Evaluating the role of the novel apoptosis inhibitor TRAILshort, in maintaining HIV persistence.

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 DESCRIPTION (provided by applicant): The interplay between a pathogenic viral infection and the human host reflects a bi-directional interaction of host sensors (e.g. TLR, RIG, etc.) which detect virus infection, host effector mechanisms which eliminate infected cells (e.g. caspase activation, expression of death inducing ligands including Fas Ligand and TRAIL), and viral adaptations which attempt to subvert the host response (e.g. virally encoded apoptosis inhibitors e.g. EBV encoded BALF1, KSHV encoded vFLIP etc.). Experimental elimination of virally encoded apoptosis inhibitors enhances host cell clearance of virally infected cells, and reduces viral persistence - as examples: inhibiting BALF1 prevents EBV persistence, inhibiting LAT inhibits HSV1 persistence, and disrupting E6 or E7 inhibits HPV persistence. HIV infection can be sensed in human cells for example by IFI-16 sensing of accumulated reverse transcripts leading to caspase 1 dependent pyroptosis, or DNA-PK sensing of integrase mediated host DNA "nicking" leading to p53 dependent apoptosis. However these sensing mechanisms do not eradicate all HIV infected cells, and HIV is as a result, persistent. HIV is not known to encode a protein which antagonizes the hosts ability to eradicate infected cells, yet accumulating evidence suggests that HIV infected cells are more resistant to cell death that corresponding uninfected cells, through an as yet undefined mechanism TNF related apoptosis inducing factor (TRAIL) is a molecule whose principal function is as an effector of immune surveillance, and it has been implicated in the pathogenesis of malignancies, as well as viral infections including HIV. Concerning the role of TRAIL in HIV, considerable data indicate that TRAIL is dysregulated during HIV infection in vivo, and there is ample evidence that treatment of cells from ART suppressed HIV- infected patients with exogenous TRAIL, reduces the number of latently infected cells, as measured by undetectable levels of replication competent virus in quantitative co-culture assays. The current research proposal concerns a novel TRAIL splice variant, which we have discovered and named TRAILshort, which is produced during HIV infection in vitro and in vivo. TRAILshort binds to TRAIL receptor 2 (TRAILR2), yet does not transmit a cell death signal, whereas TRAIL binding to TRAILR2 rapidly results in apoptotic death. Moreover, expression of TRAILshort, prevents TRAIL from engaging TRAILR2, demonstrating that TRAILshort acts as a dominant negative inhibitor of TRAIL mediated killing. Because NK cells and CD8+ CTL kill target cells by TRAIL (in addition to other mechanisms such as Granzyme B), it follows that TRAILshort expression reduces both NK cell and CD8 T cell cytotoxicity. Our underlying hypothesis is that TRAILshort production during HIV infection prevents TRAIL dependent HIV clearance mechanisms which allows HIV infected cells to persist. This hypothesis is supported by novel preliminary data included in this application, where shRNA mediated knockdown of TRAILshort does not alter uninfected T cell survival, yet selectively enhances killing of HIV infected cells, thereby reducing HIV replication by 3.5 logs, and decreasing the number of cells containing HIV DNA. The long-term goal of our work is to fully understand and manipulate TRAILshort production in order that normal immune mechanisms can contribute to clearance of virally infected cells, thereby providing an additional tool with which to contribute towards a cure for HIV. This will be fully explored by: (i) studying the regulation of TRAILshort, in order to identify means of inhibiting its production. (ii) Optimize lentivirus knock down of TRAILshort in primary CD4 T cells from HIV infected patients, and test whether HIV reactivation with TRAILshort knockdown, plus or minus co-culture with autologous CD8 T cells, or NK cells, reduces HIV reservoir size ex vivo (iii) Evaluate TRAILshort expression in HIV elite controllers, partial controllers and non-controllers, and determine the association of TRAILshort expression with HIV reservoir size.