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. 2004 Nov;78(22):12566-75.
doi: 10.1128/JVI.78.22.12566-12575.2004.

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin

William Stedman  1 , Zhong Deng, Fang Lu, Paul M Lieberman
Affiliations

ORC, MCM, and histone hyperacetylation at the Kaposi's sarcoma-associated herpesvirus latent replication origin

William Stedman et al. J Virol. 2004 Nov.

Abstract

The viral genome of Kaposi's sarcoma-associated herpesvirus (KSHV) persists as an extrachromosomal plasmid in latently infected cells. The KSHV latency-associated nuclear antigen (LANA) stimulates plasmid maintenance and DNA replication by binding to an approximately 150-bp region within the viral terminal repeats (TR). We have used chromatin immunoprecipitation assays to demonstrate that LANA binds specifically to the replication origin sequence within the KSHV TR in latently infected cells. The latent replication origin within the TR was also bound by LANA-associated proteins CBP, double-bromodomain-containing protein 2 (BRD2), and the origin recognition complex 2 protein (ORC2) and was enriched in hyperacetylated histones H3 and H4 relative to other regions of the latent genome. Cell cycle analysis indicated that the minichromosome maintenance complex protein, MCM3, bound TR in late-G(1)/S-arrested cells, which coincided with the loss of histone H3 K4 methylation. Micrococcal nuclease studies revealed that TRs are embedded in a highly ordered nucleosome array that becomes disorganized in late G(1)/S phase. ORC binding to TR was LANA dependent when reconstituted in transfected plasmids. DNA affinity purification confirmed that LANA, CBP, BRD2, and ORC2 bound TR specifically and identified the histone acetyltransferase HBO1 (histone acetyltransferase binding to ORC1) as a potential TR binding protein. Disruption of ORC2, MCM5, and HBO1 expression by small interfering RNA reduced LANA-dependent DNA replication of TR-containing plasmids. These findings are the first demonstration that cellular replication and origin licensing factors are required for KSHV latent cycle replication. These results also suggest that the KSHV latent origin of replication is a unique chromatin environment containing histone H3 hyperacetylation within heterochromatic tandem repeats.

