Aims To understand the mechanisms of Early Growth Response Protein 1 (Egr-1) induction upon HSV-1 lytic infection and its roles in regulating viral gene expression and replication. Study Design Rabbit corneal cell line SIRC and other cell lines were infected by HSV-1 to investigate the Egr-1 induction and its occupancy on the viral genome in different conditions. UV-inactivated HSV-1 and a recombinant virus over-expressing Egr-1 were generated to evaluate the regulatory effects on viral gene expression and replication during the infection. Methodology Egr-1 induction triggered by viral infection was determined by Western Blot analyses and immune-fluorescent microscopy. Real-time RT-PCR and a novel Cignal™ Reporter Assay were used for quantitative measurement of Egr-1 expression. Chromatin Immuno-precipitation (ChIP) was performed to address the Egr-1 occupancy to the viral regulatory sequences and the influence on viral replication was assessed by plaque assays. Results Our results indicated that Egr-1 expression requires viral gene expression since the UV-inactivated HSV-1 failed to produce Egr-1 protein. Blockade of viral replication did not block the Egr-1 protein synthesis, supporting the hypothesis that HSV-1 replication was not essential for Egr-1 production. Chromatin immune-precipitation (ChIP) and RT-PCR assays demonstrated that induced Egr-1 was able to interact with key regulatory elements near HSV-1 immediate-early (IE) genes and promote viral gene expression. Recombinant virus overexpressing Egr-1 revealed that Egr-1 enhanced the viral replication and the release of infectious virus. Conclusion Together these results concluded that HSV-1 triggers the expression of an important host transcription factor Egr-1 via a unique mechanism and benefit the viral gene expression and replication.

The herpes simplex virus (HSV) virion host shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules, leading to inhibition of protein synthesis. This ability to inhibit a critical cellular function suggested that vhs could be used as a suicide gene in certain gene therapy applications. To investigate whether vhs might be useful for treatment of AIDS, we tested the ability of both HSV type 1 (HSV-1) and HSV-2 vhs to inhibit replication of human immunodeficiency virus (HIV). Replication of HIV was substantially inhibited when an infectious HIV proviral clone was cotransfected into HeLa cells together with vhs under the control of the cytomegalovirus (CMV) immediate-early promoter. HSV-2 vhs was more active than HSV-1 vhs in these experiments, consistent with previously published studies on these genes. Since expression of vhs from the CMV promoter is essentially unregulated, we also tested the ability of vhs expressed from the HIV long terminal repeat (LTR) promoter to inhibit HIV replication. Wild-type HSV-1 vhs inhibited HIV replication more than 44,000-fold in comparison to a mutant vhs gene encoding a nonfunctional form of the Vhs protein. Production of Vhs in transfected cells was verified by Western blot assays. A larger amount of Vhs was observed in cells transfected with plasmids expressing vhs from the HIV LTR than from the CMV promoter, consistent with the greater inhibition of HIV replication observed with these constructs. Mutant forms of Vhs were expressed at higher levels than wild-type Vhs, most likely due to the ability of wild-type Vhs to degrade its own mRNA. The strong inhibitory activity of the vhs gene and its unique biological properties make vhs an interesting candidate for use as a suicide gene for HIV gene therapy.Cells. HeLa-CD4 ϩ -LTR-␤-gal cells (25) are CD4 ϩ and contain one integrated copy of HIV-1 LTR linked to a ␤-galactosidase gene. HLtat cells (13, 46) contain the HIV-1 LTR promoter and produce a high level of a synthetic one-exon tat gene. HL2/3 cells (7) produce high levels of Gag, Env, Tat, Rev, and Nef proteins. They do not produce detectable viral reverse transcriptase or detectable amounts of mature virions. All cell lines were obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases: HeLa cells from Richard Axel, HeLa-CD4 ϩ -LTR-␤-gal cells from Michael Emerman, and HLtat and HL2/3 cell lines from Barbara K. Felber and George N. Pavlakis.HeLa, HeLa-CD4 ϩ -LTR-␤-gal, HLtat, and HL2/3 cells were grown in Dulbecco's modified Eagle's medium with 4.5 g of glucose (Mediatech, Washington, D.C.) per liter. The medium was supplemented with 10% fetal bovine serum (Sigma Chemical Company, St. Louis, Mo.) and gentamicin (50 mg/liter; GIBCO/BRL, Life Technologies, Gaithersburg, Md.). The cells were grown at 37°C in 5% CO 2 .Plasmids. Plasmid pNL4.3, containing a full-length, replication-competent HIV proviral DNA in pUC18, was obtained through the AIDS Research...