Fig 2: NCOA2 stabilizes RTA protein expression.(A) Effect of NCOA2 on RTA expression. First, 293T cells were cotransfected with 1 μg of RTA expression plasmid and increasing amounts of NCOA2 expression vector (0, 0.5, 1, 2 μg). The expression of RTA proteins was examined by immunoblotting with the indicated antibodies. (B) The 293T cells were treated as in (A). RTA mRNA was detected using RT-qPCR with the indicated primers. (C) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then, cells were transfected with the Flag-tagged RTA expression plasmid. The expression of NCOA2 and RTA was determined by immunoblotting with the indicated antibodies. (D) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then cells were transfected with the Flag-tagged RTA-△PARS-II expression plasmid. The expression of NCOA2 and RTA-△PARS-II was determined by immunoblotting with the indicated antibodies. (E) The N-terminal truncation of NCOA2 regulates the stability of RTA. First, 293T cells were cotransfected with HA-tagged RTA expression vector with or without NCOA2 and its truncations. Then, RTA expression levels were examined by western blotting using anti-HA antibodies. (F) Effect of NCOA2 on the expression of the RTA mutant with deletion of the PARS II region. First, 293T cells were cotransfected with 1 μg of Flag-tagged RTA-△PARS-II expression plasmid and increasing amounts of NCOA2 expression vector (0, 0.5, 1, 2 μg). Then, the expression of RTA-△PARS-II proteins was examined by immunoblotting with the indicated antibodies. (G) The 293T cells were treated as in (F), and the mRNA level of RTA-△PARS-II was detected using RT-qPCR with the indicated primers. (H) Measurement of RTA stability in the absence and presence of NCOA2. First, 293T cells were transfected with Flag-tagged RTA with or without the NCOA2 expression plasmid for 36 h. Then, cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA. Tubulin was used as a control for equivalent sample loading. (I) The relative levels of RTA from immunoblots (H) were quantified by densitometry and normalized to the Tubulin level. The band intensities on the exposed film were plotted graphically. (J) Measurement of RTA-△PARS-II stability in the absence and presence of NCOA2. First, 293T cells were transfected with Flag-tagged RTA-△PARS-II with or without the NCOA2 expression plasmid for 36 h. Then, cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA-△PARS-II. Tubulin was used as a control for equivalent sample loading. The band intensities on the exposed film are plotted graphically (K). (L) First, 293T cells were transfected with RTA expression plasmid with sh-NCOA2 or sh-GFP. Then, the cells were treated with 100 μg/ml of CHX and analyzed at different time points as indicated by immunoblotting for RTA. Tubulin was used as a control for equivalent sample loading. The band intensities on the exposed film were plotted graphically (M).

Fig 3: NCOA2 inhibits proteasome-mediated degradation of RTA.(A) NCOA2 inhibits the degradation of RTA. First, 293T cells were cotransfected with the indicated expression constructs for 36 h and then treated with 0.5 μM MG132 or 0.1% DMSO for another 6 h. The cells were lysed and used for western blotting with the indicated antibodies. The band intensities of RTA were plotted graphically. Data are from a minimum of three experimental replicates with a standard deviation. (B) NCOA2 inhibits the ubiquitination of RTA. The same lysates as in (A) were subjected to immunoprecipitation using anti-Flag antibody. Purified proteins, along with input samples, were analyzed by western blotting with anti-ubiquitin antibodies. (C) shRNA-NCOA2 and shRNA-GFP plasmids were transfected into 293T cells for 12 h. Then cells were transfected with the Flag-tagged RTA expression plasmid for 24 h and then treated as in (A). Cells were lysed and used for western blotting with the indicated antibodies. The band intensities of RTA were plotted graphically. Data were from a minimum of three experimental replicates with a standard deviation. (D) Same lysates as in (C) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. (E) First, 293T cells were cotransfected with NCOA2 and the RTA-△PARS-II mutant, and then, they were treated as in (A). The expression of the RTA-△PARS-II mutant was analyzed by western blotting. The band intensities of RTA were plotted graphically. (F) The same lysates as in (E) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. (G) 293T cells were transfected with shRNA-NCOA2 or shRNA-GFP plasmids, and then cells were transfected with RTA-△PARS-II mutant and treated as in (A). The expression of RTA-△PARS-II mutant was analyzed by western blotting. The band intensities of RTA were plotted graphically. (H) The same lysates as in (G) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. Statistical significance was analyzed with a two-tailed Student’s t-test (*P < 0.05 or **P < 0.01).

Fig 4: Working model for the role of NCOA2 in regulating the stability of RTA.After interacting with RTA, MDM2 promotes the ubiquitination and degradation of RTA, which inhibits KSHV lytic replication. NCOA2 competes with MDM2 to interact with RTA, which blocks the interaction between RTA and MDM2 and then inhibits the degradation of RTA. The abundance of RTA is increased by NCOA2. In turn, RTA promotes the expression of NCOA2, which forms a positive feedback loop between NCOA2 and RTA and promotes KSHV lytic replication.

Fig 5: Ectopic expression of NCOA2 enhances KSHV lytic reactivation.(A) iSLK.RGB cells were stably transfected with lentiviruses containing a Flag-tagged NCOA2 expression plasmid or an empty vector plasmid, named iSLK.RGB-NCOA2 and iSLK.RGB-Vector, respectively. The overexpression of NCOA2 was detected by western blotting. (B) iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells were treated with dox at different time points as indicated. Fluorescence microscopy images of EGFP-positive cells among iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells. (C) Flow cytometry analysis of EGFP-positive cells among iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells. (D) Quantitation of the percent of EGFP-positive cells from (C). (E) NCOA2 overexpression increases the transcription of viral genes. RNA was extracted from dox-induced cells at 48 hours post-induction (hpi) to investigate the transcriptional level of NCOA2 and several KSHV genes: RTA, PAN, ORF57, ORF65 and LANA. (F) NCOA2 overexpression increases the expression of viral genes. The expression levels of RTA protein and ORF64 protein were determined by immunoblotting with the indicated antibodies. (G) NCOA2 overexpression increases virus production. Culture supernatants from dox-induced iSLK.RGB-Vector and iSLK.RGB-NCOA2 cells at 48 hpi were quantified by qPCR for KSHV copy numbers. (H) BCBL1 cells were stably transfected with lentiviruses containing a NCOA2 expression plasmid or an empty vector plasmid, named BCBL1-NCOA2 and BCBL1-Vector, respectively. The overexpression of NCOA2 was detected by western blotting. (I) NCOA2 overexpression increases virus production in BCBL1 cells. BCBL1-NCOA2 and BCBL1-Vector cells were treated with VPA for 24 h, and the KSHV copy numbers from culture supernatants were quantified by qPCR. Data in D, E, G, and I represent the mean +/- SD of 3 replicates pooled from three independent experiments. Data were analyzed with a two-tailed Student’s t-test (*P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001).