Host hindrance to HIV replication

Transcription

1 F. Barré-Sinoussi Nobel Prize 2008 Host hindrance to HIV replication Gianfranco Pancino Regulation of Retroviral Infections Unit

2 Synopsis 1. Short overview on the known restriction factors 2. Update on SAMHD1 restriction mechanisms 3. New data on p21-mediated lentiviral restriction

3 Lentiviruses integrate the host cell genome and establish a persistent infection HIV target cells HIV replication cycle CD4 + T lymphocytes Monocytes/macrophages Dendritic cells from R. A. Weiss, Nature 2001

4 The balance of HIV replication Virulence factors HIV Dependency Factors Restriction Factors

5 HIV-dependency factors Brass AL. Science, 2008

6 HIV restriction factors Antagonistic virulence factors APOBEC3 proteins TRIM5α proteins BST-2/Tetherin SAMHD1 Others (?) Vif Vpu Vpx Vpr, Nef (?) Dominant and autonomous antiviral activity Antagonized by viral proteins Sequence evolution in antagonism with viral genes Species specificity

7 Retroviral restriction factors Uncoating and reverse transcription Post-entry blocks Ca TRIM5α APOBEC3G Vif From Suzuki and Craigie Nature Reviews Microbiology 2007 Exit block Tetherin Vpu H. G. Göttlinger, Nature 2008

8 HIV restriction by APOBEC3G HIV restriction by BST-2 Harris R S et al. J. Biol. Chem. 2012;287: by American Society for Biochemistry and Molecular Biology

9 A role for retroviral restriction factors in the control of HIV-1 infection in vivo? Controversial data on associations of APOBEC3G, TRIM5α, BST2 expression and/or polymorphisms with the protection (HESN) or with the control of HIV-1 infection (viral load, CD4+ T cell count) PRO Jin et al J Virol 2005 Biasin et al JID 2007 Ulenga et al JID 2008 Vázquez-Pérez et al Retrovir, 2009 Price et al. AIDS 2010 Pillai SK et al. PNAS 2011 An et al. J Virol 2004 An et al. J Infect Dis 2009 van Manen et al. PLoS Pathog 2008 CON Cho et al, J Virol 2006 Gandhi et al J Virol 2008 Sewram et al JID 2009 Reddy et al. AIDS 2010 Mous K et al. PloS One 2012 Do et al. J Infect Dis, 2005 Itaya, S. J Infect Dis 2010 Potential utility for new therapeutic approaches

10 SAMHD1 A block to retrovirus reverse transcription in non cycling cells Laguette N, et al. SAMHD1 is the dendritic- and myeloid-cell-specific HIV-1 restriction factor counteracted by Vpx. Nature Hrecka K, et al. Vpx relieves inhibition of HIV-1 infection of macrophages mediated by the SAMHD1 protein. Nature. 2011

11 SIV and HIV-2 require Vpx to replicate in macrophages and dendritic cells p24 (ng/ml) p27 (ng/ml) 1000 HIV-2 Gh-1 Late RT Vpx is required for HIV-2 and SIVmac reverse transcription in myeloid cells ,1 0, Days post-infection Vpx WT Dvpx Vpx is not essential for HIV-1 replication in myeloid cells ,1 HIV-1 BaL But helps. 0, Days post-infection Goujon et al. Retrovirology 2007

12 Vpx targets the HIV restriction factor SAMHD1 Vpx induces proteasomal degradation of SAMHD1 dntp ( 32- P) Laguette N et al. Nature 2011, Hrecka K et al. Nature 2011 SAMHD1 degrades dntp PPP SAMHD1 Goldstone et al., Nature Lahouassa H et al. Nat Immunol Harris R S et al. J. Biol. Chem Powell et al., J Biol Chem. 2011

13 SAMDH1 phosphorylation is the key for anti-hiv activity

14 Loss of SAMHD1 anti-hiv activity following phosphorylation in cycling cells Phosphorylated SAMHD1 loses its antiviral but not its dntpase activity Cribie A. et al Cell Reports 2013 White TE. et al Cell Host & Microbe 2013

15 SAMHD1: OPEN QUESTIONS (1) Which is the function of SAMHD1 that mediates viral restriction? Hint: SAMHD1 exhibits nuclease activity against ss DNA and RNA and RNA in DNA/RNA hybrids. N. Beloglazova N. et al. J Biol Chem 2013 What is the contribution of the SAMHD1-mediated cellular dntp reduction in HIV-restriction activity?

