, ICP 4, ICP 8 and DNA polymerase, but not alkaline exonuclease, localized in areas outside the condensed chromatin structures. DAPI staining revealed the presence of DNA in these areas and, as such structures were never seen when uninfected cells had entered mitosis, it is suggested that this extrachromosomal DNA is of viral origin. These studies therefore suggest that ICP 4 is associated with progeny virus DNA and that while its intranuclear localization is initially at non-viral sites, as DNA replication proceeds so ICP 4 is recruited into areas of virus DNA transcription and replication.

Herpes simplex virus immediate-early protein ICP4 in murine models of latency.

Herpes simplex virus-1 (HSV) infection of trigeminal and dorsal root ganglia was established in mice via corneal scarification and footpad injection, respectively. Trigeminal and dorsal root ganglia were removed during the acute and latent stages of infection and processed for the immediate-early HSV polypeptide ICP4 (VP175) using both a monoclonal reagent and polyclonal antiserum and the avidin biotin complex immunoperoxidase method. ICP4 (VP175) antigen was readily detected during the acute infection of both dorsal and trigeminal ganglia, but not during latency. This antigen could again be detected by reactivation of the latent virus by explanation and organ culture. The detection of ICP4 (VP175) during latency in a rabbit model but not in a murine model may correlate with biologic differences in each system (e.g., rabbits spontaneously reactivate). Alternatively, the discrepancy could reflect viral strain characteristics or may imply that ICP4 (VP175) need not be constitutively expressed (at detectable levels) in all models of latent infection.

Cells that constitutively express the herpes simplex virus immediate-early protein ICP4 allow efficient activation of viral delayed-early genes in trans.

To study the role of herpes simplex virus type 1 immediate-early proteins in the transcriptional activation of herpes simplex virus genes, we isolated stably transformed cells expressing herpes simplex virus type 1 ICP4, an immediate-early protein known from previous studies to be necessary for delayed-early and late transcription. These cells efficiently expressed six delayed-early herpes simplex virus genes introduced by viral superinfection, in the absence of de novo viral protein synthesis. In contrast, the delayed-early gene encoding alkaline exonuclease and the late gene encoding the capsid protein VP5 were expressed at much lower levels. Expression of a second late gene, that for glycoprotein C, was undetectable under the same experimental conditions. These results suggest that many, but not all, delayed-early genes are efficiently activated by ICP4; in addition, they demonstrate that although the late gene for VP5 is detectably activated by ICP4, its full expression requires additional factors.