Hexamethylene bisacetamide stimulates herpes simplex virus immediate early gene expression in the absence of trans-induction by Vmw65

Abstract

Hexamethylene bisacetamide (HMBA) and DMSO are known to induce differentiation of cultured erythroleukaemic cells and to enhance the reactivation of latent herpes simplex virus (HSV) after explantation of ganglia. We report that the presence of these compounds in cell culture medium overcomes the replication defect of in1814, an HSV-1 mutant with an insertion mutation that inactivates the virion transinducing factor, Vmw65 (VP16). The effect of HMBA was not cell type-specific and was attained even by a short exposure (1-5 to 5 h) to the agent early after infection. 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 RNA levels in inl814-infected cells approached the values observed in wild-type HSV-1-infected cells in the absence of HMBA. Transport of viral DNA to the cell nucleus was not affected by HMBA. The results suggest that HMBA-and DMSOmediated enhancement of reactivation from latency is due to an increase in IE RNA production. In addition, these studies demonstrate a primary effect of HMBA on gene regulation which may bea paradigm for initial events during erythroleukaemic cell differentiation.

General and Specific Alterations in Programming of Global Viral Gene Expression during Infection by VP16 Activation-Deficient Mutants of Herpes Simplex Virus Type 1

“…For example, the poorly replicating viral mutant strain in1814 contains a fouramino-acid insertion in VP16 that prevents interaction with Oct-1 and is unable to activate IE transcription in standard infection conditions (1). By mechanisms that are not yet well understood, HMBA compensates for the loss of activation by VP16 and permits viral replication and production (17).…”

Herpes simplex virus type 1 immediate early gene expression is stimulated by inhibition of protein synthesis.

“…The Moloney murine leukaemia virus (Momulv) LTR promoter was substituted for the ICP0 promoter of in1814 to yield in1820, a mutant which behaved as if devoid of ICP0 in all cell types tested except BHK, and a temperature-sensitive mutation in the ICP4 coding sequences, cloned from tsK, was introduced into in1820 (Preston et al, 1997). The resultant virus, in1820K, fails to produce functionally significant amounts of the three major transactivator proteins VP16, ICP0 and ICP4 when most cell types are infected at temperatures above 38 mC, yet it can be propagated to high titres in BHK cells at 31 mC in the presence of hexamethylene bisacetamide [HMBA, an agent which complements in1814 (McFarlane et al, 1992)]. Mutant in1820K can be used to infect many cell types, except BHK, at relatively high m.o.i.…”

Reactivation of latent herpes simplex virus from explanted dorsal root ganglia

“…They were cultured for 1, 2, 3, 4, 5, 7 or 21 days at 37 °C in BHK medium containing 10% NCS, 10% fetal calf serum (FCS), antibiotics and 2-5 gg/ml amphotericin B. Supernatants from DRG of 10 mice were removed at each time point to be assayed in the presence of 3 mMhexamethylene bisacetamide (HMBA; Sigma) for release of reactivated virus. In the presence of HMBA, in1814 plaques on BHK cells with the same efficiency as wt HSV-1 (McFarlane et al, 1992) but the drug has no effect on the efficiency of plaque formation by wt HSV-1 and so was used routinely in the assay, irrespective of the virus type. Alternatively, cultured DRG were also fixed at the daily intervals in 4% paraformaldehyde, embedded and serially sectioned (5 Ixm) onto slides coated with aminopropyltriethoxysilane (Maddox & Jenkins, 1987), as previously described (Ecob-Prince et al, 1993a), to detect virus-specific antigen and mRNA produced by virus reactivating in the explanted DRG.…”