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Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

Zhang SM, Zhang H, Yang TY, Ying TY, Yang PX, Liu XD, Tang SJ, Zhou PK - Int. J. Biol. Sci. (2014)

Bottom Line: On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription.Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1.We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Radiation Toxicology and Oncology; Beijing Institute of Radiation Medicine, 100850, Beijing, China;

ABSTRACT
HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

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DNA-PKcs modulates HIV transcription via its kinase activity and by forming a complex with CDK9, Cyclin T1 and Tat. A, The impact of DNA-PKcs expression levels on HIV transcription. MT2 cells were transfected with or without a DNA-PKcs over-expression plasmid, shRNA plasmid, HIV-LTR, RL-CMV and flag-Tat plasmids, as indicated. B, The plasmids, as indicated, were transfected into MT2 cells and DMSO (control) or NU7026 (DNA-PKcs kinase inhibitor) were added to the cell culture 12 h after transfection. The dual luciferase activity was assayed 48 h after transfection for both A and B. C, After treatment with or without recombinant His-tagged Tat protein (2 µg/ml) for 4 h, the total cell lysates were immunoprecipitated with normal IgG, anti-DNA-PKcs, anti-Cyclin T1 or anti-CDK9 antibody, and a western blot was performed using the indicated antibodies. D, MT2 cells were seeded into six-well plates with coverslips and transfected with a CFP-Tat plasmid and then fixed and stained as described in the Materials and Methods. To increase the adherence of the MT2 cells to the coverslips, the coverslips were pretreated with 0.2% gelatin overnight, prior to seeding. Anti-DNA-PKcs (red), anti-Cyclin T1 (green) and nuclear staining by DAPI were performed. E, A model describing the assembly of the DNA-PKcs/Tat/Cyclin T1/CDK9 complex at the HIV-1 5' LTR and their mutual interaction and regulation. Please see the text for details. Three independent experimental replicates were performed for each group. **P ≤0.01.
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Figure 6: DNA-PKcs modulates HIV transcription via its kinase activity and by forming a complex with CDK9, Cyclin T1 and Tat. A, The impact of DNA-PKcs expression levels on HIV transcription. MT2 cells were transfected with or without a DNA-PKcs over-expression plasmid, shRNA plasmid, HIV-LTR, RL-CMV and flag-Tat plasmids, as indicated. B, The plasmids, as indicated, were transfected into MT2 cells and DMSO (control) or NU7026 (DNA-PKcs kinase inhibitor) were added to the cell culture 12 h after transfection. The dual luciferase activity was assayed 48 h after transfection for both A and B. C, After treatment with or without recombinant His-tagged Tat protein (2 µg/ml) for 4 h, the total cell lysates were immunoprecipitated with normal IgG, anti-DNA-PKcs, anti-Cyclin T1 or anti-CDK9 antibody, and a western blot was performed using the indicated antibodies. D, MT2 cells were seeded into six-well plates with coverslips and transfected with a CFP-Tat plasmid and then fixed and stained as described in the Materials and Methods. To increase the adherence of the MT2 cells to the coverslips, the coverslips were pretreated with 0.2% gelatin overnight, prior to seeding. Anti-DNA-PKcs (red), anti-Cyclin T1 (green) and nuclear staining by DAPI were performed. E, A model describing the assembly of the DNA-PKcs/Tat/Cyclin T1/CDK9 complex at the HIV-1 5' LTR and their mutual interaction and regulation. Please see the text for details. Three independent experimental replicates were performed for each group. **P ≤0.01.

