Canonical and Variant Forms of Histone H3 Are Deposited onto the Human Cytomegalovirus Genome during Lytic and Latent Infections

Cells and infections.

TERT (telomerase reverse transcriptase)-immortalized human fibroblasts (TERT-HFs) and normal human dermal fibroblasts (NHDFs; Clonetics) were maintained in Dulbecco's modified Eagle medium (DMEM; Sigma). THP-1 cells (TIB-202; ATCC) were maintained in RPMI 1640 medium (Invitrogen). Medium was supplemented with 10% (vol/vol) fetal bovine serum (FBS; Sigma) and 1× penicillin-streptomycin with l-glutamine (G1146; Sigma). Primary CD34⁺ cells derived from cord blood (2C-101; Lonza) were maintained in hematopoietic progenitor growth medium (HPGM; Lonza) supplemented with recombinant human thrombopoietin (TPO), recombinant human stem cell factor (SCF), and recombinant human Flt3 ligand (all from Peprotech), as described previously (53). Cells were infected with HCMV in minimal volume for 60 min, followed by addition of medium to normal culture volumes. For UV inactivation, virus stocks were exposed to a 245-nm light source at 0.12 J/cm³ for 2 min on ice using a UV Stratalinker 2400 (Stratagene) prior to infection, as previously described (54).

Inhibitors and antibodies.

Where indicated in the figure legends and on the figures, valproic acid (VPA) (1 mM; Sigma) was added 3 h prior to infection of THP-1 cells, and phosphonoacetic acid (PAA) (100 μg/ml; Sigma) was added 24 h postinfection of NHDFs. The following antibodies were from commercial sources: anti-FLAG M2 (F1804; Sigma), anti-histone H3 (pan-H3) (ab1791; Abcam), anti-beta-actin (ab8226; Abcam), anti-tubulin (DM 1A; Sigma), anti-histone H2B (07-371; Millipore), anti-lamin A/C (sc-7292; Santa Cruz Biotechnology), anti-UL44 (CA006-100; Virusys), anti-hemagglutinin (HA) (HA.11; Covance), anti-histone H3.1/2 (ABE154; Millipore), anti-histone H3.3 (09-838; Millipore), and anti-Daxx (D7810; Sigma). Monoclonal antibodies against IE1 (1B12), pp28 (CMV157), and pp71 (2H10-9) have been described previously (55–57).

Epitope-tagged cell lines.

Fibroblast and THP-1 cell lines expressing either histone H3.1 or H3.3 with C-terminal Flag and HA tags from the elongation factor 1α (EF1α) promoter were generated by lentiviral transduction. Tagged cDNAs for H3.1 and H3.3 were amplified by PCR from pOZ-H3.1 and pOZ-H3.3 (58) (a gift from Yoshihiro Nakatani, Harvard Medical School) and cloned into the EcoRI/BamHI sites of pSIN-EF2-puro (Addgene plasmid 16580) (59). Recombinant lentivirus was produced by cotransfection of 293Y cells with pSIN-EF2-puro-eH3, pMD-VSV-G (where VSV-G is vesicular stomatitis virus G protein), and pΔR8.91 (a gift from Nathan Sherer, University of Wisconsin—Madison). TERT-HFs and THP-1 cells were transduced with recombinant lentivirus in the presence of 8 μg/ml Polybrene, and populations of stably transduced cells were selected with puromycin.

Western blotting.

Cells were lysed in radioimmunoprecipitation assay (RIPA) buffer with protease and phosphatase inhibitors or 1% SDS containing 2% β-mercaptoethanol, and equal amounts of lysates were separated by SDS-PAGE, transferred to Optitran membranes (GE Healthcare), and analyzed by Western blotting as previously described (60).

Subnuclear fractionations.

