A Screen to Indentify Novel Regulators of HIV Latency
Steven E. Kauder, J. David Gladstone Institutes
In HIV-infected patients, virus from a reservoir of latently infected cells quickly repopulates the system after the cessation of antiretroviral treatment. To better understand the molecular mechanisms of HIV latency and eliminate this reservoir, our laboratory has developed J-Lat cells, a model system for the study of HIV latency in vitro. In these cells, the production of virus is reversibly suppressed at the level of transcription and can be measured by GFP fluorescence. As in latently infected CD4+ T cells recovered from patients, in J-Lat cells the majority of virus integrations have occurred within actively transcribed genes.
Few gene products or molecular mechanisms that regulate HIV latency in cells have been described. Here, we propose a novel approach to identify the full spectrum of genes involved in latency. A library of siRNAs will be expressed in J-Lat cells; those cells in which HIV expression is reactivated will be identified by GFP fluorescence. As this is the first reported attempt to identify regulators of HIV latency by phenotype, we expect to identify novel factors in this screen.
Among the genes isolated, we expect three functional groups to be the most prominent: (1) Those that interact directly with the proviral promoter and repress HIV transcription. Chromatin immunoprecipitation will be used to identify proteins that bind the HIV promoter during latency and are modified or dissociate upon virus reactivation. (2) Those that modulate signaling pathways involved in HIV transcription. Modulation of these pathways by these genes can be observed by standard assays of signaling molecules. (3) Those that decrease the transcription of cellular genes proximal to the latent provirus. A reduction in transcription of these genes may decrease interference with provirus transcription, leading to reactivation. Expression of these cellular genes will be determined in cells with reactivated HIV.
The genes identified in this screen can then be utilized in future studies with primary cells recovered from infected patients and in the development of specific inhibitors to reactivate latent virus. Only then, with the depletion of the latent reservoir, can clearance of HIV from infected patients be contemplated.