Rapid and quantitative assessment of KSHV LANA-mediated DNA replication

Rapid and quantitative assessment of KSHV LANA-mediated DNA replication Kaposi’s sarcoma-associated herpesvirus (KSHV) latency-associated nuclear antigen (LANA) mediates DNA replication of terminal repeat (TR) DNA to enable viral episome persistence in latently infected cells. Southern blotting is routinely used to detect LANA-replicated DNA. We developed and validated a real-time PCR assay for TR-associated DNA and compared it with Southern blot analysis. Both PCR and Southern blot detected LANA-replicated DNA, but the PCR assay was more rapid and did not require radioisotope. PCR detection at 24 and 72 hours post-transfection demonstrated rapid loss of transfected TR DNA. LANA, and to a lesser extent a moderately deficient LANA mutant, reduced the rate of DNA loss through addition of replicated TR DNA and reduction in the loss of non-replicated DNA, the latter of which is consistent with LANA’s nuclear segregation function. Therefore, this work develops a rapid, sensitive, and quantitative PCR (qPCR) assay to detect KSHV LANA-replicated DNA and demonstrates that LANA reduces TR DNA loss after transfection through replication and nuclear partitioning of TR DNA. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Rapid and quantitative assessment of KSHV LANA-mediated DNA replication

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Springer Vienna
Copyright © 2011 by Springer-Verlag
Biomedicine; Infectious Diseases; Medical Microbiology; Virology
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