Cells were collected and washed once in cold phosphate-buffered saline (PBS). An aliquot was lysed in 1% SDS containing 2% β-mercaptoethanol as total lysate, and the remaining cells were pelleted and resuspended in cold CSK buffer (10 mM PIPES [piperazine-N,N′-bis(2-ethanesulfonic acid)], pH 6.8, 100 mM NaCl, 300 mM sucrose, 2 mM MgCl₂, 1 mM EGTA, and 1 mM dithiothreitol [DTT]) containing 0.5% Triton X-100. Samples were incubated on ice for 5 min and then pelleted at 5,000 × g for 3 min at 4°C. Supernatant was collected as the soluble fraction, and the pellets were washed once in cold CSK buffer and then resuspended in CSK buffer. RNase-free DNase (M6101; Promega) was added to 100 units/ml, and samples were incubated at 37°C for 2 h. Ammonium sulfate was added to a final concentration of 0.25 M, and samples were incubated on ice 5 min prior to centrifugation at 5,000 × g for 3 min at 4°C. Supernatant was collected as the chromatin fraction, and pellets were washed once in CSK buffer containing 2 M NaCl and resuspended in urea buffer (8 M urea, 10 mM Tris, pH 8.0) as the nuclear matrix fraction. All buffers were supplemented with protease and phosphatase inhibitors just prior to use.

Immunoprecipitations.

Immunoprecipitations were performed as previously described (61) with minor modifications. Briefly, cells were collected and washed once with PBS and once with hypotonic buffer (15 mM HEPES, pH 7.5, 20 mM KCl, 5 mM MgCl₂). Cell pellets were resuspended in hypotonic buffer supplemented with 0.02% NP-40, 10 μg/ml RNase A, and protease and phosphatase inhibitors and incubated at 4°C with rotation for 15 min. NaCl was added to a final concentration of 400 mM with agitation to extract nuclear proteins, and extracts were incubated at 4°C with rotation for 15 min prior to centrifugation at 15,000 × g for 30 min at 4°C. Supernatant was incubated with 2 μg of antibody overnight at 4°C with rotation. Antibody complexes were collected with protein A+G magnetic beads (88802; Thermo) and washed for 5 min three times with hypotonic buffer containing 400 mM NaCl. Bound proteins were eluted in 1× SDS loading buffer containing β-mercaptoethanol and boiled before being run on SDS-PAGE gels.

ChIP assays.

Chromatin immunoprecipitation (ChIP) assays were performed as previously described (54). Briefly, infected cells were collected, washed once with PBS, and fixed with 1% formaldehyde for 8 min at room temperature, followed by addition of glycine to 125 mM to quench the reaction. Nuclei were then isolated, and chromatin was sheared by sonication. Chromatin from approximately 1 million cells per reaction mixture was incubated with 2 to 4 μg of ChIP-grade antibodies or an equal amount of isotype control IgG overnight at 4°C. Chromatin/antibody complexes were collected using Magna ChIP protein A+G magnetic beads (16-663; Millipore) for 2 h at 4°C. Beads were then washed, and cross-links were reversed. DNA was isolated with a QIAquick PCR cleanup kit (28106; Qiagen) and quantified by quantitative PCR (qPCR) using iTaq Universal SYBR green Supermix (172-5124; Bio-Rad) with primers specific to the major immediate early promoter (MIEP) (62), IE1 (63), LUNA promoter (64), LUNA (65), B2.7 RNA (65), or cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (66). Precipitating DNA was normalized to input levels from the same lysates using the ΔC_T (where C_T is threshold cycle) method (67). Statistical analysis was performed using Mstat, version 6.1, software.

DNA and mRNA analysis.

Total genomic DNA was isolated using a genomic DNA minikit (IB47202; IBI). Equal amounts of total DNA were analyzed by quantitative PCR using iTaq Universal SYBR green Supermix (172-5124; Bio-Rad) with primers specific to viral (IE1) (63) or cellular (GAPDH) (66) DNA. RNA isolation and quantitative reverse-transcription PCR (qRT-PCR) were performed as previously described (54). Viral gene expression was normalized to cellular gene expression using the ΔC_T method (67).

siRNA transfections.

