Herpes simplex virus immediate-early promoters are responsive to virus and cell trans-acting factors.

The promoters for each of the immediate-early genes from herpes simplex virus type 1 were cloned and fused to a chloramphenicol acetyltransferase cassette. These chimeric genes were used as targets in a transient expression assay to determine how the immediate-early gene products ICP4 and ICP0 and the virion-associated stimulatory protein Vmw65 affected their expression in HeLa and Vero cells. The basal level of expression from these cassettes differed significantly depending on the extent of 5'-flanking sequence and the cell line that served as host. The promoters from IE-4 and IE-0 behaved in a qualitatively similar fashion independent of the host cell. However, the promoter for ICP27 had a unique response pattern: in Vero cells it acted as an alpha gene promoter, whereas in HeLa cells its response was more like that of a beta gene promoter. The promoter sequences for ICP22 and ICP47 behaved as the IE-4 and IE-0 promoters did in HeLa cells, but their response to the effector molecules in Vero cells was unlike that of other alpha gene promoters we have studied. Evidence is also presented for a role for ICP27 in autoregulation.     

Varicella-zoster virus complements herpes simplex virus type 1 temperature-sensitive mutants.

Varicella-zoster virus (VZV) can complement temperature-sensitive mutants of herpes simplex virus. Of seven mutants tested, two, carrying mutations in the immediate-early ICP4 and ICP27 proteins, were complemented. This complementation was not seen in coinfections with adenovirus type 5 or cytomegalovirus. Following transfection into CV-1 cells, a DNA fragment containing the VZV short repeat sequence complemented the ICP4 mutant. These data demonstrate a functional relationship between VZV and herpes simplex virus and have allowed localization of a putative VZV immediate-early gene.     

Quantitative analysis of cytoplasmic actin gene promoter and nuclear polyhedrosis virus immediate-early promoter activities in various tissues of silkworm Bombyx mori using recombinant Autographa californica nuclear polyhedrosis virus as vector

Cassettes harboring luciferase reporter driven by Bombyx mori cytoplasmic actin gene promoter (A3) (671 bp) and B. mori nuclear polyhedrosis virus immediate-early promoter (IE-1) (580 bp) were transferred to the bacmid AcΔEGT to generate the recombinant Autographa californica nuclear polyhedrosis viruses, AcNPVA3Luc and AcNPVIELuc, respectively. Recombinant baculoviruses were injected into the hemocoele of newly ecdysed 5th instar larvae. The activities of the A3 and IE-1 promoters in various tissues were measured by luciferase activity assay and normalized by the copy number of recombinant virus. Results showed that the activity of the A3 promoter was approximately 10-fold higher than the IE-1 promoter. The promoter activities of A3 and IE-1 were highest in the silk gland, followed by fat body, middle gut, Malpighian tubule, and hemocyte. In silk gland, activity of the two promoters was highest in posterior silk gland, followed by middle and anterior silk glands. The difference in promoter activities reflects the growth speed of tissue in silkworm larvae. The activity of the A3 promoter remained unchanged and was not inhibited significantly by viral factors at least 3–4 d post injection of rAcNPV.     

DNA binding by the herpes simplex virus type 1 ICP4 protein is necessary for efficient down regulation of the ICP0 promoter.

The herpes simplex virus type 1 ICP4 and ICP0 polypeptides are immediate-early proteins that positively and negatively regulate expression of other viral genes in trans. ICP4 has recently been shown to bind DNA bearing the consensus sequence 5'-ATCGTCNNNN(T/C)CG(A/G)C-3', present upstream of a number of viral genes. To test the hypothesis that this DNA-binding activity is involved in ICP4-mediated gene regulation, site-specific mutagenesis was employed to mutate the version of this sequence in the promoter of the ICP0 gene. The mutation eliminated detectable binding of ICP4 to the promoter as measured in vitro by a gel electrophoresis band shift assay. The ability of the mutated ICP0 promoter to direct synthesis of a reporter gene was also investigated in a transient transfection assay. Whereas ICP4 was found to transactivate the wild-type ICP0 promoter two- to threefold, the mutated promoter was transactivated seven- to ninefold. In assays containing the ICP0 transactivator gene, ICP4 down regulated the wild-type promoter far more efficiently than the mutated promoter. Finally, both the wild-type and mutated ICP0 promoters exhibited a similar response to ICP4 in transfections that included a vector expressing the viral transactivator protein VP16. These experiments suggest that the sequence-specific DNA-binding activity of ICP4 is an essential element of its role as a negative regulator of gene expression.     

Varicella-zoster virus open reading frame 4 protein is functionally distinct from and does not complement its herpes simplex virus type 1 homolog, ICP27.

Varicella-zoster virus (VZV) open reading frame 4 (ORF4) encodes a putative immediate-early protein which is homologous to herpes simplex virus type 1 (HSV-1) ICP27 on the basis of gene location and similarity in amino acid sequence. In transient expression assays, however, ORF4 and ICP27 exhibit different properties. ICP27 alone has little activity on target plasmids, but it acts as a transactivator or a transrepressor in the presence of other HSV-1 transactivators. In contrast, ORF4 directly transactivates plasmids containing homologous or heterologous promoters and has no apparent transrepressing activity. To further illuminate the functional similarities and differences between ORF4 and ICP27, Vero cell lines which express ORF4 under the inducible metallothionein promoter were constructed. Cell lines expressing functionally active ORF4 protein upregulated the expression of transfected VZV target plasmids but were unable to efficiently complement HSV-1 ICP27 mutants. These results indicate that, despite structural similarities, VZV ORF4 and HSV-1 ICP27 behave differently in transient expression assays and may play different roles in virus replication.