title: Rapid and quantitative assessment of KSHV LANA-mediated DNA replication.
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.
authors.affiliation: Channing Laboratory and Departments of Medicine, Brigham and Women's Hospital and Harvard Medical School, 181 Longwood Avenue, Boston, MA 02115, USA.
authors.forename: Erika
authors.initials: E
authors.lastname: De León Vázquez
authors.forename: Kenneth M
authors.initials: KM
authors.lastname: Kaye
mesh_terms: D000956:Antigens, Viral / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D004279:DNA, Viral / Q000502:physiology*; D015967:Gene Expression Regulation, Viral / Q000502:physiology; D019288:Herpesvirus 8, Human / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D020079:Terminal Repeat Sequences / Q000235:genetics / Q000502:physiology; D014779:Virus Replication / Q000502:physiology*
chemical_list: D000956:Antigens, Viral; D004279:DNA, Viral; D009687:Nuclear Proteins; C109540:latency-associated nuclear antigen