Herpes Simplex Virus Type 1 Immediate-early Gene Research Articles

Identification and control of the cis-acting elements of the immediate early gene of equid herpesvirus type 1.

Consensus cis-acting DNA sequences upstream of the immediate early (IE) gene of equid herpesvirus type 1 (EHV-1, strain Ab4) were identified. One copy of the conserved motif TAATGARATTC, which is the binding site for the host cellular factor Oct-1 and herpes simplex virus type 1 (HSV-1) virion protein, VmW65, complex, was identified at positions -630 to -620. Using transient transfections and chloramphenicol acetyltransferase assays the IE promoter of EHV-1 was shown to be trans-activated by VmW65 within the region -685 to +73. Ultraviolet light-inactivated EHV-1 was able to stimulate the expression of the IE gene of EHV-1 as well as HSV-1, indicating that EHV-1 possesses a protein equivalent to VmW65. The ubiquitous equid herpesvirus type 2 (EHV-2), which is not known to be a primary pathogen, was also able to trans-activate the EHV-1 and HSV-1 IE genes. Further work is being performed in order to identify the nature of the EHV-1 and EHV-2 trans-activating proteins.

Transactivation of the HIV-1 LTR by HSV-1 immediate-early genes

Herpes simplex virus type 1 (HSV-1) activates transcription from the long terminal repeat (LTR) promoter region of the human immunodeficiency virus type 1 (HIV-1). HSV-1 immediate-early (IE) genes ICPO and ICP4 are thought to be important mediators of this process, which is known to involve the induction of the cellular activators NF- κB and Sp1. We demonstrate that ICPO and ICP4 transactivation of the LTR is largely dependent on the presence of NF- κB and Sp1 binding sites. However, in Jurkat CD4-positive lymphocytes, HSV-1 activates LTR constructs lacking all NF- κB or Sp1 binding sequences. This effect is still evident when all sequences upstream of the TATA motif are removed. Such enhancer-independent transactivation can be produced by cotransfection of ICPO and ICP4. Thus HSV-1 IE genes transactivate the HIV-1 LTR both through the induction of NF- κB and Sp1 and through another as yet undefined cellular factor.

Deletion of the VP16 open reading frame of herpes simplex virus type 1.

VP16 (also called Vmw65 and alpha TIF) is a structural protein of herpes simplex virus type 1 (HSV-1) that trans-induces HSV-1 immediate-early gene transcription. This report describes an HSV-1 VP16 deletion mutant that was constructed and propagated in a cell line transformed with a VP16 expression vector. The VP16 deletion mutant replicated like wild-type HSV-1 during infection of the VP16-expressing cell line. Deletion mutant virions propagated in this cell line contained wild-type, cell-derived VP16 protein that was recruited during virion assembly and was functional for immediate-early gene trans-induction. The mutant failed to replicate during subsequent infection of cells that do not express VP16, as determined in plaque assays and single-step replication assays. The deletion mutant induced nearly normal levels of viral DNA synthesis and capsid production during these infections, but it induced slightly lower levels of viral DNA encapsidation and appeared by transmission electron microscopy to be defective in further steps of virion maturation. A genetic revertant of the deletion mutant that was restored for VP16-coding sequences exhibited fully wild-type replication properties in both VP16-expressing and nonexpressing cells. The absence of VP16 protein synthesis at late times of HSV-1 infection prevents the production of infectious progeny virus and correlates with a profound defect in HSV-1 particle assembly.

Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus.

The herpes simplex virus type 1 (HSV-1)-mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human alpha 2 interferon (HuIFN-alpha 2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIV alpha 1 cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIV alpha 1 cells, HuIFN-alpha 2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIV alpha 1 cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIV alpha 1 cells. No major differences could be detected in the expression of HSV-1 immediate-early (IE) genes IE175 and IE110 (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIV alpha 1 cells to account for the inability of HSV-1 to induce HIV-1 in HNHIV alpha 1 cells. However, major differences were observed in the binding pattern of NF-kappa B-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-kappa B-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIV alpha 1 cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes.

