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. 2016 Oct 28;90(22):10309-10320.
doi: 10.1128/JVI.01220-16. Print 2016 Nov 15.

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

Emily R Albright  1 , Robert F Kalejta  2
Affiliations

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

Emily R Albright et al. J Virol. .

Abstract

Chromatin is the nucleoprotein complex that protects and compacts eukaryotic genomes. It is responsible for a large part of the epigenetic control of transcription. The genomes of DNA viruses such as human cytomegalovirus (HCMV) are devoid of histones within virions but are chromatinized and epigenetically regulated following delivery to the host cell nucleus. How chromatin is initially assembled on viral genomes and which variant forms of the core histone proteins are deposited are incompletely understood. We monitored the deposition of both ectopically expressed and endogenous histones H3.1 and H3.2 (collectively, H3.1/2) and H3.3 during lytic and latent HCMV infections. Here, we show that they are deposited on HCMV genomes during lytic and latent infections, suggesting similar mechanisms of viral chromatin assembly during the different infection types and indicating that both canonical and variant core histones may be important modulators of infecting viral genomes. We further show that association of both H3.1/2 and H3.3 occurs independent of viral DNA synthesis or de novo viral gene expression, implicating cellular factors and/or virion components in the formation of chromatin on virion-delivered genomes during both lytic and latent infections. IMPORTANCE It is well established that infecting herpesvirus genomes are chromatinized upon entry into the host cell nucleus. Why or how this occurs is a mystery. It is important to know why they are chromatinized in order to better understand cellular pathogen recognition (DNA sensing) pathways and viral fate determinations (lytic or latent) and to anticipate how artificially modulating chromatinization may impact viral infections. It is important to know how the genomes are chromatinized in order to potentially modulate the process for therapeutic effect. Our work showing that HCMV genomes are loaded with canonical and variant H3 histones during both lytic and latent infections strengthens the hypothesis that chromatinization pathways are similar between the two infection types, implicates virion or cellular factors in this process, and exposes the possibility that histone variants, in addition to posttranslational modification, may impact viral gene expression. These revelations are important to understanding and intelligently intervening in herpesvirus infections.

