Conceived and designed the experiments: JG ESR. Performed the experiments: JG. Analyzed the data: JG QC HCJ. Contributed reagents/materials/analysis tools: JL. Wrote the paper: JG ESR.
The authors have declared that no competing interests exist.
Apoptosis of virus infected cells can restrict or dampen full blown virus propagation and this can serve as a protective mechanism against virus infection. Consequently, viruses can also delay programmed cell death by enhancing the expression of anti-apoptotic proteins. Human Bcl-2 is expressed on the surface of the mitochondrial membrane and functions as the regulator of the delicate balance between cell survival and apoptosis. In this report, we showed that the replication and transcription activator (RTA) encoded by KSHV ORF 50, a key regulator for KSHV reactivation from latent to lytic infection, upregulates the mRNA and protein levels of Bcl-2 in 293 cells, and TPA-induced KSHV-infected cells. Further analysis revealed that upregulation of the cellular Bcl-2 promoter by RTA is dose-dependent and acts through targeting of the CCN9GG motifs within the Bcl-2 promoter. The Bcl-2 P2 but not the P1 promoter is primarily responsive to RTA. The results of ChIP confirmed the direct interaction of RTA protein with the CCN9GG motifs. Knockdown of cellular Bcl-2 by lentivirus-delivered small hairpin RNA (shRNA) resulted in increased cell apoptosis and decreased virion production in KSHV-infected cells. These findings provide an insight into another mechanism by which KSHV utilizes the intrinsic apoptosis signaling pathways for prolonging the survival of lytically infected host cells to allow for maximum production of virus progeny.
Kaposi's Sarcoma-Associated Herpesvirus (KSHV), the etiological factor associated with Kaposi's Sarcoma is also known as human herpesvirus 8 (HHV 8). Several other malignancies such as primary effusion lymphoma, and multicentric Castleman's disease have also been known to associate with KSHV
RTA, encoded by KSHV ORF 50, is an immediate early protein and functions as the critical regulator for the shift of KSHV life cycle from latency to lytic activation. Studies have previously shown that RTA is able to activate the transcription of many viral genes, including K1
Apoptosis is a major antiviral cellular response against viral infection. The B-cell leukemia/lymphoma 2 (Bcl-2) family of proteins controls the intrinsic mitochondrial pathway of cellular apoptosis
Bcl-2 protein levels can be regulated transcriptionally and post transcriptionally. Furthermore, the human Bcl-2 gene contains both the P1 and P2 promoters
We now show that RTA can transactivate the cellular Bcl-2 promoter in a dose-dependent manner. RTA upregulates Bcl-2 through targeting of CCN9GG-like RTA responsive elements (RREs) within the promoter and that the P2 promoter is important for RTA-mediated transcriptional regulation of cellular Bcl-2. These findings provide insights into new mechanisms by which RTA transactivates cellular Bcl-2 and so contributes to enhanced efficiency of KSHV lytic replication.
Bcl-2 mRNA transcripts and protein levels were examined to specifically detect the RTA-mediated cellular Bcl-2 upregulation (
(A) Quantification of Bcl-2 transcripts by RT-PCR. Total RNA was ectracted from 10 million HEK 293 and DG 75 cells tranfected with increasing amount of RTA plasmid (0, 5, 10, 15 µg). A total of 2 µg of RNA was used to synthesize cDNA. Real-time PCR was performed by using a power SYBR green PCR Master Mix kit with GAPDH as control. Standard deviations were illustrated by error bars. (B) Western blot (WB) analysis of endogenous Bcl-2 in the RTA-expressing HEK 293. GAPDH was used for internal control. Quantification of the relative densities of Bcl-2 was plotted below the blots.
KSHV lytic replication can be initiated by chemical inducers, like TPA and sodium butyrate
(A) Quantification of Bcl-2 transcripts by RT-PCR. (B) Western blot analysis of endogenous Bcl-2 in induced and uninduced BC3, BCBL1, BJAB cells.
In addition to RT-PCR analysis, we wanted to corroborate our results and so performed Northern blot analysis to verify Bcl-2-specific mRNA in RTA expressing HEK 293 cells and in induced KSHV-infected B cells by using a Bcl-2-specific DNA probe.
Total RNA was extracted from each cell (HEK 293, RTA-expressing HEK 293, induced and uninduced BC3, BCBL1, BJAB cells). Blots were first probed against Bcl-2 mRNA, then reprobed with a GAPDH probe. Density of Bcl-2 signal divided by that of GAPDH control was used to quantify the levels of Bcl-2 mRNA expression. Western blot analysis was used to detect expression of RTA transcript to show the dose effect.
