The LANA^SIM motif is important for maintenance of KSHV episome.

<p>(<b>A</b>) Deletion of LANA^SIM does not affect the TR-binding ability of LANA but lose the capacity of LANA-mediated TR maintenance. HEK293 cells were co-transfected TR-puromycin plasmid with wild type or different mutants of myc-tagged LANA or vector alone as indicated. The equal transfection efficiency is monitored by GFP co-expression. At 48 hr post-transfection, cells were subjected to ChIP assays with normal mouse IgG or mouse anti-myc(9E10) antibodies followed by real-time quantitative PCR with TR primer (<i>upper panel</i>). The level of TR per transfection was compared to vector alone and shown as input. The results of TR-binding ability of LANA were presented after normalization with the protein levels of LANA. For colony formation assays of LANA-mediated TR maintenance, two thousands transfected cells at 2 day post transfection were seed and selected with 2 µg/ml puromycin for 14 days (<i>lower panel</i>). The average of colonies quantified from duplicate experiments is shown in the figure. The expression of wild type LANA and its mutants are presented by western blot assay at the middle panel. (<b>B</b>) Dominant negative (DN) mutant of LANA with SIM deletion reactivates KSHV lytic replication. HEK293/Bac36 stable cells were transfected with different dosage (10, 20 µg) of expressing plasmids as indicated in the figure. At 48 hr post-transfection, the relative virion per ml supernatant of transfected cells was detected by quantitative PCR with TR as target. GAPDH was used as internal control. The expression of wild type LANA and its mutants are presented by western blot assay at the bottom panel. (<b>C</b>) Reporter assays of the Rta promoter in the presence of LANA under normoxic and hypoxic conditions. HEK293 and BJAB cells were individually co-transfected HRE-containing Rta promoter driven Luciferase reporter (pRta-Luc) with wild type (WT) LANA-myc or its mutants (ΔSIM, ΔAD, or ΔAD,SIM). At 24 hr post-transfection, cells were exposed with or without 1% oxygen for overnight before harvest. The results were presented by the RLU (relative luciferase unit) fold compared to pRta-Luc with vector alone. Data is presented as means±SD of three independent experiments. (<b>D</b>) Knockdown of KAP1 or Sin3A blocks LANA^SIM-mediated inhibition of LANA on Rta promoter. HEK293 cells with constitutive knockdown of KAP1 (shKAP1), Sin3A (shSin3A) or a scrambled negative control (shCtrl) were co-transfected pRta-Luc with LANA-myc (WT or ΔSIM) or vector alone. The pRta-Luc parental vector (pGL2-basic) alone was used as a control. Cells were collected at 48 hr post-transfection and analyzed by reporter assays as described in panel C.