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Figures

FIG 1
FIG 1
eH3-expressing cell lines support HCMV lytic infection and latency. (A) Lysates from TERT-HF (HF) and THP-1 cells nontransduced (NT) or transduced with recombinant lentivirus expressing epitope-tagged histone H3.1 (eH3.1) or H3.3 (eH3.3) were analyzed by Western blotting with the indicated antibodies. *, tagged H3; **, endogenous H3. (B and C) Nontransduced (NT) and eH3.1 or eH3.3 transduced TERT-HFs or THP-1 cells were fractionated and analyzed by Western blotting with the indicated antibodies. T, total lysate; S, soluble; C, chromatin; M, nuclear matrix. (D) Lysates from nontransduced (NT) and eH3.1 or eH3.3 transduced TERT-HFs infected with AD169 at an MOI of 1 and harvested at the indicated hour postinfection (hpi) were analyzed by Western blotting with the indicated antibodies. (E) Nontransduced (NT) and eH3.1 or eH3.3 transduced THP-1 cells were infected with AD169 (AD) at an MOI of 1 in the absence (−) or presence (+) of 1 mM VPA. At 18 h postinfection, lysates were collected and analyzed by Western blotting with the indicated antibodies. All data are representative of results from at least three experiments.
FIG 2
FIG 2
eH3.1 and eH3.3 associate with the HCMV genome during lytic infection of fibroblasts. (A) ChIP assays for the indicated loci using antibodies for total H3, HA, or IgG in parental TERT-HFs infected with AD169 at an MOI of 1 for 2 h. (B and C) ChIP assays for the indicated loci using an antibody for total H3 in eH3.1- and eH3.3-expressing TERT-HFs infected with AD169 at an MOI of 1 for 2 or 72 h, as indicated. (D and E) ChIP assays for the indicated loci using an antibody for the HA epitope tag in eH3.1- and eH3.3-expressing TERT-HFs infected with AD169 at an MOI of 1 for 2 or 72 h, as indicated. Gray bars represent signal from IgG controls. Data represent the means ± standard errors of the means from at least 4 experiments. *, P < 0.05; ns, not significant (P > 0.1), by a Wilcoxon rank sum test. LUNAp, LUNA promoter.
FIG 3
FIG 3
eH3.1 and eH3.3 associate with the HCMV genome during latent infection of THP-1 cells. (A) ChIP assays for the indicated loci using antibodies for total H3, HA, or IgG in parental THP-1 cells infected with AD169 at an MOI of 3 for 18 h. (B and C) ChIP assays for the indicated loci using antibodies for total H3 (B) and eH3 (HA) (C) in eH3.1- and eH3.3-expressing THP-1 cells infected with AD169 at an MOI of 3 for 18 h. Gray bars represent signal from IgG controls. Data represent the means ± standard errors of the means from three experiments. *, P < 0.05; ns, not significant (P > 0.1), by a Wilcoxon rank sum test.
FIG 4
FIG 4
Endogenous H3.1/2 and H3.3 associate with the HCMV genome during lytic infection of fibroblasts. (A) Nuclear extracts from eH3.1 and eH3.3 transduced TERT-HFs were subjected to immunoprecipitations (IPs) with the indicated antibody and analyzed for tagged histones by Western blotting (WB). (B to D) ChIP assays for the indicated loci using antibodies against total H3 (panH3), H3.1/2, H3.3, or an IgG control in NHDFs infected with AD169 at an MOI of 1 for 2, 6, or 72 h, as indicated. Data represent the means ± standard errors of the means from at least three experiments. *, P < 0.05; **, P < 0.01; ns, not significant (P > 0.1), by a Wilcoxon rank sum test.
FIG 5
FIG 5
Endogenous H3.1/2 and H3.3 associate with the HCMV genome during latent infection. (A and B) ChIP assays for the indicated loci using antibodies against total H3 (panH3), H3.1/2, H3.3, or an IgG control in THP-1 cells infected with AD169 or TB40/E at an MOI of 1 for 18 h. (C and D) ChIP assays for the indicated loci using antibodies against total H3 (panH3), H3.1/2, H3.3, or an IgG control in primary CD34+ cells infected with AD169 or TB40/E at an MOI of 1 for 18 h. Data represent the means ± standard errors of the means from at least four experiments. *, P < 0.05; **, P < 0.01, by a Wilcoxon rank sum test.
FIG 6
FIG 6
Daxx promotes histone deposition onto latent HCMV genomes. (A) Lysates from THP-1 cells transfected with a scrambled (siCtrl) or Daxx (siDx1 and siDx2)-specific siRNA for 48 h were analyzed for Daxx knockdown by Western blotting. Tubulin served as a loading control. (B) RNA from siScr- and siDx-treated THP-1s infected with HCMV AD169 at an MOI of 1 for 18 h was analyzed for IE1 transcripts by qRT-PCR. Viral gene expression was normalized to cellular GAPDH and is shown relative to that in the siScr-treated control. (C) Viability of THP-1 cells transfected with a scrambled or Daxx-specific siRNA for 48 h was measured using a CellTiter-Glo assay. Data are shown as relative viability compared to that of the siScr control. (D to G) THP-1 cells treated with a scrambled or Daxx-specific siRNA for 48 h were infected with AD169 at an MOI of 1 for 18 h. ChIP assays were then performed with the indicated antibodies, and the precipitating DNA was analyzed by qPCR for the MIEP, transcribed regions of IE1, LUNA promoter (LUNAp), or the cellular GAPDH gene. Data represent IgG-subtracted percent input relative to the level of the siScr control. All data in panels B to G are means ± standard errors of the means from four experiments. *, P < 0.05; **, P < 0.01; ns, not significant (P > 0.2), by a Student's t test.
FIG 7
FIG 7
Incorporation of H3.1/2 or H3.3 into HCMV chromatin does not require viral transcription. (A) THP-1 cells infected with live (AD) or UV-inactivated (UV) AD169 at an MOI of 1 for 18 h were harvested and analyzed for viral and cellular transcripts by qRT-PCR. Viral transcripts were normalized to the level of cellular GAPDH and are shown relative to live AD169-infected controls. Data are the means ± standard errors of the means from three experiments. *, P < 0.05, by a Wilcoxon rank sum test. (B) ChIP assays for the indicated loci using antibodies against total H3 (panH3), H3.1/2, H3.3, or an IgG control in THP-1 cells infected with live (black bars) or UV-inactivated (striped bars) AD169 as described for panel A. Data represent the means ± standard errors of the means from three experiments. *, P < 0.05; ns, not significant (P > 0.1), by a Wilcoxon rank sum test.
FIG 8
FIG 8
Incorporation of H3.1/2 or H3.3 into HCMV chromatin does not require viral DNA synthesis. (A) NHDFs infected with AD169 at an MOI of 1 in the absence (−) or presence (+) of 100 μg/ml PAA were harvested at the indicated hour postinfection (hpi), and viral (IE1) and cellular (GAPDH) DNA were quantitated by qPCR. Viral DNA was normalized to cellular DNA and is shown relative to the 2-hpi time point. Data represent the means ± standard errors of the means from four experiments. *, P < 0.05, by a Wilcoxon rank sum test. (B) ChIP assays for the indicated loci using antibodies against total H3 (panH3), H3.1/2, H3.3, or an IgG control from NHDFs infected with AD169 at an MOI of 1 for 72 h in the absence (black bars) or presence (striped bars) of 100 μg/ml PAA. Data represent the means ± standard errors of the means from four experiments. *, P < 0.05; ns, not significant (P > 0.1), by a Wilcoxon rank sum test.
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