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. 2011;6(8):e23892.
doi: 10.1371/journal.pone.0023892. Epub 2011 Aug 25.

Upregulation of cellular Bcl-2 by the KSHV encoded RTA promotes virion production

Jianming Gao  1 , Qiliang Cai, Jie Lu, Hem Chandra Jha, Erle S Robertson
Affiliations

Upregulation of cellular Bcl-2 by the KSHV encoded RTA promotes virion production

Jianming Gao et al. PLoS One. 2011.

Abstract

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 CCN(9)GG 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 CCN(9)GG 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Bcl-2 is upregulated in HEK 293 and DG 75 cells tranfected with RTA.
(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.
Figure 2
Figure 2. Bcl-2 is upregulated in TPA and butyrate induced BC3, BCBL1 cells.
(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.
Figure 3
Figure 3. Northern blot analyses of Bcl-2 RNA.
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.
Figure 4
Figure 4. RTA transactivated the Bcl-2 promoter in a dose-dependent manner.
(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.
Figure 5
Figure 5. The CCN9GG-like KSHV RTA responsive element (RRE) and P2 promoter region are indispensable for activation of Bcl-2 transcription by RTA.
(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.
Figure 6
Figure 6. The CCN9GG motifs contribute to Bcl-2 promoter activation by RTA.
(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 Fig. 4 legend. Deletion of the first and second CCN9GG motifs led to a significant decrease in RTA-mediated Bcl-2 promoter activity. (C) A ChIP assay was carried out using induced or uninduced BCBL1 cells. RTA-DNA complexes were specifically precipitated with or without RTA antibody. The precipitated DNA was recovered by PCR with primers covering RRE 1 and 2 (upper panel). Real-time PCR was performed to quantify the immunoprecipitated promoter DNA.
Figure 7
Figure 7. RTA-mediated Bcl-2 upregulation is important for prolonging the survival of lytically infected host cells and to enhance production of virus progeny.
(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.
Figure 8
Figure 8. Hypothetical model showing RTA mediated upregulation of cellular Bcl-2.
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.
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