Polycomb Repressive Complex 2 Targets Murine Cytomegalovirus Chromatin for Modification and Associates with Viral Replication Centers

Regulation of viral transcription by chromatin structure has emerged as a fundamental determinant in the establishment of lytic and latent herpesvirus infections. The Polycomb group (PcG) of epigenetic repressors promotes heterochromatin formation by trimethylating histone H3 on lysine-27 (H3K27me3) and regulates development, stem cell renewal and differentiation and the cell cycle. These cellular processes are tightly coupled to the molecular switch between lytic and latent herpesvirus infections. Using chromatin immunoprecipitation analysis, we observed enrichment of H3K27me3 at the major immediate-early (MIE) locus of murine cytomegalovirus (MCMV) very early following infection of permissive fibroblasts. As lytic replication progressed, we observed a loss of H3K27me3 enrichment concomitant with the appearance of H3K4me3. However, late during infection, as viral replication centers are established, we observed a significant increase in PcG protein association with chromatin. Additionally, in co-immunofluorescence assays using confocal microscopy, we detected strong enrichments for PcG protein within the viral replication compartment, suggesting an association between viral DNA synthesis machinery and PcG proteins. Together, our results suggest a novel, dynamic interaction between PcG epigenetic repressors and MCMV genomes

Human Cytomegalovirus 5-Kilobase Immediate-Early RNA Is a Stable Intron

Immediate-early viral gene products of human cytomegalovirus (HCMV) are derived from several genomic loci and largely serve to establish a cellular environment conducive to viral replication. We have further examined an unusual immediate-early transcript known as the 5-kb RNA, concluding that it is a stable intron encoded by HCMV. The 5-kb RNA is highly AT rich in sequence and lacks open reading frames likely to be translated into protein. We confirmed the absence of polyadenylation of the transcript and showed that it is primarily nuclear localized during viral infection. We mapped the 5′ end of the 5-kb RNA to a consensus splice donor site and localized the 3′ end in the vicinity of a splice acceptor site. In transfection studies, we showed that the 5-kb RNA can be spliced from a heterologous primary transcript. Using bacterial artificial chromosome technology, we constructed a viral recombinant containing a mutation in the 5′ splice donor site that defines the 5′ end of the RNA and found that this mutation eliminates expression of the 5-kb RNA during viral infection. This mutant grows in human fibroblasts without complementation. Taken together, these data support the conclusion that the 5-kb RNA is a stable intron expressed by HCMV

Viral DNA replication kinetics in mouse fibroblasts.

<p>MCMV DNA content on total DNA collected from MCMV infected cells analyzed by Q-PCR at 1.5, 3, 12, 18 and 24 hpi as indicated. Each graph displays the mean value and S.E.M. for, with data from three independent assays. The results are presented as the relative IE1-3 DNA amount normalized to a cellular reference, Dlx1.</p

Measurement of H3K27me3 enrichment at the MIE locus of MCMV.

<p>(A) Graphical illustration of the MIE locus of MCMV. Solid black boxes represent regions probed for H3K27me3 & H3K4me3 enrichment. Regions denoted in figure: (1) Represents the Enhancer 2 region, (2) the transcriptional start site (TSS) of the IE1-3 transcriptional unit and (3) represents Exon 1 within the ORF of the IE1-3 locus. +1 transcriptional start sites are indicated with black arrows. Enhancer regions are marked in hatched boxes. IE1-3 exons are depicted as gray arrows. (B) H3K27me3 is enriched at the MIE locus at pre-IE times during MCMV infection. ChIPs using anti-H3K27me3 antibody were analyzed by Q-PCR using primer/probe sets specific for the indicated loci at 1.5, 3, 6 and 12 hpi as indicated. Each graph displays the mean value and S.E.M. for each queried locus, with data from three independent ChIPs. The results are presented as the IgG subtracted %Input of the queried locus normalized to the IgG subtracted β-Actin %Input. Samples with values that vary significantly from the negative control, Dlx1,are indicated by asterisks (* <i>P</i> value<0.05, ** <i>P</i> value<0.01; ANOVA).</p

Western blot analysis of PRC2 association with chromatin during MCMV infection.

<p>(A) Validation of chromatin fractionations. Mouse fibroblasts were fractionated as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029410#s4" target="_blank">Materials and Methods</a>. 25 µg of protein from the soluble (S) and chromatin (C) fractions were probed for the chromatin-associated proteins SUZ12, HDAC1 and HP1. The blot was then stripped and re-probed for the ER-associated protein GRP78. (B) Chromatin fractions prepared from vehicle-treated (DMSO), cycloheximide (100 µg/ml) or phosphonoacetic acid (200 µg/ml) treated, mock-infected (M) or MCMV-infected fibroblasts at the indicated times. Blots were probed with antibodies against the PRC2 components EZH2 and SUZ12, or HDAC1 and HP1 for a loading control and fractionation quality control, respectively.</p

Validation of ChIPs for (A) H3K27me3 and (B) H3K4me3.

<p>Chromatin from uninfected mouse fibroblasts was immunoprecipitated using anti-H3K27me3 antibody. The specificity of the ChIPs was assessed by measuring the %Input by Q-PCR at HoxC11, Dlx1 and β-Actin. Each graph displays the mean value and S.E.M. for each queried locus, with data from four independent ChIPs. *** <i>P</i> value<0.001.</p

Q-PCR Primers and Probes.

<p>Q-PCR Primers and Probes.</p

Co-immunofluorescence assay for PRC2 enrichment within MCMV replication compartments.

<p>At 18 hpi, mock-infected or MCMV-infected fibroblasts were fixed and incubated with antibodies against M44 and (A) EZH2, (B) SUZ12 or (C) H3K27me3. (D) Purified rabbit IgG served as an isotype control. The first column of panels displays DAPI staining for nuclei. The second column of panels displays M44 staining, marking MCMV replication compartments. The third column of panels displays staining EZH2, SUZ12, H3K27me3 or isotype IgG. The fourth column of panels displays a merged image of the first three channels. The fifth column of panels displays a high-magnification image of the merged image from the fourth column. All images are 40×, unless otherwise indicated.</p

Immunofluorescence assay for PRC2 proteins during MCMV infection.

<p>At 12 hpi, mock-infected or MCMV:mCherry-infected fibroblasts were fixed and probed with antibodies against the PRC2 components, EZH2 or SUZ12. Large arrowheads indicate MCMV infected cells that also exhibit increased immunofluorescence signal for EZH2 or SUZ12, while small arrows indicate uninfected neighboring cells. All images are at 40× magnification.</p

Measurement of H3K4me3 enrichment at the MIE locus of MCMV.

<p>ChIPs using anti-H3K4me3 antibody were analyzed by Q-PCR using primer/probe sets specific for the indicated loci at 1.5, 3, 6 and 12 hpi as indicated. Each graph displays the mean value and S.E.M. for each queried locus, with data from three independent ChIPs. The results are presented as the IgG subtracted %Input of the queried locus normalized to the IgG subtracted β-Actin %Input. Samples with values that vary significantly from the negative control, HoxC11, are indicated by asterisks (* <i>P</i> value<0.05; ANOVA).</p