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

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?

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

 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). P21 (cip-1/waf-1)

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

si-RNA mediated silencing of p21 increases HIV-1 replication R5-tropic HIV-1 X4-tropic HIV-1 Control siRNA cells CD4 GFP 0.9% 2.3% 3.5% 10% p21 siRNA cells

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

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 Methods

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

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

Integrated HIV-1 DNA mRNA transcripts Late RT transcripts n=5 * p21 inhibitor control 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

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 Cyclin-dependent kinase 9 CDK9/p-TEFb

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 Methods

GST Pol II CTD-Phos (Ser-2) p21 siRNA control siRNA p21 inhibits enzymatic activity of CDK9 p21 siRNA control siRNA p= relative signal intensity (CTD-P (Ser-2)/ GST) Negative control

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

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 Conclusions Chen et al, JCI, March 2011 Buzon et al, JVI, July 2011

Acknowledgements 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 Funding Doris Duke Charitable Foundation NIH/NIAID Harvard Catalyst Gates Foundation Mark and Lisa Swartz Foundation University of California, Davis Robert Weiss University of Lausanne, CH Amalio Telenti Lee Moffitt Cancer Center, Tampa, FL Douglas Cress VGTI Florida, Port St. Lucie, FL Nicholas Chomont