Small RNA-mediated Silencing. *M Mechanism for gene regulation involving i small noncoding RNA. * Innate host cell defense mechanism against viruses

Transcription

1 HIV-1 gene expression is a key regulator of AIDS progression Tat Cellular Factors HIV-1 gene expression Cellular Factors Activation Repression High viremia Transcriptional latency AIDS progression Reservoir

2 Small RNA-mediated Silencing *M Mechanism for gene regulation involving i small noncoding RNA * Innate host cell defense mechanism against viruses mirna

3 MicroRNAs : metabolism and activity Virus-encoded mirna Suppressor of silencing (EBV, KSHV, SV40, -Tat (HIV-1) HCMV, MHV and HIV-1) Dicer -P19 (Tombusvirus) -Tas (HFV) -NS35 (Ebola) Exportin 5 mirna duplex pri-mirna Drosha pre-mirna RISC (Argonaute 2) RNA Polymerase II Nucleus P-bodies - mir-32 : - HFV replication - mir-122a : HCV replication mrna Cytoplasm Translational Repression Adapted from Sullivan & Ganem, Mol. Cell 2005

4 Dicer and Drosha are Potent Repressors of HIV-1 Replication in PBMCs from Infected Donors sirna : sirna : Dicer GAPDH Drosha GAPDH /ml) HIV-1 p2 24 antigen (pg/ Sc-Dicer Dicer Sc-Drosha Drosha Days post-co-culture Triboulet et al Science 315:

5 Dicer and Drosha antiviral activity can result from: *Cellular mirna as well as vmirna able to target HIV-1 RNA and block their expression *mirna regulating cellular genes required for virus replication *mirna independent

6 A small set of cellular microrna are regulated by HIV HIV-1 p24 ant igen (pg/ml) Days post-infection Infected Control Days post-infection mir-20 mir-210 mir-297 5S RNA (INF/NI) Log2( 5 mir-122a 4 mir-206 mir mir-34a 2 mir-370 mir-373* 1 mir mir-383 mir mir mir Days post-infection mir-18 mir-20 mir-17-3p

7 HIV-1 down-regulates the polycistronic microrna cluster 17/ Fluoresce ence (A.U) N.INF INF day0 N.INF day21 INF day21 0 mir-17-3p mir-17-5p mir-18a mir-19a mir-19b-1 mir-20a mir-92-1 mirna C13orf25 mir-17/92 mir-17-5p/-3p mir-18a mir-19a mir-19b-1 mir-20a mir-92-1

8 Impossible d'afficher l'image. Votre ordinateur manque peut-être de mémoire pour ouvrir l'image ou l'image est endommagée. Redémarrez l'ordinateur, puis ouvrez à nouveau le fichier. Si le x rouge est toujours affiché, vous devrez peut-être supprimer l'image avant de la réinsérer. HIV-1 Down Regulates mir-17-5p & mir-20a that Target PCAF, a Key Regulator of HIV-1 Gene Expression sirna/mirna : Anti-miRNA : anti-pcaf anti-tubulin anti-pcaf anti-tubulin 1,2 12 Re elative luciferase activity ,8 0,6 0,4 0,2 Re elative luciferase activity Sc PCAF 3'UTR 17-5p 20 sirna/mirna 0 Control 17-5p 20 Anti-miRNA

9 Dicer and Drosha antiviral activity can result from: *Cellular mirna as well as vmirna able to target HIV-1 RNA and block their expression *mirna regulating cellular genes required for virus replication

10 HIV-1 mrnas are associated with P-bodies MS2-GFP-NLS Myc-Ago2 merge GFP MS2 MS2-GFP-NLS MS2-GFP-NLS Myc-Ago2-PAZ9 merge LTR (x12) LTR HIV-1 reporter RNA MS2-GFP-NLS Flag-Dcp1a merge

11 Impossible d'afficher l'image. Votre ordinateur manque peut-être de mémoire pour ouvrir l'image ou l'image est endommagée. Redémarrez l'ordinateur, puis ouvrez à nouveau le fichier. Si le x rouge est toujours affiché, vous devrez peut-être supprimer l'image avant de la réinsérer. Targeting of HIV-1 RNA to P-bodies is small RNA-Dependent pnl4-3 Myc-Ago2 Myc-Ago2 PAZ9 P19-HA BSS A5 A4b A4a A4c A1 A2 A3 gag-pol vif vpr rev1 tat1 D1 D2 D3 D4 vpu A7 rev2 env tat2 nef IP anti-myc WB anti-myc SJ4.7A WB anti-ha pnl4-3 Myc-Ago2 Myc-Ago2 PAZ9 P19-HA Extracts IP H 2 O Input IP p PSP Myc-Ago2 Myc-Ago2 PAZ Tat 2 Tat 1 Nef 5 Nef 4 Nef 3 Rev 2 Rev 1 MW 1 2 3

12 Conclusion We provide evidence for a physiological role of the mirna-silencing machinery in controlling HIV-1 replication Interplay exists between HIV-1 replication and the microrna pathway

13 How is our work relevant to post-integration latency and the viral reservoir? *Physiological suppression of microrna-silencing pathway by HIV-1 during virus replication. Triboulet et al Science 315: *Suv39H1 and HP1 are responsible for chromatin-mediated HIV-1 transcriptional silencing and post integration latency. Du Chene et al EMBO J. 26: *The Proteasome Regulates HIV-1 Transcription by Both Proteolytic and Non-Proteolytic Mechanisms. Lasso I, Latrielle D, et al Mol Cell. 25 :

14 IGH Robinson Triboulet Rosemary Kiernan Christine Chable-Bessia Irina Lassot Isaure Du Chéné Daniel Latreille Pierre Corbeau Yea-Lih Lin ICGEB Alessandro Marcello IGMM Edouard Bertrand Eugenia Basyuk CHU Jacques Reynes Vincent Baillat IPMC (Nice) Bernard Mari Pascal Barbry NIAID. NIH. (Bethesda) Kuan-Teh Jeang Yamina Bennasser EU HFSP