Mentions: We have proven the interaction between Tat and DNA-PKcs and the regulation of DNA-PKcs by Tat. We next asked if DNA-PKcs modulates HIV-1 transcription by binding to Tat. To verify this hypothesis, we transfected the HIV-LTR reporter RL-CMV together with myc-Tat, YFP-DNA-PKcs, Si-lacZ or Si-DPK plasmids into MT2 cells. As shown in Fig. 6A, the overexpression of DNA-PKcs strongly inhibits HIV transcription, while knockdown of DNA-PKcs greatly stimulates HIV transcription. To further determine if the kinase activity of DNA-PKcs is involved in HIV transcription, we treated MT2 cells transfected with the HIV-LTR reporter plasmid with NU7026, a DNA-PKcs-specific inhibitor. The results demonstrated that inhibition of DNA-PKcs kinase activity drastically suppresses HIV transcription (Fig. 6B). In HIV-1-infected T cells, Tat forms a ternary complex with Cyclin T1 and CDK9 to initiate transcription 1-3. To further investigate if DNA-PKcs forms a larger complex comprised of Tat, Cyclin T1 and CDK9, we performed three immunoprecipitation experiments using anti-DNA-PKcs, anti-Cyclin B1 and anti-CDK9 antibodies in the presence or absence of the Tat protein. As shown in Fig. 6C, DNA-PKcs interacts directly with CDK9 but not Cyclin T1, though Tat does not influence the affinity between DNA-PKcs and CDK9. Although Cyclin T1 co-localizes with DNA-PKcs, there is no direct interaction between the two proteins. These results imply that the DNA-PKcs protein suppresses HIV transcription by interacting with Tat and CDK9 and stimulates HIV transcription via its kinase activity.


Interaction between HIV-1 Tat and DNA-PKcs modulates HIV transcription and class switch recombination.

Zhang SM, Zhang H, Yang TY, Ying TY, Yang PX, Liu XD, Tang SJ, Zhou PK - Int. J. Biol. Sci. (2014)

DNA-PKcs modulates HIV transcription via its kinase activity and by forming a complex with CDK9, Cyclin T1 and Tat. A, The impact of DNA-PKcs expression levels on HIV transcription. MT2 cells were transfected with or without a DNA-PKcs over-expression plasmid, shRNA plasmid, HIV-LTR, RL-CMV and flag-Tat plasmids, as indicated. B, The plasmids, as indicated, were transfected into MT2 cells and DMSO (control) or NU7026 (DNA-PKcs kinase inhibitor) were added to the cell culture 12 h after transfection. The dual luciferase activity was assayed 48 h after transfection for both A and B. C, After treatment with or without recombinant His-tagged Tat protein (2 µg/ml) for 4 h, the total cell lysates were immunoprecipitated with normal IgG, anti-DNA-PKcs, anti-Cyclin T1 or anti-CDK9 antibody, and a western blot was performed using the indicated antibodies. D, MT2 cells were seeded into six-well plates with coverslips and transfected with a CFP-Tat plasmid and then fixed and stained as described in the Materials and Methods. To increase the adherence of the MT2 cells to the coverslips, the coverslips were pretreated with 0.2% gelatin overnight, prior to seeding. Anti-DNA-PKcs (red), anti-Cyclin T1 (green) and nuclear staining by DAPI were performed. E, A model describing the assembly of the DNA-PKcs/Tat/Cyclin T1/CDK9 complex at the HIV-1 5' LTR and their mutual interaction and regulation. Please see the text for details. Three independent experimental replicates were performed for each group. **P ≤0.01.
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Figure 6: DNA-PKcs modulates HIV transcription via its kinase activity and by forming a complex with CDK9, Cyclin T1 and Tat. A, The impact of DNA-PKcs expression levels on HIV transcription. MT2 cells were transfected with or without a DNA-PKcs over-expression plasmid, shRNA plasmid, HIV-LTR, RL-CMV and flag-Tat plasmids, as indicated. B, The plasmids, as indicated, were transfected into MT2 cells and DMSO (control) or NU7026 (DNA-PKcs kinase inhibitor) were added to the cell culture 12 h after transfection. The dual luciferase activity was assayed 48 h after transfection for both A and B. C, After treatment with or without recombinant His-tagged Tat protein (2 µg/ml) for 4 h, the total cell lysates were immunoprecipitated with normal IgG, anti-DNA-PKcs, anti-Cyclin T1 or anti-CDK9 antibody, and a western blot was performed using the indicated antibodies. D, MT2 cells were seeded into six-well plates with coverslips and transfected with a CFP-Tat plasmid and then fixed and stained as described in the Materials and Methods. To increase the adherence of the MT2 cells to the coverslips, the coverslips were pretreated with 0.2% gelatin overnight, prior to seeding. Anti-DNA-PKcs (red), anti-Cyclin T1 (green) and nuclear staining by DAPI were performed. E, A model describing the assembly of the DNA-PKcs/Tat/Cyclin T1/CDK9 complex at the HIV-1 5' LTR and their mutual interaction and regulation. Please see the text for details. Three independent experimental replicates were performed for each group. **P ≤0.01.
Mentions: We have proven the interaction between Tat and DNA-PKcs and the regulation of DNA-PKcs by Tat. We next asked if DNA-PKcs modulates HIV-1 transcription by binding to Tat. To verify this hypothesis, we transfected the HIV-LTR reporter RL-CMV together with myc-Tat, YFP-DNA-PKcs, Si-lacZ or Si-DPK plasmids into MT2 cells. As shown in Fig. 6A, the overexpression of DNA-PKcs strongly inhibits HIV transcription, while knockdown of DNA-PKcs greatly stimulates HIV transcription. To further determine if the kinase activity of DNA-PKcs is involved in HIV transcription, we treated MT2 cells transfected with the HIV-LTR reporter plasmid with NU7026, a DNA-PKcs-specific inhibitor. The results demonstrated that inhibition of DNA-PKcs kinase activity drastically suppresses HIV transcription (Fig. 6B). In HIV-1-infected T cells, Tat forms a ternary complex with Cyclin T1 and CDK9 to initiate transcription 1-3. To further investigate if DNA-PKcs forms a larger complex comprised of Tat, Cyclin T1 and CDK9, we performed three immunoprecipitation experiments using anti-DNA-PKcs, anti-Cyclin B1 and anti-CDK9 antibodies in the presence or absence of the Tat protein. As shown in Fig. 6C, DNA-PKcs interacts directly with CDK9 but not Cyclin T1, though Tat does not influence the affinity between DNA-PKcs and CDK9. Although Cyclin T1 co-localizes with DNA-PKcs, there is no direct interaction between the two proteins. These results imply that the DNA-PKcs protein suppresses HIV transcription by interacting with Tat and CDK9 and stimulates HIV transcription via its kinase activity.

