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

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.

SUBMITTER: Stedman W

PROVIDER: S-EPMC525046 | biostudies-literature | 2004 Nov

REPOSITORIES: biostudies-literature

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

Stedman William W Deng Zhong Z Lu Fang F Lieberman Paul M PM

Journal of virology 20041101 22

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. Th ...[more]

PMID: 15507644

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Project description:The maintenance of latent Kaposi's sarcoma-associated herpesvirus (KSHV) genomes is mediated in cis by their terminal repeats (TR). A KSHV genome can have 16 to 50 copies of the 801-bp TR, each of which harbors a 71-bp-long minimal replicator element (MRE). A single MRE can support replication in transient assays, and the presence of as few as two TRs appears to support establishment of KSHV-derived plasmids. Why then does KSHV have such redundancy and heterogeneity in the number of TRs? By determining the abilities of KSHV-derived plasmids containing various numbers of the TRs and MREs to be established and maintained in the long term, we have found that plasmids with fewer than 16 TRs or those with tandem repeats of the MREs are maintained inefficiently, as shown by both their decreased abilities to support formation of colonies and their instability, resulting in frequent rearrangements yielding larger plasmids during and after establishment. These defects often can be overcome by adding the Epstein-Barr virus (EBV) partitioning element, FR (i.e., family of repeats), in cis to these plasmids. In addition we have found that the spacing between MREs is important for their functions, too. Thus, two properties of KSHV's origin of latent replication essential for the efficient establishment and maintenance of viral plasmids stably are (i) the presence of approximately 16 copies of the TR, which are needed for efficient partitioning, and (ii) the presence of at least 2 MRE units separated by 801 bp of center-to-center spacing, which are required for efficient synthesis.KSHV is a human tumor virus that maintains its genome as a plasmid in lymphoid tumor cells. Each plasmid DNA molecule encodes many origins of synthesis. Here we show that these many origins provide an essential advantage to KSHV, allowing the DNAs to be maintained without rearrangement. We find also that the correct spacing between KSHV's origins of DNA synthesis is required for them to support synthesis efficiently. The identification of these properties illuminates plasmid replication in mammalian cells and should lead to the development of rational means to inhibit these tumorigenic replicons.

Dataset Information

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

Publications

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

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