1 / 17

Mathias Lichterfeld, M.D., Ph.D. Massachusetts General Hospital

An intrinsic inhibitor of p-TEFb and HIV-1 transcriptional elongation in CD4 T cells from elite controllers. Mathias Lichterfeld, M.D., Ph.D. Massachusetts General Hospital. Elite Controllers. No detectable viremia in the absence of HAART approx 1/300 HIV-1 infected patients

isaura
Download Presentation

Mathias Lichterfeld, M.D., Ph.D. Massachusetts General Hospital

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. An intrinsic inhibitor of p-TEFb and HIV-1 transcriptional elongation in CD4 T cells from elite controllers Mathias Lichterfeld, M.D., Ph.D. Massachusetts General Hospital

  2. Elite Controllers • No detectable viremia in the absence of HAART • approx 1/300 HIV-1 infected patients • Model for spontaneous control of HIV-1 • Correlates of immune protection ? • HIV-specific T cells • Intrinsic resistance of CD4 T cells?

  3. X4-tropic HIV-1 p=0.0065 p<0.0001 p=0.0017 p=0.015 p<0.0001 n=12 n=15 n=17 n=15 R5-tropic HIV-1 p=0.0001 p<0.0001 p=0.15 p=0.0005 p<0.0001 n=21 n=21 n=17 n=11 Reduced susceptibility of CD4 T cells from elite controllers to HIV-1 infection

  4. P21 (cip-1/waf-1) • Cyclin dependent kinase inhibitor expressed in T cells and macrophages • Can serve as tumor suppressor • Contributes to resistance of macrophages and hematopoietic stem cells against HIV-1 (Bergamaschi et al, JVI 2009; Zhang et al, JCI 2007).

  5. HLA-DR- CD4+ cells p<0.0001 p<0.0001 p<0.0001 p<0.0001 HLA-DR+ CD4+ cells p<0.0001 p<0.0001 p<0.0001 p<0.0001 Upregulation of p21 in CD4 cells from elite controllers

  6. 5 5 5 5 10 10 10 10 4 4 4 4 10 10 10 10 3 3 3 3 10 10 10 10 2 2 2 2 10 10 10 10 0 0 0 0 2 2 2 2 3 3 3 3 4 4 4 4 5 5 5 5 0 0 0 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 si-RNA mediated silencing of p21 increases HIV-1 replication R5-tropic HIV-1 X4-tropic HIV-1 3.5% 0.9% Control siRNA cells CD4 10% 2.3% p21 siRNA cells GFP

  7. p21 inhibits early viral replication steps Late RT transcripts mRNA transcripts Integrated HIV-1 DNA * * * * HIV-1 negatives EC HIV-1 negatives EC HIV-1 negatives EC n=6 n=10 n=7 n=9 n=9 n=9 control p21 inhibitor at day 0 p21 inhibitor at day -2

  8. Methods • Ex vivo isolation of CD4 T cells from EC, HIV-1 negatives and progressors • Direct infection with VSV-G pseudotyped HIV-1 without prior in vitro activation • PCRs-based detection of late RT, 2-LTR and integrated HIV-1 DNA after 48 hours

  9. Intrinsic restriction of HIV-1 integration in ex-vivo infected CD4 T cells from elite controllers

  10. Restriction of HIV-1 integration in CD4 T cells from EC is unrelated to p21 EC EC EC HIV- EC - p21 HIV- EC - p21 HIV- EC - p21 HIV- - p21 HIV- - p21 HIV- - p21

  11. Late RT transcripts Integrated HIV-1 DNA mRNA transcripts * p21 inhibitor control n=5 n=5 n=5 p21 independently inhibits HIV-1 mRNA transcription from proviral DNA • Infection with VSV-G pseudotyped, single-cycle HIV-1 • Addition of p21 inhibitor after 48 hours

  12. Cyclin-dependent kinase 9 CDK9/p-TEFb • Host protein complex expressed in all CD4 T cells • Phosphorylates RNAPII • Required for elongation of HIV-1 mRNA transcripts • CD4 cells lacking p-TEFb/CDK9 are resistant to HIV-1

  13. Methods • Isolation of CDK9 from CD4 T cells treated with p21 siRNA or control siRNA • In vitro kinase to assess ability of CDK9 to phosphorylate RNAPII • Detection of phosphorylated RNAPII by Western blot

  14. p =0.0075 1.0 0.8 0.6 relative signal intensity (CTD-P (Ser-2)/ GST) 0.4 0.2 0.0 p21 siRNA control siRNA p21 inhibits enzymatic activity of CDK9 Pol II CTD-Phos (Ser-2) GST p21 siRNA control siRNA Negative control

  15. p21 inhibits transcriptional elongation of HIV-1 • Transfection of CD4 T cells with control or p21-specific siRNA • Quantitative assessment of proximal, intermediate and distal HIV-1 mRNA segments p=0.007 p=0.06 proximal intermediate distal HIV-1 mRNA fragments n=7 n=7 n=7

  16. Conclusions • Intrinsic resistance of ex-vivo infected and in-vitro activated CD4 cells from EC against HIV • Inhibition of HIV-1 integration in ex-vivo infected CD4 T cells from EC is unrelated to p21 • HIV-1 restriction of in-vitro activated CD4 T cells from EC involves p21-dependent inhibition of CDK9/ transcriptional elongation of HIV-1 mRNA • Evidence for blockages of multiple HIV-1 replication steps in CD4 T cells from EC Chen et al, JCI, March 2011 Buzon et al, JVI, July 2011

  17. Acknowledgements University of California, Davis Robert Weiss MGH ID Division/Ragon Institute Maria Buzon Chun Li Katherine Seiss Jill Beamon Mary F. Carrington Patrick Burke Xu Yu Florencia Pereyra Abraham Brass Bruce Walker Maha Al-Mozaini Jennifer Rychert Eric Rosenberg University of Lausanne, CH Amalio Telenti Lee Moffitt Cancer Center, Tampa, FL Douglas Cress VGTI Florida, Port St. Lucie, FL Nicholas Chomont Funding Doris Duke Charitable Foundation NIH/NIAID Harvard Catalyst Gates Foundation Mark and Lisa Swartz Foundation

More Related