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Rapid and quantitative assessment of KSHV LANA-mediated DNA replication

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Abstract

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.

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Acknowledgments

This work was supported by grants (KMK) from the National Cancer Institute (CA082036 and CA082036S1) and from the US Department of Defense (PR093491). Bo Zhao provided advice regarding CD21 promoter amplification.

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Authors and Affiliations

  1. Channing Laboratory and Departments of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 181 Longwood Avenue, Boston, MA, 02115, USA

    Erika De León Vázquez & Kenneth M. Kaye

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  1. Erika De León Vázquez
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  2. Kenneth M. Kaye
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Correspondence to Kenneth M. Kaye.

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De León Vázquez, E., Kaye, K.M. Rapid and quantitative assessment of KSHV LANA-mediated DNA replication. Arch Virol 156, 1323–1333 (2011). https://doi.org/10.1007/s00705-011-0985-0

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  • DOI: https://doi.org/10.1007/s00705-011-0985-0

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