For transient knockdown with small interfering RNAs (siRNAs), THP-1 cells were transfected with 80 pmol of siRNA per 1 million cells using TransIT-X2 (MIR6000; Mirus) according to the manufacturer's instructions. Scrambled control siRNA (siScr; D-001810-10) or siRNAs targeting Daxx (siDx1 and siDx2; J-004420-05 and J-004420-06) were purchased from Dharmacon. Viability of transfected cells was assayed at 48 h posttransfection using a CellTiter-Glo assay (G7571; Promega) according to the manufacturer's instructions.

Ectopically expressed H3.1 and H3.3 are deposited onto lytic and latent HCMV genomes.

Histones of the H3 class are found associated with HCMV genomes during lytic infection and latency, but specifically which form(s) of histone H3 is present in HCMV chromatin has not been investigated. We examined H3.1 and H3.3 incorporation into nucleosomal HCMV by chromatin immunoprecipitation (ChIP) assays. H3.1 and H3.3 differ by only 5 amino acids. Therefore, to ensure specificity, we generated stable cell lines in both human fibroblasts (a model for HCMV lytic infection) and THP-1 monocytes (a model for HCMV latent infection) that ectopically express H3.1 or H3.3 with C-terminal Flag and HA epitope tags under the control of the cellular EF1α promoter (eH3.1 or eH3.3, respectively). We selected populations of transduced cells that express equivalent levels of eH3.1 and eH3.3 representing approximately 15% and 7% of total histone H3 in fibroblasts and THP-1 cells, respectively (Fig. 1A). Ectopically expressed histones fractionated with chromatin in a manner similar to that of the endogenous histone H2B in both HFs (Fig. 1B) and THP-1 cells (Fig. 1C), consistent with previous reports indicating that the epitope tags do not interfere with chromatin incorporation (58).

HCMV delivered equivalent amounts of tegument and expressed representative immediate early (IE1), early (UL44), and late (pp28) lytic phase proteins with similar kinetics and to similar steady-state levels in eH3.1- and eH3.3-expressing fibroblasts compared to expression in parental cells (Fig. 1D). Similarly, HCMV entered eH3.1- and eH3.3-expressing THP-1 cells as efficiently as parental cells, as judged by tegument delivery of pp71 (Fig. 1E). IE1 gene expression was silenced in these cells, consistent with the establishment of latency (Fig. 1E). As previously reported (53, 54, 68), treatment with the histone deacetylase (HDAC) inhibitor VPA prevented silencing of IE1 gene expression during infection with AD169 in parental THP-1 cells as well as in eH3.1- and eH3.3-expressing THP-1 cells (Fig. 1E). Taken together, these results demonstrate that HCMV infections are not altered in either HF or THP-1 cells expressing ectopic eH3.1 or eH3.3, indicating that these cell lines are viable tools with which to interrogate histone variant deposition during HCMV infection.

ChIP assays were first performed in HCMV AD169-infected eH3.1- and eH3.3-expressing fibroblasts with an antibody recognizing an endogenous epitope conserved in both H3.1/2 and H3.3. As expected, in HCMV-infected parental fibroblasts, an antibody against histone H3, but not an antibody against the HA epitope tag, precipitated both viral and cellular loci (Fig. 2A). In eH3.1- and eH3.3-expressing fibroblasts, we found histone H3 associated with all three viral loci examined at both 2 (Fig. 2B) and 72 (Fig. 2C) h postinfection (hpi). The same lysates were used for immunoprecipitation with the HA antibody to detect loci associated with the ectopically expressed histone. Both eH3.1 and eH3.3 were associated with the HCMV and cellular genome at 2 (Fig. 2D) and 72 (Fig. 2E) hpi. Similarly, ChIP experiments in parental THP-1 cells infected with HCMV AD169 detected total H3, but not eH3 (HA), associated with both viral and cellular loci (Fig. 3A). Using ChIP experiments in HCMV AD169-infected eH3.1- and eH3.3-expressing THP-1 cells, we found histone H3 class proteins associated with all three viral loci examined during the establishment phase of latency (Fig. 3B). Consistent with our observations during lytic infection, both eH3.1 and eH3.3 were deposited onto the viral genome following latent infection of THP-1 cells (Fig. 3C). We conclude that both eH3.1 and eH3.3 are incorporated into chromatin associated with the HCMV genome during lytic infection and latency.

