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(a) β-Galactosidase staining of MRC-5 cells infected with in1382 or in1383 at 1, 3 and 6 days p.i. or at 6 days p.i. following induction by superinfection with HSV-2 strain 333 for 6 h. (b) β-Galactosidase staining of MRC-5 cells infected with in1382 at 6 days p.i. or at 6 days p.i. following superinfection with either HSV-1 ICP0 mutant dl1403, wild-type (wt) HSV-1 strain 17 or wt HSV-2 strain 333 for 4 h.
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f1: (a) β-Galactosidase staining of MRC-5 cells infected with in1382 or in1383 at 1, 3 and 6 days p.i. or at 6 days p.i. following induction by superinfection with HSV-2 strain 333 for 6 h. (b) β-Galactosidase staining of MRC-5 cells infected with in1382 at 6 days p.i. or at 6 days p.i. following superinfection with either HSV-1 ICP0 mutant dl1403, wild-type (wt) HSV-1 strain 17 or wt HSV-2 strain 333 for 4 h.

Mentions: Previous studies have shown that infection of cells in culture with IE gene-deficient mutants of HSV-1 results in the establishment of a quiescent state (Harris & Preston, 1991; Preston & Nicholl, 1997; Samaniego et al., 1998; Minaker et al., 2005). Quiescent genomes remain functional, as reactivation can be induced efficiently by provision of ICP0 in trans following superinfection with HSV or recombinant adenovirus (Zhu et al., 1990; Preston & Nicholl, 1997; Hobbs et al., 2001; Minaker et al., 2005). In order to facilitate analyses of the chromatin status of HSV DNA during quiescence and following the induction of reactivation, we used an MRC-5 cell-based system to generate material for ChIP analyses. Quiescent infection was established by infecting MRC-5 cells with replication-defective HSV-1 mutants carrying mutations in the virion transactivator VP16, ICP0 and tsICP4, and the reporter gene lacZ under the control of either the HCMV IE promoter (in1382) or the ICP0 promoter (in1383). Infection of cells at an m.o.i. of 1 at the non-permissive temperature of 39 °C resulted in transient LacZ expression from the HCMV IE or ICP0 promoter with shut-off evident at day 3 through to day 6 p.i. (Fig. 1a). Induction of reporter gene expression 6 h after HSV-2 superinfection demonstrated the retention of functional and responsive virus genomes. The inability of the HSV-1 ICP0 deletion mutant dl1403 to de-repress and activate HCMV IE promoter-driven lacZ expression from MRC-5 cells harbouring quiescent in1382 genomes indicated an essential requirement for ICP0 in genome de-repression (Fig. 1b).

Histone modifications associated with herpes simplex virus type 1 genomes during quiescence and following ICP0-mediated de-repression

Coleman HM, Connor V, Cheng ZS, Grey F, Preston CM, Efstathiou S - J. Gen. Virol. (2008)

Bottom Line: These data indicate that ICP0-mediated genome de-repression is intimately linked to enrichment of acetylated histones at virus promoters.The fold change in association of pan-acetylated histone H3 following Ad.TRE.ICP0-mediated de-repression consistently revealed promoter-specific variation, with the highest fold changes (>50-fold) being observed at the latency-associated transcript promoter and enhancer regions.Chromatin immunoprecipitation analyses using an antibody specific to the C terminus of histone H3 as a surrogate measure of nucleosome occupancy revealed little variability in the total loading of histone H3 at the various HSV promoters.

Affiliation: Division of Virology, Department of Pathology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QP, UK.

ABSTRACT
In the current study, it was shown that repressed virus genomes in quiescently infected MRC5 cells adopt a repressed histone-associated structure marked by the enrichment of deacetylated histones at a wide variety of herpes simplex virus type 1 (HSV-1) promoters. In addition, it was shown that genome de-repression, mediated by HSV-2 superinfection or delivery of ICP0 using a recombinant adenovirus vector, resulted in the enrichment of acetylated histones on HSV DNA. These data indicate that ICP0-mediated genome de-repression is intimately linked to enrichment of acetylated histones at virus promoters. The fold change in association of pan-acetylated histone H3 following Ad.TRE.ICP0-mediated de-repression consistently revealed promoter-specific variation, with the highest fold changes (>50-fold) being observed at the latency-associated transcript promoter and enhancer regions. Chromatin immunoprecipitation analyses using an antibody specific to the C terminus of histone H3 as a surrogate measure of nucleosome occupancy revealed little variability in the total loading of histone H3 at the various HSV promoters. This observation suggests that acetylation of histone H3 in response to ICP0 expression is not uniformly targeted across the HSV-1 genome during ICP0-mediated de-repression.

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