Differentiation of the U937 macrophage cell line removes an early block of HSV-1 infection.

In the human macrophage-like cell line U937, which is resistant to infection with herpes simplex virus type 1 (HSV-1), it was previously shown that resistance can be overcome by inducing differentiation of the cells by treatment with phorbol 12-myristate 13-acetate (PMA). The present data show that differentiation, and not PMA treatment alone, enabled HSV-1 replication, because vitamin D3 and mezerein were also able to cause U937 cells to differentiate to a state permissive for HSV-1 infection. Additionally, a portion of the undifferentiated cells underwent a productive infection when treated with PMA 2 days after infection, suggesting persistence of HSV-1 in these cells. The nonpermissiveness of the undifferentiated cells was further defined. Resistance did not involve differences in virus uptake, because the amounts of viral DNA in the infected cells and nuclei of differentiated and undifferentiated U937 cells were not significantly different early after infection. However, only very low levels of RNA for HSV-1 immediate-early, early, and late genes could be detected in the undifferentiated U937 cells by Northern blot analysis compared with the differentiated U937 cells. These data suggest that the primary block in HSV-1 replication in undifferentiated U937 cells occurred after transport of the viral DNA to the cell nucleus but prior to steady-state accumulation of viral RNA for immediate-early genes.

Purification of a set of cellular polypeptides that bind to the purine-rich cis-regulatory element of herpes simplex virus immediate early genes.

Expression of herpes simplex virus type 1 (HSV1) immediate early (IE) genes is activated by a polypeptide component of the mature virion termed viral protein 16 (VP16). Stimulation of IE expression by VP16 operates via two cis-regulatory sequences: TAATGARAT, and the purine-rich hexanucleotide sequence GCGGAA. VP16 does not bind directly to either of the IE cis-regulatory sequences. Rather, these elements appear to represent binding sites for host cell proteins. Herein, we report the purification of a host cell factor that binds to the GCGGAA motif. We show further that this factor is capable of binding in vitro to an oligomerized form of the hexanucleotide sequence GAAACG, which is common to a variety of virus- and interferon-inducible genes. The GAAACG repeats of interferon- and virus-inducible genes, and the GA-rich repeats of HSV1 IE genes confer similar functional properties when appended to the promoter of a heterologous gene. These observations raise the possibility that HSV1 may activate its IE genes in a manner that exploits one of the components used by mammalian cells to combat virus infection.

Trans-activation of Herpes Simplex virus type 1 immediate early genes is specifically inhibited by human recombinant interferons

The effect of human recombinant interferons (IFNs) alpha and gamma on the transcription of Herpes Simplex virus type-1 (HSV-1) immediate early (IE) genes has been studied. Human amniotic cells (WISH) were transfected with a chimaeric plasmid containing the regulatory region of the HSV-1 IE genes controlling the expression of a reporter gene (CAT). Subsequently cells were infected with live HSV-1 to trans-activate the IE sequences. When cell samples were treated with IFNα or IFNγ prior to viral infection, transcription of the CAT gene was greatly inhibited.

Transcriptional Activity of the Herpes Simplex Virus Genome during Establishment, Maintenance, and Reactivation of in vitro Virus Latency