Endogenous H3.1 and H3.3 are deposited onto lytic and latent HCMV genomes.

Ectopically expressed, epitope-tagged histone variants allow for easy detection and facile discrimination of histone variants and eliminate concerns emanating from the different binding affinities inherent to different antibodies. However, such ectopically expressing cells use constitutively active promoters and poly(A) addition sequences such that the de novo synthesis of canonical histones such as H3.1 can occur during cell cycle stages (e.g., G₁) when they would not normally be generated efficiently. Such de novo synthesis perturbs normal free histone pools, and therefore results with ectopically expressed canonical histones (like H3.1) must be interpreted with caution. Our data indicate that H3.1 can be deposited on viral genomes when it is ectopically expressed. However, evidence that it is truly deposited (e.g., during a natural infection) requires an examination of the endogenous H3.1/2 protein.

We therefore sought to confirm our results from eH3.1- and eH3.3-expressing cell lines indicating that both variants could be deposited on the viral genome during lytic and latent infection by examining the deposition of endogenous histone H3 variants. During the course of our work, ChIP-grade antibodies that recognize H3.1/2 or H3.3 became commercially available. The H3.1/2 antibody recognizes a linear epitope found in both H3.1 and H3.2 while the H3.3 antibody recognizes an epitope unique to H3.3. We independently verified the specificity of these antibodies with immunoprecipitation experiments in eH3.1- and eH3.3-expressing fibroblasts (Fig. 4A) and then used them for ChIP experiments during lytic and latent HCMV infections.

As expected, ChIP assays with a pan-histone H3 antibody detected histone H3 at all viral and cellular loci tested at all time points in HCMV AD169-infected fibroblasts (Fig. 4B, C, and D). Likewise, ChIP assays with antibodies to the endogenous proteins detected both H3.1/2 and H3.3 association with the HCMV genome during lytic infection (Fig. 4B, C, and D). At first glance the data seem to imply that H3.1/2 shows a stronger association than H3.3 with the HCMV genome at early times during lytic infection (Fig. 4B and C), a difference largely neutralized at a late (72 hpi) time point (Fig. 4D). However, this interpretation is confounded by the possibility that differences in antibody affinities (not genome association) could explain the higher recovery of viral sequences with the H3.1/2 antibody than with the H3.3 antibody. Thus, the conservative conclusion is that during lytic infection, H3.1/2 and H3.3 are deposited onto the HCMV genome. However, our data raise the intriguing possibility of an earlier and more robust association of H3.1/2 histones than of H3.3 with HCMV genomes.

The incompletely differentiated myeloid lineage cells where HCMV establishes latency also chromatinize infecting viral genomes (64, 69, 70). We found that both H3.1/2 and H3.3 associated with HCMV AD169 latent genomes, similarly to lytic infection, at all three loci examined in THP-1 cells (Fig. 5A). Clinical strains such as TB40/E encode the viral UL138 gene that enforces latency maintenance by maintaining a repressive epigenetic signature at the viral MIEP (54). HCMV-TB40/E-infected THP-1 cells also showed H3.1/2 and H3.3 association with latent viral genomes (Fig. 5B), indicating that viral genes unique to clinical strains do not appear to affect the identity of histones deposited during the establishment of latency. While THP-1 cells are an invaluable, trusted, and widely used working model for HCMV latency, primary CD34⁺ hematopoietic progenitor cells represent a more physiologic cell type for latency studies. Our ChIP experiments indicate that both H3.1/2 and H3.3 are associated with latent AD169 (Fig. 5C) and TB40/E (Fig. 5D) genomes in primary CD34⁺ cells. We conclude that both H3.1/2 and H3.3 are deposited onto latent HCMV genomes.

