Single Molecule Analysis of Replicated DNA Reveals the Usage of Multiple KSHV Genome Regions for Latent Replication

PLoS Pathogens

Settapong Kosiyatrakul¹  Carl L. Schildkraut¹  Maria E. McDowell²  Subhash C. Verma²  Erle S. Robertson³  Qiliang Cai³  Jie Lu³ 

[1] Department of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, United States of America;Department of Microbiology & Immunology, University of Nevada, Reno, School of Medicine, Center for Molecular Medicine, Reno, Nevada, United States of America;Department of Microbiology and Tumor Virology Program of the Abramson Cancer Center, Perelman School of Medicine at the University of Pennsylvania, Philadelphia, Pennsylvania, United States of America

关键词: DNA replication;    Viral replication;    Genomics;    Genomic signal processing;    Kaposi's sarcoma-associated herpesvirus;    Probe hybridization;    Genome complexity;    Cell cycle;    cell division;   

DOI  :  10.1371/journal.ppat.1002365

学科分类：生物科学（综合）

来源: Public Library of Science

【 摘 要 】

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.

【 授权许可】

CC BY