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Rapid and quantitative assessment of KSHV LANA-mediated DNA replication

Authors :
Kenneth M. Kaye
Erika De León Vázquez
Source :
Archives of Virology. 156:1323-1333
Publication Year :
2011
Publisher :
Springer Science and Business Media LLC, 2011.

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.

Details

ISSN :
14328798 and 03048608
Volume :
156
Database :
OpenAIRE
Journal :
Archives of Virology
Accession number :
edsair.doi.dedup.....0144e67e89e65a53ff44a6850b458b5b
Full Text :
https://doi.org/10.1007/s00705-011-0985-0