Since TPA-induced KSHV-infected cells and RTA expressing cells showed higher levels of Bcl-2 mRNA and protein expression, we further explored the molecular mechanism of Bcl-2 promoter up-regulation by RTA. To analyze the role of RTA on regulating the Bcl-2 promoter, a fixed amount of full length Bcl-2 promoter luciferase construct (pGL3-Bcl-2)
(A, B) Ten million HEK 293 cells or DG75 cells were transfected with 10 µg of PGL3-Bcl-2, and 0, 5, 10, 15, or 20 µg pcDNA-RTA. Cell lysates were prepared at 24 h posttransfection for the luciferase assay. Data from three independent groups are exemplified with mean and standard deviation to show the fold activation by comparison of the promoter activity in the presence of RTA with the value of pGL3B alone. Western blot was used to confirm the transfection efficiency with GAPDH served as control for equal protein loading.
To identify the potential motifs indispensable for the upregulation of cellular Bcl-2 by RTA, a series of truncated promoter luciferase contructs were generated and tested by reporter analysis in both HEK 293 and DG75, as shown in
(A) Illustration of the wild type and mutant Bcl-2 promoters. Nine CCN9GG motifs in Bcl-2 promoter are shown. Numbers represent the nucleotide positions upstream of the initiating ATG. (B, C) Ten million HEK 293 cells or DG75 cells were transfected with 10 µg of indicated mutant Bcl-2 promoter reporter plasmids, and 5, 10, 15, or 20 µg pcDNA-RTA. Cell lysates were prepared at 24 h posttransfection for the luciferase assay. The deletion of P1 promoter did not result in a major decrease in promoter activity and ever resulted in an increase in promoter activity in HEK 293 cells. However, deletion of either RREs or P2 promoter resulted in complete loss of Bcl-2 promoter activity in the presence of RTA.
The two promoters P1 and P2
To confirm the role of the CCN9GG motifs in Bcl-2 regulation, we performed reporter assays by using the pGL3-ΔRRE1,2 luciferase construct in which the first and second CCN9GG motifs were deleted (
(A) Scheme showing pGL3-ΔRRE1,2 and pGL3-ΔP1 construct used in this study. (B) Fix amounts (10 µg) of indicated mutant Bcl-2 promoter luciferase constructs were transfected or cotransfected into ten million HEK 293 cells with 10, 20 µg of pcDNA-RTA respectively. Cell lysates were prepared and the luciferase assay was carried out as above mentioned in
On the basis of the results we observed above, we hypothesized that RTA could take advantage of the Bcl-2 pathway to promote cell survival which may result in enhanced virus progeny production. Hence, we wanted to determine whether upregulation of cellular Bcl-2 by RTA may in fact affect cellular apoptosis. We constructed stable Bcl-2 knockdown cells (BC3-shBcl2, BJAB-shBcl2) and their control cells (BC3-shC,B JAB-shC) by lentivirus infection and puromycin selection (
(A) Bcl-2 knockdown cells and control cells with GFP expression were monitored by fluorescence microscopy showing successful lentivius infection. (B) Western blots (WB) showing expression of Bcl-2 in BJAB-shC, BJAB-shBcl-2, BC3-shC, and BC3-shBcl-2 cells. GAPDH served as control. (C) Supernatants of RTA and butyrate-induced BC3-shC, and BC3-shBcl2 cells were prepared after induction 48 h for PCR to analyze the production of KSHV progeny. KSHV genomic DNA isolated from BC3 cells served as control. (D) Different cellular apoptosis pattern in induced and uninduced BJAB-shBcl2 and BC3-shBcl2 cell lines. The first sub-G1 peak represents apoptotic cell.
Similar to other large DNA herpesviruses, KSHV has two major life cycles referred to as the latent and lytic replication cycles. Latency exists in most of the KSHV infected cells which harbor the viral episomes
Apoptosis of infected cells can restrict or dampen full blown virus replication and this can serve as a protective defense mechanism against virus infection. Consequently, viruses can also delay programmed cell death by enhancing the expression of antiapoptotic proteins
Human Bcl-2 is expressed on the surface of the mitochondrial membrane and functions as a major regulator of the delicate balance between cell survival and apoptosis
To determine the mechanisms by which RTA activates Bcl-2, we identified potential responsive elements within the Bcl-2 promoter. Interestingly, this promoter contains nine CCN9GG-like motifs and these CCN9GG-like motifs were recently identified KSHV RRE present in five gene promoters of KSHV
Depletion of Bcl-2 clearly increases the level of cellular apoptosis, and decreases virus progeny production in induced BC3-shBcl2 cells compared with that of the BC3-shC cells. This provides strong evidence that upregulation of cellular Bcl-2 by RTA can definitely contribute to KSHV lytic replication. The utilization of the Bcl-2 pathway by RTA would be a responsible strategy for prolonging the survival of lytically infected cells and thus allow enhanced production of virus progeny.