Daxx promotes histone deposition onto latent HCMV genomes.

Daxx is an H3.3 chaperone (34) and participates in the transcriptional repression of HCMV lytic phase genes during latency (53, 68). We therefore asked if Daxx was responsible for depositing H3 histones on latent HCMV genomes. Transient transfection of two independent siRNAs targeting Daxx into THP-1 cells reduced Daxx steady-state protein levels (Fig. 6A) and, as previously shown (53, 68), increased viral IE1 transcription (Fig. 6B) while not altering cell viability (Fig. 6C). ChIP assays revealed decreased levels of total H3 and variant H3.3 at the MIEP (Fig. 6D) and within the transcribed region of IE1 (Fig. 6E) in Daxx-depleted cells compared to levels in cells transfected with a scrambled siRNA control. These loci are transcriptionally repressed during latency. However, Daxx depletion did not impact total H3 or H3.3 incorporation at the viral LUNA promoter (Fig. 6F) or the cellular GAPDH gene (Fig. 2G), loci that are transcriptionally active during latency. Our observations indicate that Daxx promotes H3.3 incorporation at transcriptionally repressed but not transcriptionally active viral loci during HCMV latency.

Interestingly, Daxx depletion impaired H3.1/2 deposition at all loci tested (Fig. 6D to G) even though Daxx is not known to directly deposit or to bind H3.1/2 (34, 61). We consider it unlikely for H3.1/2 deposition to require prior H3.3 incorporation because H3.1/2 deposition in the absence of Daxx is impaired at loci where H3.3 deposition is not (Fig. 6F and G) and because other data actually suggest that H3.1/2 deposition occurs prior to H3.3 (Fig. 4). Thus, the impaired incorporation of H3.1/2 into HCMV chromatin in Daxx-depleted THP-1 cells likely indicates either an alteration of Daxx histone-binding specificity or an effect on other histone chaperone complexes. We have not examined the requirement of Daxx for histone deposition during lytic infection of fibroblasts because under these conditions Daxx is rapidly degraded by tegument-delivered pp71 (71).

Incorporation of H3.1/2 and H3.3 requires neither viral transcription nor viral DNA synthesis.

The naked DNA introduced into nuclei by infection with viruses such as HCMV does not appear to mimic a normal cellular DNA substrate for histone deposition. Regions of naked cellular DNA are generated by the processes of transcription and replication. During transcription, nucleosomes are removed from templates to permit the passage of RNA polymerases and then added back to recently transcribed DNA to reestablish chromatin (19). To determine whether viral transcription was required for the incorporation of either H3.1/2 or H3.3, we infected THP-1 cells with UV-inactivated HCMV AD169 that failed to accumulate the B2.7 RNA (Fig. 7A) normally transcribed during latency (65). Similar levels of endogenous H3.1/2 and H3.3 were found associated with three different loci of live and UV-inactivated virus (Fig. 7B), indicating that viral transcription is not required for the initial chromatinization of HCMV genomes.

During DNA replication, nucleosomes are removed from templates to permit the passage of DNA polymerases and then added back to recently replicated DNA to reestablish chromatin (72). We infected HFs with HCMV AD169 in the presence or absence of the viral DNA polymerase inhibitor PAA and demonstrated that the drug efficiently inhibited viral DNA replication (Fig. 8A). Similar levels of endogenous H3.1/2 and H3.3 were found associated with all three viral loci in the presence or absence of PAA (Fig. 8B). In all cases, recovery from the histone antibodies was greater than that of the control IgG antibody although the increase did not always reach statistical significance. We conclude that viral DNA replication is not required for the chromatinization of HCMV genomes. In summary, our evidence demonstrates that both H3.1/2 and H3.3 are deposited onto lytic and latent HCMV genomes through processes that require neither viral transcription nor DNA replication.