An Investigation of the Effect of Hexamethylene Bisacetamide on Herpes Simplex Virus Gene Expression

A number of functionally related chemicals have been identified which overcome the replication defect of in1814, an HSV-1 mutant with a 12bp insertion mutation that inactivates the virion transinducing factor, Vmw65. Hexamethylene bisacetamide (HMBA) and dimethylsulphoxide (DMSO) are potent inducers of terminal differentiation in cultured erythroleukaemic cells, and have also been shown to increase the speed and efficiency of reactivation of latent HSV-1 and HSV-2 after explantation of ganglia. The work presented in this thesis demonstrates that the presence of 3-5mM HMBA in the cell culture medium, the optimum concentration for induction murine erythroleukaemic cell (MELC) differentiation, increases the titre of in1814 on human foetal lung (HFL) cells 500-fold, enabling the mutant to initiate infection almost as efficiently as wild type HSV-1. The titre of 1814R, the rescued "revertant" virus, was virtually unaffected by the presence of 5mM HMBA. Related compounds DMSO and hypoxanthine, which induce MELC differentiation, also significantly increased the titre of i n 1814. A number of known metabolites of HMBA did not increase the titre of in1814, either alone or in combination with various concentration of HMBA. The effect of HMBA is not cell type specific, as it was reproduced in a number of cell lines including HFL, BHK and HeLa. Pre-treatment of cells with HMBA prior to infection had no effect on the subsequent titre of in1814, demonstrating that HMBA does not act by inducing stable cellular changes. HMBA exposure was shown to be required only transiently, immediately after adsorption, indicating that HMBA functions at the beginning of the virus life cycle. RNA dot blot analysis revealed that the presence of HMBA resulted in an increase in immediate early (IE) RNA accumulation after infection of cells in the presence of cycloheximide, such that the RNA levels in in1814-infected cells approached values observed in wild type HSV-1-infected cells in the absence of HMBA. The observation that HMBA increases IE-RNA levels in the presence of cycloheximide, suggests that its target is a pre-existing cellular or viral component(s). Southern blot analysis of nuclear DNA isolated from HFL cells infected with in1814 in the presence or absence of HMBA, demonstrated that the transport of viral DNA to the cell nucleus was not affected by HMBA. A range of plasmids with the chloroamphenicol acetyl transferase (CAT) gene under the control of various viral promoters were introduced in HFL cells by lipofection. In this transient expression system, no detectable promoter or sequence specificity was identified in the mode of action of HMBA since increased CAT activity was observed from IE, early and late HSV-1 promoters and from the human cytomegalovirus (HCMV) and SV40 enhancers, in the presence of 5mM HMBA. The question of specificity was subsequently addressed using a range of mutant viruses which allowed direct investigation into the effect of HMBA on different promoters in the viral genome. These results demonstrated that HMBA does not substantially compensate for the absence of Vmw110 or adenovirus (Ad) El a, nor does it act as or induce an active cellular homologue of Vmw175. Further analyses revealed that the HCMV enhancer and S V40 promoter/enhancer are responsive to activation by HMBA when integrated into the HSV genome. Gel retardation analysis revealed that HMBA does not promote IEC formation with the TAATGARAT motif nor does it act as a "linker molecule", allowing mutant Vmw65, extracted from in1814 virions, to interact with Oct-1 and TAATGARAT. Therefore HMBA does not act as or induce a cellular homologue of Vmw65. In addition, HMBA-treatment of nuclear extracts did not promote complex formation at other protein binding motifs involved in regulation of transcription. The HMBA effect on the titre of in1814 was not attributable to its demethylating properties as treatment with 5-azacytidine, an inhibitor of DNA methylation, did not increase the titre of in1814. Addition of 811M 5-azacytidine actually decreased the titre of in 1814, especially when present both prior to and after the addition of the virus, whereas little effect was seen on the titre of 1814R. HMBA-mediated modulation of protein kinase C (PKC) activity did not appear to be responsible for the effect on the titre of in1814, as an inhibitor of HMBA-induced MELC differentiation, 12-O-tetradecanoylphorbol- 13-acetate (TPA), did not antagonize the HMBA effect on in 1814 titre. In addition, exposure to leupeptin, which is known to inhibit the conversion of membrane bound inactive PKC to the soluble activated form, also failed to antagonize the effect of HMBA on the titre of in 1814 . The addition of 5mM HMBA to the cell culture medium increased the efficiency of calcium phosphate-mediated transfection approximately threefold, for both wild type HSV-1 and in1814 viral DNA