In summary, our study which investigated the interaction between KSHV RTA and cellular Bcl-2 showed that RTA can transactivate Bcl-2 transcription through targeting of the RRE CCN9GG motifs within the Bcl-2 promoter. We also demonstrated that the P2 promoter region instead of the P1 promoter region was highly responsive to RTA. The results of ChIP confirmed the direct interaction of RTA protein with the CCN9GG motifs. Knockdown of cellular Bcl-2 by lentivirus-delivered small hairpin RNA (shRNA) resulted in increased cell apoptosis and decreased virion production in KSHV-infected cells. RTA can upregulate Bcl-2-mediated block to cellular apoptosis, and so prolong cell survival leading a further increase in production of virus progeny (
P2 promoter and the CCN9GG-like RREs are indispensable for upregulation of Bcl-2 by RTA. Enhanced expression of Bcl-2 prolongs cell survival and virus progeny production. RRE, RTA responsive element.
Plasmid pcDNA-RTA is an expression vector which encodes the full-length RTA. The Bcl-2 gene full-length promoter reporter plasmid, pGL3-Bcl-2 was a kind gift from Véronique Bourgarel-Rey (Aix-Marseille Université, Marseille Cedex 05, France). The truncated reporter plasmids pGL3-ΔRREs, pGL3-ΔP1, pGL3-ΔP2 and pGL3-ΔRRE1,2 were obtained by PCR-based site mutagenesis (Primers see
Primer | Squence |
Note |
Bcl-2-F1 |
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Real-time PCR |
Bcl-2-R1 |
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GAPDH-F1 |
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Real-time PCR |
GAPDH-R1 |
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Bcl-2-F2 |
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Northern probe |
Bcl-2-R2 |
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GAPDH-F2 |
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Northern probe |
GAPDH-R2 |
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Bcl-2Δ1 |
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ΔRREs clone |
Bcl-2Δ2 |
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Bcl-2Δ3 |
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ΔP2 clone |
Bcl-2Δ4 |
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Bcl-2Δ5 |
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ΔP1 clone |
Bcl-2Δ6 |
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Bcl-2Δ7 |
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Bcl-2Δ8 (Δ1) |
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Bcl-2Δ9 |
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ΔRRE1,2 clone |
Bcl-2Δ10 |
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Bcl-2Δ11 |
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Bcl-2Δ12 (Δ1) |
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K9-F |
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PCR for virus progeny |
K9-R |
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ChIP-F |
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ChIP assay |
ChIP-R |
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Restriction sites are underlined.
RTA antibody was generated from a hybridoma (Lan et al. Institute Pasteur, Shanghai, China). The Bcl-2 mouse monoclonal antibody is a product of Santa Cruz Biotechnology, Inc (Santa Cruz, CA). The GAPDH mouse antibody was obtained from Novus Biologicals, LLC (Littleton, CO).
Human embryonic kidney fibroblast 293 was obtained from Jon Aster (Brigham and Women's Hospital, Boston, MA). BJAB is a KSHV and EBV negative B cell line and was provided by Elliott Kieff (Harvard Medical School, Boston, MA). DG75 is a KSHV negative B cell line. BC3 and BCBL1 are KSHV-positive body cavity-based lymphoma-derived cell lines obtained from the NIH AIDS Research and Reference Reagent Program
HEK 293 and DG75 cells were transfected by using a Bio-Rad Gene Pulser II electroporator. Experimental method for transfection by electroporation was previously described
After treatment, the cells were harvested and lysed in a RIPA buffer. The protein concentration of the lysates was measured by Bradford assay. Samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to polyvinylidene difluoride (PVDF) membranes. Western boltting was performed using anti-Bcl2, anti-RTA, or anti-GAPDH antibody and goat anti-mouse IgG secondary antibody conjugated to IR dye 800 (Rockland Immunochemicals, Inc., Gilbertsville, PA). After the membranes were washed, the signals were scanned with Odyssey infrared image (LI-COR, Inc., Lincoln, NE).
