Histone deacetylase 3, not histone deacetylase 2, interacts with the major immediate early locus of human cytomegalovirus

Huang et al. Virology Journal 2011, 8:151 http://www.virologyj.com/content/8/1/151 SHORT REPORT Open Access Histone deacetylase 3, not histone deacetylase 2, interacts with the major immediate early locus of human cytomegalovirus Ying Huang1†, Qiyi Tang2*†, Michael Nguyen1, Kalpana Dulal1, Weijia Wang1 and Hua Zhu1* Abstract Evidence suggests that genome chromatinization and the posttranslational modification of histones are involved in the regulation of viral gene expression, including the human cytomegalovirus (HCMV). We performed a ChIP-onChip assay to determine whether histone deacetylases (HDACs) interact with HCMV genomic DNA on a global level. Surprisingly, we found that HDAC3, but not HDAC2, interacts not only with the major immediate early (MIE) promoter but also with the entire MIE locus, suggesting a heterogeneous interaction of HDAC3 with HCMV DNA. The interaction of HDAC3 with the MIE region is related to inhibition of viral replication because HDAC3 inhibitors enhanced HCMV replication. Human cytomegalovirus (HCMV) is a ubiquitous virus and infects a majority of the general population (50-90%) [1]. The fact that the incidence of cytomegalic inclusion disease (CID) is intimately related to viral burden suggests that the inhibition of viral production by the specific repression of viral gene expression will reduce the occurrence of CID [2]. Understanding the mechanism of HCMV gene regulation is the pre-requisite for developing drugs that interfere with viral replication by repressing viral gene expression. The HCMV major immediate early (MIE) gene products, IE1 and IE2, are among the first de novo-expressed viral proteins that are required for subsequent viral gene expression and hence viral replication [3]. IE1 and IE2 mRNAs are encoded by the major IE locus that spans from 169 to 175 kbp of the viral genome and are produced by alternative splicing and differential polyadenylation [4-6]. IE1 and IE2 share the first 85 amino acids [1]. MIE genes are controlled by a strong promoter/enhancer that contains many regulatory elements [3,7]. * Correspondence: ; † Contributed equally 1 Department of Microbiology and Molecular Genetics, UMDNJ-New Jersey Medical School, 225 Warren Street, Newark, NJ, 07101, USA 2 Department of microbiology/AIDS program, Ponce School of Medicine, 395 Zona Industrial Reparadara-2, Ponce, PR, 00716-2348, USA Full list of author information is available at the end of the article Different cellular mechanisms have been found to play roles in inhibiting viral gene expression, and one of the most prominent ones is gene silencing through viral DNA chromatinization (also called chromatin remodeling) [8], a procedure carried out by histone or histonerelated proteins, such as histone acetylase (HAT) and histone deacetylase (HDAC). Several posttranslational modifications of histone proteins have been defined to be involved in chromatin remodeling, including acetylation by HAT, deacetylation by HDAC, SUMOylation by SUMO (Small Ubiquitin-like Modifier)-related pathway, deSUMOylation by SENP (a SUMO-specific protease) family enzymes, phosphorylation by kinase pathways, and methylation via methylases [9-13]. Those enzymatic pathways orchestrate to regulate cellular gene transcription and are termed as epigenetic codes [10,13]. Viral gene transcription requires cellular machinery, which is probably also regulated by cellular gene regulatory pathways. Histones are abundant nuclear proteins and have been shown to bind with HCMV genomic DNA [14]. Therefore, it was reasonable to propose that chromatin remodeling of viral DNA takes place in the nucleus, which speculation was validated when the fact that HDAC inhibitors can promote cytomegalovirus production was also confirmed [15-17]. How HCMV strips off the cellular proteins in order for the virus to replicate its own DNA is not fully understood. Recent studies have shown that HCMV and murine CMV © 2011 Huang et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Huang et al. Virology Journal 2011, 8:151 http://www.virologyj.com/content/8/1/151 (MCMV) major immediate early proteins, IE1 and IE2 (or IE3 for MCMV), interact with HDAC1, 2, and 3, and HDAC inhibitors enhance viral production [17-21], and dynamic chromatin modification of the MIE promoter and other viral promoters has been shown. However, interaction of HDACs with the viral genome has not been clearly demonstrated [22]. In this study, we performed chromatin immunoprecipitation followed by microarray on an HCMV DNA chip (ChIP-on-chip) assay to demonstrate the interaction of HCMV DNA with HDACs. To our surprise, we found that HDAC3, but not HDAC2, interacts specifically with the MIE locus, which suggests a heterogeneous interaction of HDAC3 with HCMV genomic DNA. In addition, we found that the interactions of HDAC3 with the MIE locus might relate to the modulation of viral replication because HDAC3 inhibitors can significantly enhance viral growth. The chromatinization of viral DNA after its having entered the nucleus has been noted not only in latently infected viruses such as EBV and KSHV (the genomes of which are tethered to cellular chromosomes) but also in the lytic infection of HCMV [8,23,24]. On the other hand, histone proteins have not been found in herpesvirus virons [25]. Therefore, the chromatinization of HCMV DNA must be temporary and dynamic. We wonder 1) whether the HDACs are bound to the HCMV DNA, and 2) if so, where they interact and whether the interaction is homogenous or heterogeneous. In order to answer these questions, we performed a ChIP-on-chip assay. The human foreskin fibroblast cells (HFF) were infected with HCMV at an MOI of 5. The cells were fixed at 24 hours postinfection with 1% paraformaldehyde. The chromatin immunoprecipitation (ChIP) inputs were prepared and performed using the commercial kit (EZ ChIP, Upstate Cell Signal Solutions), according to the manufacturer’s protocol. The antibodies used for ChIP assays include anti-HDAC2 (clone 3F3), antiHDAC3 (clone 3G6, Upstate USA, Inc.), and normal IgG (as a negative control). To generate an HCMV genomic microarray for the ChIP-on-chip assay, an entire HCMV (Toledo strain) genomic DNA was subdivided into 593 small DNA fragments and amplified by PCR (primers are listed in Additional file 1, Table S1). Each PCR fragment was ~500 bp long with 100 bp overlapping the adjacent fragments. The PCR products were verified by agarose gels, purified, quantified, and printed on glass slides, as described [26]. Each DNA fragment was spotted in triplicate on each array. The printing quality of the array was controlled by hybridizing the array with a Cyanine 3-dUTPlabeled random 9-mer, and the slides were scanned Page 2 (...truncated)