16 SAMHD1: OPEN QUESTIONS (2) Why HIV-1 did not conserve Vpx? Hypothese: To prevent activation of the immune system upon DC infection (Manel and Littman, Cell 2011) HIV-1 may have evolved the capacity to achieve sufficient replicative potential in macrophages in spite of SAMHD1

17 p21 Cip1/WAF1 a metabolic barrier to lentiviral reverse transcription in macrophages Zhang J. et al. Primitive hematopoietic cells resist HIV-1 infection via p21 Waf1/Cip1/Sdi1. J Clin Invest Bergamaschi A, et al. The CDK inhibitor p21 Cip1/WAF1 is induced by FcγR activation and restricts the replication of human immunodeficiency virus type 1 and related primate lentiviruses in human macrophages. J Virol. 2009

18 p21 induction by Fcg crosslinking inhibits HIV-1 replication in macrophages Copies per 100ng DNA Copies/100 ng DNA p21mrna (Fold change) HIV-1-luc/VSV-G Bioluminescence Luciferase Late RT 3 p21 expression , , ,5 0 US IVIg 0 US IVIg Luciferase, cps x HIV-1/VSV-G Ctrl si-p21 si-ctrl US S Late RT US S Ctrl si-p21 si-neg Bergamaschi A. et al. J Virol 2012 p21 silencing increases reverse transcription levels

19 Exogenous dns rescue p21-mediated restriction of HIV-1 p21 knockdown increases dntp levels in macrophages 600 Luc activity (rlu x10 3 ) US IVIg US+0.25mM dns IVIg+0.25mM dns US+0.5mM dns IVIg+0.5mM dns US+1mM dns IVIg+1mM dns Intracellular datp

20 p21 inhibits the expression of the dntp synthesis enzyme RNR2 WB: ap21 WB: arnr1 WB: arnr1 WB: ap53r2 WB: apcna p21 RNR2 RNR1 p53r2 PCNA p21 mrna (Fold change) RNR2 mrna (Fold change) WB: agapdh GAPDH 0 US sictl IVIg sictl US sip21 IVIg sip21 0

21 copie/100 ng DNA (Fold change) p21 inhibits HIV-1 RT by down-regulating RNR2 WB: arnr2 WB: ap53r2 WB: aactin RNR2 p53r2 Actin Luc activity (fold change) US sictl IVIg sictl Luciferase US sirnr2 IVIg sirnr2 HIV-1/VSV-G Copies/100ng DNA (fold change) US sictl IVIg sictl Late RT US sirnr2 IVIg sirnr2 RNR2 knock-down inhibits HIV-1 reverse transcription 1,8 Late RT 1,5... And suppress HIV recovery in p21 KD macrophages 1,2 0,9 0,6 0,3 0

22 p21 and SAMHD1: complementary ways to restrict HIV-1 reverse transcription by dntp starvation p21 RNR2 NTP RNR2 RNR1 dntp HIV-1 RT SAMHD1

23 Acknowledgements Anna Bergamaschi Awatef Allouch Pierre Versmisse Annie David Chiraz Hamimi Asier Saez-Cirion Unité de Régulation des Infections Rétrovirales Resistance Team

24 ACKNOWLEDGEMENTS HIC STUDIES Institut Cochin INSERM U1016 Florence Margottin- Goguet CatherineTransy Hichem Lahouassa University of Rochester School of Medicine (US) Baek Kim Sarah Amie Institut de Génétique humaine CNRSUPR1142 Monsef Benkirane Nadine Laguette