Total RNA extraction from cells (HEK 293, RTA-transfected HEK 293, DG75, RTA-transfected DG75, induced and un-induced BC3, BCBL1, BJAB cells), generation of cDNA, preparation of reaction mixture and RT-PCR amplification condition were described previously with minor modifications
A human Bcl-2 gene DNA probe of 539 bp and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) probe of 628 bp were generated by PCR using HEK 293 cells cDNA as template. Primer 3 software was used to design the primers (listed in
Ten million HEK 293 and DG75 cells were cotransfected with 10 µg of the reporter plasmids and increasing amounts of RTA plasmids respectively. Cell lysates were prepared at 24 h posttransfection. Twenty five microliters of luciferase assay substrate was mixed with 40 µl cell lysate, and a LMaxII384 luminometer (Molecular Devices, Inc., Sunnyvale, CA) was used to measure luminescence level. The results showed the mean and standard deviation (SD) of the data of three independent experiments. Western blot analysis was used to confirm the expression of the proteins.
At 48 h post-induction, BCBL1 cells were fixed by adding 37% formaldehyde to a final concentration of 1% for 10 min. The cross-linking reaction was stopped by adding glycine to a final concentration of 0.125 M for 5 min. Cells were washed three times with 1× ice-cold PBS and resuspended in cell lysis buffer [5 Mm PIPES (pH 8.0), 85 mM KCl, 0.5% NP-40] containing 1 mM PMSF and 1 µg/ml protease inhibitors for 10 min at 4°C. After centrifugation, the cell pellets were resuspended in nuclear lysis buffer [50 mM Tris/HCl (pH 8.1), 10 mM EDTA, 1% SDS] and incubated for 10 min at 4°C. The resulting solution was diluted 5-fold with dilution buffer [16.7 mM Tris/HCl (pH 8.1), 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100, 1 mM PMSF, 1 µg/ml protease inhibitors] and sonicated. After centrifugation, the supernatant was pre-cleared with mouse IgG and immobilized Protein A. Immunoprecipitation was performed by incubation at 4°C with anti-RTA antibody or mouse IgG antibody. Immune complexes were collected by a further incubation with immobilized Protein A for 1–2 h at 4°C. The beads were washed four times with IP buffer [25 mM Tris/HCl (pH 7.2), 150 mM NaCl] and resuspended in elution buffer (1% SDS/0.1 M NaHCO3). After centrifugation, the supernatant was neutralized with 1 M Tris/HCl (pH 7.2). RNase A and NaCl were added, followed by incubation at 65°C overnight to reverse the cross-linked DNA–protein complex. Proteinase K was added, followed by incubation at 55°C for 1–2 h. DNA extraction was carried out using phenol: chloroform and analysed by PCR using specific primers covering RRE1 and RRE 2 in the full length reporter vector. The DNA was quantified by SYBR green real-time PCR. For calculation of the relative DNA amount from quantitative real-time PCR, the Ct (threshold cycle) value of antibody-precipitated sample was normalized by the Ct value of the input, and the normalized Ct value of induced and uninduced groups were compared.
Bcl-2 small hairpin RNA (shBcl-2) and control shRNA (shC) were synthesized by Sigma (St. Louis, MO) as previously described
To investigate whether Bcl-2 upregulation can increase the production of viral progeny, the KSHV encoded ORF9 was amplified by PCR using viral DNA in TPA and butyrate induced BC3-shC and BC3-shBcl2 cells. Cells were induced and further incubated for 2 days at 37°C under 5% CO2. The supernatant was then collected and passed through a 0.45-µm-pore-size filter, and viral particles were spun down at 23,500 rpm for 20 min. Intact cells were discarded to prevent possible lysis and contamination from cellular viral DNA. The pellet was resuspended in 50 µl of 0.2× PBS, heated to 95°C for 15 min, and switched to 56°C for 1 h with proteinase K treatment (10 mg/ml). The enzyme was then destroyed by treatment at 95°C for 30 min. A 10-µl portion of virus lysate was used for PCR amplification of the KSHV-specific region of ORF9. The primers for amplification of ORF9 are listed in
Cellular apoptosis patterns were examined in induced and uninduced BC3-shBcl2, BJAB-shBcl2 and their control cell lines, BC3-shC and BJAB-shC using propidium iodide (PI) staining and flow ctyometry analysis using previously published protocols from our lab
We are grateful to Véronique Bourgarel-Rey (Aix-Marseille Université, Marseille Cedex 05, France) for providing the Bcl-2 full-length promoter luciferase construct, pGL3-Bcl-2. E.S.R. is a scholar of the Leukemia and Lymphoma Society of America.