Search

Vaccinia virus recombinants containing the sequences from herpes simplex virus type 1 (HSV-1) encoding the immediate early (IE)(α) proteins ICP4 and ICP0, under the control of a mutated vaccinia virus 11 K late promoter, were constructed. A cDNA copy of the gene encoding ICP0 and an ICP4-encoding genomic segment were each inserted into the vaccinia virus genome at the thymidine kinase (TK) locus by homologous recombination. Steady-state analyses revealed that RNAs homologous to the IE-0 and IE-4 sequences accumulated in cells infected by recombinants with the kinetics of a typical vaccinia late mRNA. Western blot analyses demonstrated that the expression level of both ICP0 and ICP4, produced by the recombinant viruses, was comparable to that in HSV-1-infected cells at late times postinfection. Both proteins synthesized in cells infected by the recombinants were located in the nucleus as revealed by immunofluorescence. Althoughin vitro studies reveal that extracts from vaccinia-virus-infected cells lose the ability to transcribe genes that contain RNA polymerase II promoters (Puckett and Moss 1983), Ce11 35, 441–448) both ICP0 and ICP4 expressed by the recombinant viruses can transactivate plasmids containing a reporter gene driven by the promoters for the HSV-1 TK and glycoprotein C genes. Nuclear extracts prepared from cells infected with the vaccinia virus vector expressing ICP4 exhibited sequence-specific DNA-binding activity.

The IE-0 gene of herpes simplex virus type 1 (HSV-1) contains two introns and encodes ICP0, a powerful transcriptional activator. We have isolated a cDNA clone that encodes ICP0 from a lambda gt10 cDNA library constructed from RNAs made from HSV-1-infected HeLa cells. DNA sequence analysis of this clone confirmed the predicted intron/exon boundaries (L. J. Perry, F. J. Rixon, R. D. Everett, M. C. Frame, and D. J. McGeoch, J. Gen. Virol. 67:2365-2380, 1986). Following transfection, a plasmid containing the cDNA copy of IE-0 directed the synthesis of ICP0, which was appropriately compartmentalized and distributed in the nucleus, as revealed by immunofluorescence. A transient expression assay was used to demonstrate that this cDNA copy retained the ability to transactivate the HSV-1 promoters for the IE-0 gene (an immediate-early gene), the thymidine kinase gene (an early gene), and the glycoprotein C gene (a late gene). The product of this cDNA clone cooperated with ICP4 to activate expression from the thymidine kinase gene promoter in a synergistic manner. The availability of a functional cDNA copy encoding ICP0 provides the opportunity to express this protein in vector systems that do not recognize eucaryotic donor and acceptor splicing signals to overexpress ICP0.

In-frame codon insertion and deletion mutants were constructed in a plasmid containing the sequence that encodes ICP0, a transcriptional activator of herpes simplex virus type 1 (HSV-1). The effect of these mutations was analyzed in a transient expression assay using the promoters for, the IE-0 gene (an immediate early (alpha) gene), the thymidine kinase gene (an early (beta) gene), and the glycoprotein C gene (a late (gamma) gene) fused to reporter cassettes that encoded either beta-galactosidase or chloramphenicol acetyl transferase. Assays were performed in the presence or absence of a plasmid encoding ICP4, the major regulatory protein of HSV-1. Our results demonstrate that ICP0-mediated transactivation varied depending on the position of the insertion in the gene. One region of this protein was consistently shown to be required for full activation of each promoter examined either in the presence or in the absence of ICP4. This region overlaps with a cysteine-rich region and coincides with a transactivator domain identified in another extensive mutational analysis of this sequence. Analysis of the deletion mutants generated in this study demonstrated that the carboxy-terminal regions were required for activation in certain circumstances and that this varied depending on the promoter being assayed and the cell type in which the analysis was performed.

Vaccinia virus recombinants expressing the herpes simplex virus type 1 (HSV-1) genes encoding ICP0 or ICP4 were used to identify the precise target antigen(s) of murine anti-viral cytotoxic T lymphocytes (CTL) specific for the non-structural immediate early proteins. These studies revealed that HSV-1-specific CTL, restricted to class I major histocompatibility complex genes of the H-2k haplotype but not the H-2d or H-2b haplotypes, would lyse autologous cells expressing ICP4. HSV-1-specific CTL derived from various mice strains failed to lyse target cells expressing ICP0. Calculation of the frequencies of H-2k-restricted virus-specific CTL demonstrated that approximately a third of the total HSV-1-specific response was directed against ICP4. Immunization of mice with either recombinant vaccinia virus or transfected L cells expressing ICP4 induced HSV-1-specific lymphoproliferation and delayed hypersensitivity but CTLs were not induced. More importantly, such immunized animals were unable to resist or control a subsequent challenge with virulent HSV-1.

We have previously shown that adenovirus recombinants expressing functional ICP0 reactivate latent herpes simplex virus type 2 (HSV-2) in an in vitro latency system. This study demonstrated that ICP0, independent of other HSV gene products, is sufficient to reactivate latent HSV-2 in this in vitro system. To assess the effects of defined mutations in the sequence encoding ICP0 (IE-0) on reactivation, seven in-frame insertion and three in-frame deletion mutants were moved into an adenovirus expression vector. Each recombinant directed the synthesis of stable ICP0 of the correct size. The transactivation activity of the mutated sequences in these recombinants was similar to that when they were tested in plasmids. When these recombinants were examined for their ability to reactivate in the in vitro latency system, mutants with dramatic defects in transactivation (Ad-0/125, Ad-0/89, Ad-0/2/7, and Ad-0/88/93) were unable to reactivate latent HSV-2 independent of the multiplicity of infection. An exception to this correlation was the finding that Ad-0/89, which transactivated poorly, was able to reactivate latent virus after prolonged incubation whereas other transactivation-deficient mutants could not. Moreover, the presence of ICP4 did not compensate for the inability of any of the recombinants tested to reactivate HSV-2. These results show that (i) the transactivation domains of ICP0 are also used in reactivation, (ii) the presence of another essential HSV regulatory protein ICP4 does not alter the pattern of reactivation by ICP0, and (iii) mutations in some regions of IE-0 previously shown to affect viral growth and plaque formation did not alter its ability to reactivate in this in vitro system.

ICP0, one of the five immediate-early (IE) gene products of herpes simplex virus (HSV), is a potent activator of transcription. To assess the biological activities of ICP0 and to explore its mechanisms of action, two helper-independent recombinant adenoviruses were constructed. In each recombinant, the E1 region was substituted with the ICP0-encoding genomic segment under the control of either the adenovirus major late promoter (MLP-0) or the HSV IE-0 promoter (0PRO-0). Infection of HeLa cells or 293 cells (a human embryo kidney cell line expressing adenovirus 5 E1a and -b functions) with the MLP-0 recombinant results in the synthesis of more IE-0 mRNA and ICP0 protein than did infection with the 0PRO-0 recombinant. Although 293 cells infected with MLP-0 accumulate 5- to 10-fold more IE-0 mRNA late in the infection than cells infected with HSV, the level of the protein product, ICP0, increased only slightly. In 293 cells, both recombinants could replicate, albeit at a slower rate and with lower final yields than wild-type adenovirus. Neither virus replicates its DNA in HeLa cells, and thus ICP0 cannot substitute for adenovirus E1a; however, the level of ICP0 that accumulates in MLP-0-infected HeLa cells was comparable to that of HSV-infected HeLa cells. In a functional test, we demonstrated that the adeno-ICP0 recombinant viruses can transactivate a transfected TK-CAT cassette, indicating that the ICP0 is biologically active.