We previously have described a model of in vitro herpes simplex virus (HSV) latency in which latent infection was (i) established with human leukocyte interferon (IFN-alpha) in combination with (E)-5-(2-bromovinyl)-2'-deoxyuridine (BVDU) or 9-[(2-hydroxyethoxy)methyl]guanine (acyclovir); (ii) maintained after termination of combined inhibitor treatment by incubation at 40.5 degrees, and (iii) reactivated by either reducing the incubation temperature to 37 degrees or by superinfecting at the elevated temperature with human cytomegalovirus (HCMV). We now report the use of this system to examine the transcriptional activity of the HSV genome during establishment, maintenance, and reactivation of HSV latency in vitro. Numerous species of virus-specific polyadenylated RNAs were present during the first 3 days of combined BVDU and IFN-alpha treatment of HSV type 1 (HSV-1)-infected human fetus lung fibroblast cells. However, after 7 days of combined inhibitor treatment, only a very small quantity of virus-specific RNA could be detected utilizing the short unique region of the HSV-1 genome as probe. After terminating combined BVDU and IFN-alpha treatment and increasing the temperature from 37 to 40.5 degrees on day 7 after infection, virus-specific RNA was undetectable by RNA blot hybridization analysis; however, a small amount of HSV-specific RNA was detected in 2% of the cells by in situ hybridization. The HSV-1 transcriptional products produced after HCMV superinfection in the presence of selected inhibitors of macromolecular synthesis also were examined and demonstrated that the efficient activation of HSV-1 immediate-early gene transcription required the expression of not only immediate-early HCMV gene product(s), but also at least a subset of early-late gene products.

Promoter sequence and cell type can dramatically affect the efficiency of transcriptional activation induced by herpes simplex virus type 1 and its immediate-early gene products Vmw175 and Vmw110.

The activation of transcription of the early and late classes of viral genes during infection by herpes simplex virus type 1 (HSV-1) requires the prior expression of immediate-early (IE) gene products. The IE gene products can also activate certain cellular and heterologous viral promoters. This paper presents a thorough analysis of transactivation of the HSV-1 glycoprotein gD and simian virus 40 early promoters, and two other promoters that are hybrids of both, under a variety of experimental conditions. Two methods of transactivation (superinfection with virus and co-transfection with isolated IE genes) have been used with all four target promoters in a variety of cell types. The conclusions are: (1) promoter sequence affects the efficiency of promoter activation by infectious HSV-1 virus, but this activation is not restricted to HSV promoters; (2) cell type affects the efficiency of promoter activation by HSV-1, and this can lead to a failure to activate a promoter in one cell type but not in others in which activation is generally more efficient; (3) a promoter can be activated to different extents in co-transfection experiments using plasmids carrying isolated IE genes that express Vmw110 or Vmw175 or when both are used together; (4) the pattern of activation of a promoter by the IE gene products in cotransfection experiments varies in different cell types; (5) changes in promoter sequence can alter the pattern of activation by the different IE polypeptides, and this pattern can again differ in different cell types; (6) other apparently minor experimental variables, as might exist between the standard methods used in different laboratories, can also affect the patterns of activation observed. The results are discussed in terms of the mechanism of action of the HSV-1 IE gene products and the limitations of the co-transfection assay.

Activation of the human immunodeficiency virus by herpes simplex virus type 1

Herpes simplex virus type 1 (HSV-1) and some of its immediate-early genes stimulate expression of the human immunodeficiency virus (HIV) long terminal repeat (LTR) sequences and the replication of HIV itself. To demonstrate this, the HIV LTR was linked to the indicator gene chloramphenicol acetyltransferase (CAT) and transfected into Vero cells with or without the trans-activating gene (tat) of HIV. Infection of these cells with HSV-1 strain KOS or temperature-sensitive mutant tsB21 or tsE6 resulted in a large increase in CAT activity in the absence of tat and further augmentation in the presence of tat. This stimulation was seen at both their permissive (34 degrees C) and nonpermissive (39 degrees C) temperatures, implying either that HSV-1 infection or immediate-early gene expression is all that is required. In cotransfection assays in Vero cells, cloned HSV-1 immediate-early genes ICP0 and ICP4 stimulated CAT activity in the presence of tat, while ICP27 had no effect. On the other hand, in SW480 cells, ICP4 and, to a lesser extent, ICP0 genes caused stimulation of CAT activity in the absence of tat. Deletion mutants within the HIV LTR showed that the target for HSV stimulation is distinct from the tat-responsive area and maps near the SP1 binding sites. In Hela cells, ICP0 or ICP4 stimulated the replication of a cotransfected clone of HIV, as shown by an increase in reverse transcriptase activity in the culture supernatant.