Limits...
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.

Show MeSH

Related in: MedlinePlus

HIV-1 Tat down-regulates DNA-PKcs at the transcriptional level. A, The firefly luciferase reporter plasmid pGL3-basic containing the full-length DNA-PKcs promoter insertion (pGL3-DPKPF, Figure 1C, construct number 1) was transfected together with or without the renilla luciferase plasmid RL-CMV (internal standard) and flag-Tat into HEK293 cells. The dual luciferase activity and expression of Tat were assayed 48 h- post-transfection. B, pGL3-DPKPF and RL-CMV were transfected into HEK293 cells and recombinant Tat protein was added 24 h after the transfection. The dual luciferase activity and intracellular levels of Tat were measured 48 h- post-transfection. C,D, pGL3-DPKPF and its mutants, as indicated, were transfected with or without RL-CMV and flag-tat into HEK293 cells. The dual luciferase activity was assayed 48 h after transfection. E, A gel shift assay was performed to detect the direct interaction between Tat and the -63 to -1 region of the DNA-PKcs promoter. 1, Negative control, 32P-labeled oligo; 2, 32P-labeled oligo+0.5 µg of Tat protein; 3, 32P-labeled oligo+1 µg of Tat protein; 4, 32P-labeled oligo+2 µg of Tat (His-tagged) protein; 5, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled scrambled oligo (nonspecific competitor); 6, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled oligo (specific competitor); 7, 32P-labeled oligo+2 µg of Tat protein+anti-His antibody. F, Alignment of the HIV-1 5' LTR 1-63 region and the DNA-PKcs promoter -63 to -1 region. Upper panel: a bioinformatic prediction of transcription factor binding for the DNA-PKcs promoter -63 to -1 region. G, A model describing Tat protein binding to the core promoter region of DNA-PKcs. Please see the text for details. In A, B and C, three independent transfection were performed and values were shown with average and SD. *P ≤0.05, **P ≤0.01.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4202030&req=5

Figure 2: HIV-1 Tat down-regulates DNA-PKcs at the transcriptional level. A, The firefly luciferase reporter plasmid pGL3-basic containing the full-length DNA-PKcs promoter insertion (pGL3-DPKPF, Figure 1C, construct number 1) was transfected together with or without the renilla luciferase plasmid RL-CMV (internal standard) and flag-Tat into HEK293 cells. The dual luciferase activity and expression of Tat were assayed 48 h- post-transfection. B, pGL3-DPKPF and RL-CMV were transfected into HEK293 cells and recombinant Tat protein was added 24 h after the transfection. The dual luciferase activity and intracellular levels of Tat were measured 48 h- post-transfection. C,D, pGL3-DPKPF and its mutants, as indicated, were transfected with or without RL-CMV and flag-tat into HEK293 cells. The dual luciferase activity was assayed 48 h after transfection. E, A gel shift assay was performed to detect the direct interaction between Tat and the -63 to -1 region of the DNA-PKcs promoter. 1, Negative control, 32P-labeled oligo; 2, 32P-labeled oligo+0.5 µg of Tat protein; 3, 32P-labeled oligo+1 µg of Tat protein; 4, 32P-labeled oligo+2 µg of Tat (His-tagged) protein; 5, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled scrambled oligo (nonspecific competitor); 6, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled oligo (specific competitor); 7, 32P-labeled oligo+2 µg of Tat protein+anti-His antibody. F, Alignment of the HIV-1 5' LTR 1-63 region and the DNA-PKcs promoter -63 to -1 region. Upper panel: a bioinformatic prediction of transcription factor binding for the DNA-PKcs promoter -63 to -1 region. G, A model describing Tat protein binding to the core promoter region of DNA-PKcs. Please see the text for details. In A, B and C, three independent transfection were performed and values were shown with average and SD. *P ≤0.05, **P ≤0.01.

Mentions: To explore whether Tat regulates PRKDC at the transcriptional level, HEK293 cells transfected with pGL3-DPKPF (containing the full-length PRKDC promoter) and RL-CMV were co-transfected with a flag-Tat plasmid or treated with recombinant Tat protein. As shown in Fig. 2A and B, both gene transfection and administration of the Tat protein were capable of suppressing the PRKDC promoter activity in a dosage dependent manner. To further investigate the Tat-targeted region, we conducted a luciferase assay using pGL3-DPKPF and its truncation mutants (Fig. 2D). The results showed that Tat targets the core promoter region to suppress the PRKDC promoter (Fig. 2C). Bioinformatics methods predicted that there are two SP1 binding sites and one NF-κB binding site in this core region (Fig. 2F), and there is high homology between the core region and the HIV 5'-LTR sequence (>45%). It is well known that Tat activates HIV transcription by targeting SP1. However, the actual location of physical interaction between Tat and SP1 remains controversial 21, 22. In addition, Tat binds to the TAR (Trans-Activating Response) element of its own RNA, which is encoded by LTRs. Based on the LTR similarities, we speculated that the core promoter region could be bound by the Tat protein. Not surprisingly, in vitro EMSA results showed that Tat binds to the core promoter region of PRKDC (Fig. 2E). Therefore, Tat depresses PRKDC expression by directly binding to the core promoter region.


