Histone deacetylase 3. Part 2
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 (MCMV) major immediate early proteins, IE1 and IE2 (or IE3 for MCMV), interact with HDAC1, 2, and 3, and HDAC inhibitors enhance viral production, 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. 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. On the other hand, histone proteins have not been found in herpesvirus virons. 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), anti-HDAC3 (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. 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. 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-dUTP-labeled random 9-mer, and the slides were scanned using an Axon 4000A scanner at 532 nm. Figure 1 shows that all 593 HCMV genomic fragments were printed on the microarray relatively evenly.