Fig 1.
NCOA2 interacts with KSHV RTA.
(A) First, 293T cells were transfected with Flag-NCOA2 alone or HA-RTA alone or cotransfected with Flag-NCOA2 along with HA-RTA. Cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by western blotting with the indicated antibodies. (B) The 293T cells were transfected with Flag-RTA alone or HA-NCOA alone or cotransfected with Flag-RTA along with HA-NCOA2. Cell lysates were immunoprecipitated with anti-Flag antibody and then analyzed by western blotting with the indicated antibodies. (C) In vitro GST affinity binding assay. Bacterially expressed GST alone and GST-NCOA2 attached to GST-Sepharose beads were incubated with the purified His-tagged RTA, and the pull-down lysates were immunoblotted with anti-His or anti-GST antibodies. (D) Colocalization of NCOA2 and RTA in HeLa cells. Following transfection with Flag-RTA and HA-NCOA2, HeLa cells were fixed with 4% paraformaldehyde and then stained with anti-HA and anti-Flag antibodies. Secondary antibodies conjugated to FITC or Cy3 were used to visualize the stained RTA and NCOA proteins, respectively. Diamidino-2-phenylindole shows the nuclei of cells.
Fig 2.
The interaction between endogenous NCOA2 and RTA.
(A) NCOA2 expression in HEK293T cells and KSHV-positive human cells (iSLK.RGB, BCBL1, JSC1 and BC3) was detected by western blotting. (B) Co-IP of endogenous RTA and NCOA2 in KSHV-positive cells. Lytic replication of KSHV in these cells was induced by dox or VPA, and cell lysates were subjected to immunoprecipitation with anti-NCOA2 antibody or rabbit IgG controls. Purified proteins, along with input samples, were detected by western blotting with the indicated antibodies. (C) Endogenous NCOA2 colocalizes with endogenous RTA in the nucleus. KSHV-positive B cells that were uninduced (Un) or induced with VPA (In) were fixed and stained with anti-NCOA2 antibody and anti-RTA antibody, followed by incubation with secondary antibodies conjugated to FITC or Cy3. The right sides show a high magnified view.
Fig 3.
Mapping the interaction domains in RTA and NCOA2.
(A) Truncated versions of NCOA2 are shown schematically, including N765 (1 aa-765 aa), 765C (765 aa-1464 aa), N1007 (1 aa-1007 aa), N624 (1 aa-624 aa), 624C (624 aa-1464 aa). The N-terminal bHLH-PAS domain, the centrally located nuclear receptor (NR) boxes responsible for NR binding, the C-terminal activation domain (AD1 and AD2) and a repression domain (RD) are marked. (B) Defining the RTA-interacting domain of NCOA2. Co-IP and western blotting of 293T cells transfected with HA-tagged RTA along with a vector expressing the indicated Flag-tagged NCOA2 truncations or the full-length NCOA2. (C) Schematic diagram of the RTA protein and its deletion mutants, including N273 (1 aa-273 aa), N544 (1 aa-544 aa), RTA273-544 (273 aa-544 aa), C544-691 (544 aa-691 aa), C273-691 (273 aa-691 aa). The nuclear localization (NLS), DNA binding and dimerization and transcriptional activation domain (TAD) are marked. (D) Defining the NCOA2-interacting domain of RTA. Co-IP and western blotting of 293T cells transfected with HA-tagged NCOA2 along with Flag-tagged RTA truncations or full-length RTA. An empty vector was used as a negative control. (E) In vitro GST affinity binding assay. Bacterially expressed GST alone and GST-NCOA2 attached to GST-Sepharose beads were incubated with the purified His-tagged RTA mutation (His-C544-691). The pull-down lysates were immunoblotted with anti-His or anti-GST antibody. (F) Schematic diagram of a PARS II region deletion mutant of RTA. (G) Co-IP and western blotting of 293T cells transfected with HA-NCOA2 along with Flag-tagged PARS II region deletion mutants.
Fig 4.
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 5.
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 6.
