Inhibition of human immunodeficiency virus replication by the herpes simplex virus virion host shutoff protein

Abstract

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...

Herpes simplex virus type 1 morphogenesis and virus‐cell interactions: significance of cytoskeleton and methodological aspects

“…The genes U L 41, γ 1 34.5, U L 20 and ORF P exemplify how the virus blocks the cell stress response to viral infection (21, 26). The virion host shutoff gene (vhs) encoded by viral U L 41 ensures degradation of host cell mRNAs and rapid turnover of viral mRNAs (26, 42) in favour of viral replication and virion associated host shutoff. The U L 41‐encoded protein is a tegument protein, maybe a ribonuclease, also present in light particles and is not essential for virus replication (26).…”

“…Their attraction is due to their abilities to remain latent in their host for life, to be reactivated, to cause a variety of infections, and there is still no final cure or effective vaccination available. Insights in the herpesvirus‐cell interactions are of general cell biological interest, especially to studies of DNA and RNA biogenesis (31) and intracellular transport (32), and of considerable significance in the efforts to generate antiviral (32–34) and anticancer (35–37) treatments, vaccines (38, 39), and gene therapy of, for instance, stroke, human immunodeficiency virus (HIV) infections, cancer and graft‐versus‐host diseases (40–43). The heavy reliance of viruses on the normal functions of the host cell and the latent stage make it difficult to prepare an effective vaccine or drug that interferes with the replication of the virus without simultaneously damaging the host cell.…”

Human immunodeficiency virus type I as a target for gene therapy

“…The herpes simplex shutoff gene (vhs) encodes a protein which nonspecifically accelerates the degradation of mRNA molecules to facilitate shutoff of host protein synthesis during lytic infections. Hamouda et al recently tested the ability of vhs expressed from the viral LTR promoter to inhibit HIV-1 replication (172). They demonstrated that the protein inhibited HIV-1 replication more than 44,000-fold when compared to a nonfunctional mutant protein.…”