Kaposi Sarcoma Herpesvirus (KSHV) Latency-Associated Nuclear Antigen (LANA) recruits components of the MRN (Mre11-Rad50-NBS1) repair complex to modulate an innate immune signaling pathway and viral latency
Fig 5
Inhibition of canonical NF-κB cascade and KSHV lytic reactivation upon LANA Δ161 overexpression.
(A-B) HeLa.CNX.rKSHV and the parental HeLa.CNX cells were transfected with LANA Δ161 plasmid or empty vector for 48 hours and (B) treated with 5% RTA (vol/vol, see Materials and methods) for about 24 hours. Cells were lysed with TBS-T buffer and lysates resolved by immunoblotting. Phospho-p65 levels were digitally quantified (see Materials and methods). (C) HEK293 cells were transfected with the plasmid expressing full-length LANA, the truncated LANA isoforms or the empty vector (2 μg/well), together with an NF-κB reporter vector (200 ng/well). The luciferase activity was measured 48 hours later in duplicates and the statistical analysis was performed with two-tailed student’s t-test. Statistical significance of the difference between control (pcDNA3.1) and LANA (FL or Δ161 or Δ282) transfected samples is shown: (***) for p<0.005 and (ns) for not significant. (D) HeLa.CNX cells were transfected with the plasmid expressing vFLIP or the empty vector (1μg) and additionally with the plasmid expressing full-length LANA (1μg) or the truncated isoform (Δ161, 0.5–1μg) or the empty vector (2μg of plasmid DNA in total per condition), together with NF-κB reporter vector (200 ng/well). The luciferase activity was measured 48 hours later in duplicates and the statistical analysis was performed with two-tailed student’s t-test. Statistical significance of the difference between vFLIP alone and vFLIP-LANA (FL or Δ161) transfected samples is shown: (*) for p<0,05 and (ns) for not significant.