Bottom Line: On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription.Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1.We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Radiation Toxicology and Oncology; Beijing Institute of Radiation Medicine, 100850, Beijing, China;

ABSTRACT
HIV-1 tat targets a variety of host cell proteins to facilitate viral transcription and disrupts host cellular immunity by inducing lymphocyte apoptosis, but whether it influences humoral immunity remains unclear. Previously, our group demonstrated that tat depresses expression of DNA-PKcs, a critical component of the non-homologous end joining pathway (NHEJ) of DNA double-strand breaks repair, immunoglobulin class switch recombination (CSR) and V(D)J recombination, and sensitizes cells to ionizing radiation. In this study, we demonstrated that HIV-1 Tat down-regulates DNA-PKcs expression by directly binding to the core promoter sequence. In addition, Tat interacts with and activates the kinase activity of DNA-PKcs in a dose-dependent and DNA independent manner. Furthermore, Tat inhibits class switch recombination (CSR) at low concentrations (≤ 4 µg/ml) and stimulates CSR at high concentrations (≥ 8 µg/ml). On the other hand, low protein level and high kinase activity of DNA-PKcs promotes HIV-1 transcription, while high protein level and low kinase activity inhibit HIV-1 transcription. Co-immunoprecipitation results revealed that DNA-PKcs forms a large complex comprised of Cyclin T1, CDK9 and Tat via direct interacting with CDK9 and Tat but not Cyclin T1. Taken together, our results provide new clues that Tat regulates host humoral immunity via both transcriptional depression and kinase activation of DNA-PKcs. We also raise the possibility that inhibitors and interventions directed towards DNA-PKcs may inhibit HIV-1 transcription in AIDS patients.

Show MeSH
Related in: MedlinePlus