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Figures

FIG. 1.
FIG. 1.
Chromatin organization of the KSHV genomes. (A) Eleven primer pairs were designed to sample regions of the KSHV genome indicated by capital letters above each locus. The amplified regions are delineated by grey bars and KSHV coordinates (53). (B) Real-time PCR analysis of ChIP assays with antibodies to AcH3, AcH3K9, AcH4, MeH3K4, or MeH3K9 as indicated in each graph. Values were reported as percent input and reflect the average of at least three independent experiments. (C) The same analyses as appear in panel B, except with antibodies specific for LANA, ORC2, BRD2, CBP, or control IgG, as indicated for each graph. Letters below each bar correspond to the KSHV loci depicted in panel A.
FIG. 2.
FIG. 2.
Cell cycle-dependent histone modifications and MCM protein binding at the KSHV TR. (A) Fluorescence-activated cell sorter analysis of propidium iodide-treated BCBL1 cells grown as asynchronous cultures or arrested in late G1 with mimosine or in G2/M phase with colchicine. (B) Real-time PCR analysis of ChIP assays with antibodies to AcH4, AcH3, MeH3K4, LANA, ORC2, MCM3, or IgG at KSHV loci A, G, and K as depicted in Fig. 1A. Asynchronous (black), mimosine-arrested (grey), and colchicine-arrested (hatched) cultures are indicated in the bar graph. (C) Western blot analysis of total cellular proteins derived from asynchronous (ASYN), mimosine-arrested (G1), or colchicine-arrested (M) cells probed with antibodies to LANA or β-actin, as indicated.
FIG. 3.
FIG. 3.
Nucleosome organization at TR. (A) Southern blots of MNase I-digested BCBL1 nuclei. Nuclei were digested with a twofold serial increase in MNase I, ranging from 15 to 120 U/100 μl of reaction mixture as indicated above. Extracted DNA was probed with sequences specific for the LBS, TR, or ORF50 region as indicated above each blot. The arrow indicates mononucleosome fragments of ∼147 bp. (B) MNase I digestions of asynchronous (A), mimosine-arrested (G1), or colchicine-arrested (M) BCBL1 cells were probed with sequences specific for the TR. MNase I-digested samples were cut with NotI (even-numbered lanes) or not restricted (odd-numbered lanes) prior to electrophoresis.
FIG. 4.
FIG. 4.
Nucleosome mapping by indirect end labeling. BCBL1 cell nuclei (lanes 2 to 5) or purified DNA (lanes 6 to 8) were treated with various concentrations of MNase I nuclease and subject to indirect end-labeling analysis. The number of MNase U/100 μl of reaction mixture was as follows: 30 (lane 3), 150 (lane 4), 300 (lane 5), 6 (lane 6), 0.6 (lane 7), and 0 (lanes 2 and 8). MNase I-treated DNA was purified and subjected to restriction digestion with NotI (A and B) or DraIII (C and D). Restricted DNA was then subjected to Southern blot analysis and probed with 55-bp oligonucleotides specific for the termini generated at 5′ NotI (A), 3′ NotI (B), 5′ DraIII (C), or 3′ DraIII (D). Interpretations of data are indicated to the right of each panel. Presumed nucleosomes are indicated by grey spheres, and LANA is indicated by black spheres. Arrows indicate major MNase I cleavage sites. (E) Summary of predicted nucleosome positions for two tandem TRs.
FIG. 5.
FIG. 5.
LANA recruits ORC to TR. 293 cells transfected with 2 × TR plasmid and either LANA expression vector (black bars) or empty vector (grey bars) were then assayed by ChIP assay and real-time PCR. PCR primers specific for the LBS region of TR or the AMP gene of the plasmid were analyzed by ChIP assay with antibodies specific for LANA, ORC2, MCM3, AcH3, and IgG. Data from at least three independent ChIP experiments were normalized as the percentage of total input DNA recovered for each antibody.
FIG. 6.
FIG. 6.
HBO1 binds TR in vitro and in transfected cells. (A) DNA affinity purification assays with BCBL1-derived nuclear extracts were compared for binding to 2 × TR or BKS DNA. Eluted proteins were analyzed by Western blotting with antibodies specific for LANA, HBO1, ORC2, BRD2, CBP, MCM3, telomere repeat binding factor 2, or nonspecific control rabbit IgG as indicated and visualized by enhanced chemiluminescence. (B) ChIP assay of transiently transfected 293 cells with FLAG-HBO1 and untagged LANA expression vectors. Primer amplifications for TR and AMP genes were quantitated and presented as the increase over IgG for each IP reaction.
FIG. 7.
FIG. 7.
ORC, MCM, and HBO1 contribute to LANA-dependent DNA replication. (A) Transient DNA replication assay of 2 × TR-containing plasmid in 293 cells transfected with LANA expression vectors (+) or with control vector (−). Transfections also contained siRNA directed against ORC2, MCM5, HBO1, or control luciferase as indicated above. Replicated plasmid DNA was analyzed by DpnI resistance and Southern blotting. (B) Quantification of three independent replication assays is indicated in the bar graph. (C) Western blot analysis of ORC2, MCM5, and HBO1 siRNA knockdown experiments. Transfected cell extracts were analyzed with antibodies specific for siRNA-targeted proteins ORC2, MCM5, and HBO1 or control proteins PCNA and FLAG-LANA, as indicated to the right of each panel.
FIG. 8.
FIG. 8.
Model of the KSHV latent replication origin at TR. Four nucleosomes are predicted to bind between tandem LBS. The nucleosomes adjacent to LBS have high levels of histone H3 acetylation, maintained by HBO1 and CBP acetyltranferases and the acetyl lysine binding activity of BRD2. G1/S-arrested cells correlate with a disruption of nucleosome structure at the LBS, MCM2 binding, and the loss of H3MeK4.
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