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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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Related in: MedlinePlus

Direct interaction between Tat and DNA-PKcs. A, MT2 cells were transfected with a myc-Tat plasmid. The total cell lysates were immunoprecipitated with normal IgG, anti-myc or anti-DNA-PKcs antibody, and a western blot was performed using the indicated antibodies. B, GST and GST-Tat proteins were expressed in E. coli Rosetta-strain cells, conjugated to glutathione beads and incubated with MT2 cell lysates. C, A schematic representation of the major domains of DNA-PKcs. D, The different DNA-PKcs truncation mutant plasmids with myc-tags were co-transfected with flag-Tat into HEK293 cells. The immunoprecipitation was performed with an anti-myc antibody and the WB was analyzed with the indicated antibodies.
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Figure 3: Direct interaction between Tat and DNA-PKcs. A, MT2 cells were transfected with a myc-Tat plasmid. The total cell lysates were immunoprecipitated with normal IgG, anti-myc or anti-DNA-PKcs antibody, and a western blot was performed using the indicated antibodies. B, GST and GST-Tat proteins were expressed in E. coli Rosetta-strain cells, conjugated to glutathione beads and incubated with MT2 cell lysates. C, A schematic representation of the major domains of DNA-PKcs. D, The different DNA-PKcs truncation mutant plasmids with myc-tags were co-transfected with flag-Tat into HEK293 cells. The immunoprecipitation was performed with an anti-myc antibody and the WB was analyzed with the indicated antibodies.

Mentions: The Tat protein localizes predominantly to the cell nucleus and interacts with a number of nuclear proteins, including Cyclin T1, CDK9, Cyclin B1, etc. 1-3. DNA-PKcs also resides primarily in the nucleus to maintain genomic integrity, especially under irradiating condition 9. Based on this evidence, we presumed that there is a direct interaction between Tat and DNA-PKcs. To prove this, we performed a reciprocal co-immunoprecipitation (Co-IP) experiment using MT2 cell lysates containing ectopically expressed myc-tagged Tat, followed by a subsequent GST pull-down assay. The results demonstrated that Tat and DNA-PKcs strongly interact with each other (Fig. 3A, B).


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)

Direct interaction between Tat and DNA-PKcs. A, MT2 cells were transfected with a myc-Tat plasmid. The total cell lysates were immunoprecipitated with normal IgG, anti-myc or anti-DNA-PKcs antibody, and a western blot was performed using the indicated antibodies. B, GST and GST-Tat proteins were expressed in E. coli Rosetta-strain cells, conjugated to glutathione beads and incubated with MT2 cell lysates. C, A schematic representation of the major domains of DNA-PKcs. D, The different DNA-PKcs truncation mutant plasmids with myc-tags were co-transfected with flag-Tat into HEK293 cells. The immunoprecipitation was performed with an anti-myc antibody and the WB was analyzed with the indicated antibodies.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4202030&req=5

Figure 3: Direct interaction between Tat and DNA-PKcs. A, MT2 cells were transfected with a myc-Tat plasmid. The total cell lysates were immunoprecipitated with normal IgG, anti-myc or anti-DNA-PKcs antibody, and a western blot was performed using the indicated antibodies. B, GST and GST-Tat proteins were expressed in E. coli Rosetta-strain cells, conjugated to glutathione beads and incubated with MT2 cell lysates. C, A schematic representation of the major domains of DNA-PKcs. D, The different DNA-PKcs truncation mutant plasmids with myc-tags were co-transfected with flag-Tat into HEK293 cells. The immunoprecipitation was performed with an anti-myc antibody and the WB was analyzed with the indicated antibodies.
Mentions: The Tat protein localizes predominantly to the cell nucleus and interacts with a number of nuclear proteins, including Cyclin T1, CDK9, Cyclin B1, etc. 1-3. DNA-PKcs also resides primarily in the nucleus to maintain genomic integrity, especially under irradiating condition 9. Based on this evidence, we presumed that there is a direct interaction between Tat and DNA-PKcs. To prove this, we performed a reciprocal co-immunoprecipitation (Co-IP) experiment using MT2 cell lysates containing ectopically expressed myc-tagged Tat, followed by a subsequent GST pull-down assay. The results demonstrated that Tat and DNA-PKcs strongly interact with each other (Fig. 3A, B).

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