Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication

Subhash C. Verma, Jie Lu, Qiliang Cai, Settapong Kosiyatrakul, Maria E. McDowell, Carl L. Schildkraut, Erle S. Robertson

Research output: Contribution to journalArticle

21 Citations (Scopus)

Abstract

Kaposi's sarcoma associated herpesvirus (KSHV), an etiologic agent of Kaposi's sarcoma, Body Cavity Based Lymphoma and Multicentric Castleman's Disease, establishes lifelong latency in infected cells. The KSHV genome tethers to the host chromosome with the help of a latency associated nuclear antigen (LANA). Additionally, LANA supports replication of the latent origins within the terminal repeats by recruiting cellular factors. Our previous studies identified and characterized another latent origin, which supported the replication of plasmids ex-vivo without LANA expression in trans. Therefore identification of an additional origin site prompted us to analyze the entire KSHV genome for replication initiation sites using single molecule analysis of replicated DNA (SMARD). Our results showed that replication of DNA can initiate throughout the KSHV genome and the usage of these regions is not conserved in two different KSHV strains investigated. SMARD also showed that the utilization of multiple replication initiation sites occurs across large regions of the genome rather than a specified sequence. The replication origin of the terminal repeats showed only a slight preference for their usage indicating that LANA dependent origin at the terminal repeats (TR) plays only a limited role in genome duplication. Furthermore, we performed chromatin immunoprecipitation for ORC2 and MCM3, which are part of the pre-replication initiation complex to determine the genomic sites where these proteins accumulate, to provide further characterization of potential replication initiation sites on the KSHV genome. The ChIP data confirmed accumulation of these pre-RC proteins at multiple genomic sites in a cell cycle dependent manner. Our data also show that both the frequency and the sites of replication initiation vary within the two KSHV genomes studied here, suggesting that initiation of replication is likely to be affected by the genomic context rather than the DNA sequences.

Original languageEnglish (US)
Article numbere1002365
JournalPLoS Pathogens
Volume7
Issue number11
DOIs
StatePublished - Nov 2011

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Human Herpesvirus 8
Genome
DNA
Terminal Repeat Sequences
Replication Origin
Chromatin Immunoprecipitation
Kaposi's Sarcoma
DNA Replication
Lymphoma
Cell Cycle
Proteins
Plasmids
Chromosomes
latency-associated nuclear antigen

ASJC Scopus subject areas

  • Microbiology
  • Parasitology
  • Virology
  • Immunology
  • Genetics
  • Molecular Biology

Cite this

Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication. / Verma, Subhash C.; Lu, Jie; Cai, Qiliang; Kosiyatrakul, Settapong; McDowell, Maria E.; Schildkraut, Carl L.; Robertson, Erle S.

In: PLoS Pathogens, Vol. 7, No. 11, e1002365, 11.2011.

Research output: Contribution to journalArticle

Verma, Subhash C. ; Lu, Jie ; Cai, Qiliang ; Kosiyatrakul, Settapong ; McDowell, Maria E. ; Schildkraut, Carl L. ; Robertson, Erle S. / Single molecule analysis of replicated DNA reveals the usage of multiple KSHV genome regions for latent replication. In: PLoS Pathogens. 2011 ; Vol. 7, No. 11.
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