NCOA2 inhibits the ubiquitination of RTA by blocking the interaction of RTA with MDM2.
(A-C) NCOA2 disrupts the interaction between RTA and MDM2. 293T cells were transiently transfected with Flag-RTA and HA-MDM2 together with an increasing amount of Myc-NCOA2 (A), Myc-N624 (B) or Myc-624C (C) (0, 0.5, 1, 2 μg). Forty-eight hours after transfection, the cell lysates were collected and subjected to immunoprecipitation using an anti-Flag antibody. Purified proteins, along with input samples, were detected by western blotting with the indicated antibodies. (D) NCOA2 inhibits the proteasome-mediated degradation of RTA induced by MDM2. 293T cells were transiently transfected with Flag-RTA and HA-MDM2 together with an increasing amount of Myc-NCOA2 (0, 0.5, 1, 2 μg) for 36 h, and then, they were treated with 0.5 μM MG132 or 0.1% DMSO for another 6 h. The cells were lysed and used for western blots with the indicated antibodies. (E) NCOA2 inhibits the ability of MDM2 to increase the ubiquitination of RTA. The same lysates as in (D) were subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies. (F-G) 293T cells were transiently transfected with Flag-RTA and HA-MDM2 together with an increasing amount of Myc-N624 (F) or Myc-624C (G) (0, 0.5, 1, 2 μg) for 36 h, then cells were treated with 0.5 μM MG132 for another 6 h. Cells were lysed and subjected to immunoprecipitation using anti-Flag antibody, followed by western blotting with anti-ubiquitin antibodies.
Fig 7.
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).
Fig 8.
Knockdown of endogenous NCOA2 impairs KSHV lytic replication.
(A) iSLK.RGB cells were transfected with control siRNA and two NCOA2-specific siRNAs. The knockdown efficiency was determined by western blotting. At 24 h after transfection, cells were induced by dox for another 24 h. (B) EGFP-positive cells were analyzed by fluorescence microscopy. (C) The KSHV gene transcription level was analyzed by qPCR. (D) The expression levels of RTA and ORF64 were examined by western blotting. (E) The progeny viruses from culture supernatants were analyzed by qPCR. (F) BCBL1 cells were also transfected with control siRNA and two NCOA2-specific siRNAs. The knockdown efficiency was determined by western blotting. At 24 h after transfection, cells were induced by VPA for another 24 h. The KSHV gene transcription level (G), the expression levels of RTA and ORF64 (H) and the progeny viruses (I) were analyzed by the same approaches that were used in iSLK.RGB stable cell lines. Data in C, 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).
Fig 9.
Expression of NCOA2 is upregulated during viral lytic replication by RTA.
(A and C) The expression kinetics of NCOA2 and RTA were analyzed in iSLK.RGB cells (A) or iSLK cells (C) at indicated time points after dox treatment. (B and D) NCOA2 mRNA expression level in cells from (A) or (C). (E) Effect of RTA on NCOA2 expression. 293T cells were cotransfected with 1 μg of NCOA2 expression plasmid and increasing amounts of RTA expression vector (0, 0.5, 1, 2 μg). The protein level of NCOA2 was examined by immunoblotting. (F) NCOA2 mRNA expression level in cells from (E). (G) RTA inhibits the degradation of NCOA2. 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. Cells were lysed and used for western blots with the indicated antibodies. The band intensities of NCOA2 were plotted graphically (H). Data were from a minimum of three experimental replicates with a standard deviation. Data were analyzed by unpaired t-test. *, P < 0.05; **, P < 0.01. (I) Measurement of NCOA2 stability in the absence and presence of RTA. 293T cells were transfected with HA-tagged NCOA2 with or without Flag-tagged RTA expression plasmid for 36 h. Cells were treated with 100 μg/ml of CHX and analyzed at indicated time points by immunoblotting for NCOA2. Tubulin was used as a control for equivalent sample loading. (J) The relative levels of NCOA2 from (I) were quantified by densitometry and normalized to the Tubulin level.
Fig 10.
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.
Table 1.
Primers used in this study.