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)

HIV-1 Tat down-regulates DNA-PKcs at the transcriptional level. A, The firefly luciferase reporter plasmid pGL3-basic containing the full-length DNA-PKcs promoter insertion (pGL3-DPKPF, Figure 1C, construct number 1) was transfected together with or without the renilla luciferase plasmid RL-CMV (internal standard) and flag-Tat into HEK293 cells. The dual luciferase activity and expression of Tat were assayed 48 h- post-transfection. B, pGL3-DPKPF and RL-CMV were transfected into HEK293 cells and recombinant Tat protein was added 24 h after the transfection. The dual luciferase activity and intracellular levels of Tat were measured 48 h- post-transfection. C,D, pGL3-DPKPF and its mutants, as indicated, were transfected with or without RL-CMV and flag-tat into HEK293 cells. The dual luciferase activity was assayed 48 h after transfection. E, A gel shift assay was performed to detect the direct interaction between Tat and the -63 to -1 region of the DNA-PKcs promoter. 1, Negative control, 32P-labeled oligo; 2, 32P-labeled oligo+0.5 µg of Tat protein; 3, 32P-labeled oligo+1 µg of Tat protein; 4, 32P-labeled oligo+2 µg of Tat (His-tagged) protein; 5, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled scrambled oligo (nonspecific competitor); 6, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled oligo (specific competitor); 7, 32P-labeled oligo+2 µg of Tat protein+anti-His antibody. F, Alignment of the HIV-1 5' LTR 1-63 region and the DNA-PKcs promoter -63 to -1 region. Upper panel: a bioinformatic prediction of transcription factor binding for the DNA-PKcs promoter -63 to -1 region. G, A model describing Tat protein binding to the core promoter region of DNA-PKcs. Please see the text for details. In A, B and C, three independent transfection were performed and values were shown with average and SD. *P ≤0.05, **P ≤0.01.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 2: HIV-1 Tat down-regulates DNA-PKcs at the transcriptional level. A, The firefly luciferase reporter plasmid pGL3-basic containing the full-length DNA-PKcs promoter insertion (pGL3-DPKPF, Figure 1C, construct number 1) was transfected together with or without the renilla luciferase plasmid RL-CMV (internal standard) and flag-Tat into HEK293 cells. The dual luciferase activity and expression of Tat were assayed 48 h- post-transfection. B, pGL3-DPKPF and RL-CMV were transfected into HEK293 cells and recombinant Tat protein was added 24 h after the transfection. The dual luciferase activity and intracellular levels of Tat were measured 48 h- post-transfection. C,D, pGL3-DPKPF and its mutants, as indicated, were transfected with or without RL-CMV and flag-tat into HEK293 cells. The dual luciferase activity was assayed 48 h after transfection. E, A gel shift assay was performed to detect the direct interaction between Tat and the -63 to -1 region of the DNA-PKcs promoter. 1, Negative control, 32P-labeled oligo; 2, 32P-labeled oligo+0.5 µg of Tat protein; 3, 32P-labeled oligo+1 µg of Tat protein; 4, 32P-labeled oligo+2 µg of Tat (His-tagged) protein; 5, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled scrambled oligo (nonspecific competitor); 6, 32P-labeled oligo+2 µg of Tat protein+100-fold of unlabeled oligo (specific competitor); 7, 32P-labeled oligo+2 µg of Tat protein+anti-His antibody. F, Alignment of the HIV-1 5' LTR 1-63 region and the DNA-PKcs promoter -63 to -1 region. Upper panel: a bioinformatic prediction of transcription factor binding for the DNA-PKcs promoter -63 to -1 region. G, A model describing Tat protein binding to the core promoter region of DNA-PKcs. Please see the text for details. In A, B and C, three independent transfection were performed and values were shown with average and SD. *P ≤0.05, **P ≤0.01.
Mentions: To explore whether Tat regulates PRKDC at the transcriptional level, HEK293 cells transfected with pGL3-DPKPF (containing the full-length PRKDC promoter) and RL-CMV were co-transfected with a flag-Tat plasmid or treated with recombinant Tat protein. As shown in Fig. 2A and B, both gene transfection and administration of the Tat protein were capable of suppressing the PRKDC promoter activity in a dosage dependent manner. To further investigate the Tat-targeted region, we conducted a luciferase assay using pGL3-DPKPF and its truncation mutants (Fig. 2D). The results showed that Tat targets the core promoter region to suppress the PRKDC promoter (Fig. 2C). Bioinformatics methods predicted that there are two SP1 binding sites and one NF-κB binding site in this core region (Fig. 2F), and there is high homology between the core region and the HIV 5'-LTR sequence (>45%). It is well known that Tat activates HIV transcription by targeting SP1. However, the actual location of physical interaction between Tat and SP1 remains controversial 21, 22. In addition, Tat binds to the TAR (Trans-Activating Response) element of its own RNA, which is encoded by LTRs. Based on the LTR similarities, we speculated that the core promoter region could be bound by the Tat protein. Not surprisingly, in vitro EMSA results showed that Tat binds to the core promoter region of PRKDC (Fig. 2E). Therefore, Tat depresses PRKDC expression by directly binding to the core promoter region.

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