HIV-1 Vpu Exploits the Crosstalk Between BST2 and the ILT7 Receptor to Inhibit Innate Sensing of Infected T cells by pDCs Mariana G. Bego1, Édouard A. Côté1, Nick Aschman2, Johanne Mercier1, Winfried Weissenhorn2 and Éric A. Cohen1,3 1Institut de Recherches Cliniques de Montréal (IRCM) 2 Unit of Virus-Host Interactions, Université Grenoble Alpes 3Department of Microbiology and Immunology, Université de Montréal Poster : TUPDA0103

BST2/Tetherin: one gene (a few isoforms) and many roles Vpu 1- BST-2 is an IFN-regulated host factor that restrict the release of HIV-1 by a process that is dependent on the Vpu accessory protein 2- The protein has also been shown to act as a sensor of progeny virus release and as such trigger the production of proinflammatory cytokine through activation of NFKB 3-Apart from these functions, BST-2 was reported to act as the ligands of an inhibitory receptor express on PDC, ILT7. Engagement of BST-2 with ILT-7 inhibits IFN production when PDC are activated via TLR 7/TLR9 NF-kB Sauter, 2010

HIV-infected CD4+ T cells are more potent at stimulating pDCs Sensing and antiviral responses: 1- Env-CD4 interaction 2- Membrane fusion 3-decapsidation and release of ssRNA into TLR7+ endosome 4-triggering of a MyD 88/IRF7 signaling cascade that results in IFN-I and cytokine production Weak Most efficient way pDC sense HIV is via cell contacts Strong 1-the most efficient way PDC sense HIV-1 is when they get in contact with virus infected cells, a condition that result in IFN production. This process requires Env/CD4 interaction, membrane fusion, decapsidation and release of ssRNA into TLR 7 endsomes Env –dependent process Sensitive to Fusion inhibitors but not RT inhibitors Iwasaki, A., Immunity , 2012

Goal of the study To investigate whether HIV exploits the BST2-ILT7 regulatory axis to modulate pDC antiviral responses

HIV-1 Vpu suppresses sensing of HIV-1 infected cells by pDCs Co-culture X4 infected MT4 T cells and PBMCs TypeI IFN (U/ml) PBMC + + + + - + - + - Stimuli - TRL7 ago TLR9 ago Coculture - - - MT4-mock MT4-dU MT4-WT

HIV-1 Vpu suppresses sensing of HIV-1 infected cells by pDCs Co-culture X4 infected MT4 T cells and PBMCs TypeI IFN (U/ml) PBMC + + + + - + - + - Stimuli - TRL7 ago TLR9 ago Coculture - - - MT4-mock MT4-dU MT4-WT Relative % of IFN released dU WT Mean 52% *** X4 Mean 57% Mean 46% * ** ns Relative % of IFN released Primary T cells (X4/R5 infection) dU WT X4 R5

Two Working Models 1-In the absence of Vpu, tethered virions are more effectively presented at the cell surface for transmission and sensing by pDCs 2- Vpu modulates the levels of BST2 that can engage and activate ILT7 on pDCs

Control of HIV innate sensing by Vpu is BST2-dependent Relative % of particles released HIV-1 release assay BST2-depleted MT4 cell lines

ILT7 depletion in enriched pDCs Control of HIV innate sensing by Vpu requires the ILT7 pDC inhibitory receptor A 9 81 64 12 Non pDCs (PBMCs) pDCs (PBMCs) Enriched pDCs Enriched pDCs siILT7 % Max ILT7-PE ILT7 depletion in enriched pDCs B Soluble ILT7

Interplay between Vpu and BST2 Vpu exploits the crosstalk between BST2 and ILT7 to dampen the antiviral response of PDCs Through a sophisticated targeted regulation of specific BST2 isoforms, Vpu promotes HIV release while at the same time interfering with pDC antiviral responses through ILT7 activation This mechanism of innate immune evasion is likely to be important for an efficient early viral dissemination during acute infection

Acknowledgements Other Lab Members Collaborators Reagents Fadi Hajjar Johanne Mercier Collaborators Wei Cao, MD Anderson Cancer Center Yong-Jun Liu, MedImmune Nick Aschman & W. Weissenhorn (Univ. Grenoble Alpes and UVHCI, UVHCI-CNRS, Grenoble) Mariana Bego Édouard Bérubé-Côté Reagents Idera Pharmaceuticals AIDS Reagent Program The IRCM Clinic staff and healthy volunteers IRCM Flow Cytometry and Microscopy Cores