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Rapid and quantitative assessment of KSHV LANA-mediated DNA replication
- 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.
- Subjects :
- Gene Expression Regulation, Viral
viruses
Mutant
Biology
Virus Replication
Polymerase Chain Reaction
Article
law.invention
chemistry.chemical_compound
law
Cell Line, Tumor
Virology
Humans
Antigens, Viral
Polymerase chain reaction
Southern blot
Terminal Repeat Sequences
DNA replication
Nuclear Proteins
virus diseases
General Medicine
Transfection
biochemical phenomena, metabolism, and nutrition
Molecular biology
Real-time polymerase chain reaction
Viral replication
chemistry
DNA, Viral
Herpesvirus 8, Human
DNA
Subjects
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