As part of its infection cycle, HIV-1 integrates into the genome of CD4+ T cells. After initial infection, a subset of these cells returns to a `resting memory' state. The viral DNA is still present in the genome of these memory cells but it is silent, a phase in the viral life cycle called the latent phase. These memory cells harbor the virus and can produce infectious particles upon stimulation as long as the infected cell is alive. Latent infection, therefore, requires lifelong treatment of infected individuals to suppress rebounds in viral load. The current strategy to cure HIV-1 is to reactivate latently infected cells, which will subsequently result in their clearance. Reactivation can lead to either cell death caused by the toxic effects of viral replication or immune clearance by the presentation of viral epitopes on the surface of the infected cell. Approaches to reactivate the latent virus reservoir have focused on increasing HIV-1 gene expression, as it is thought that latency is maintained by multiple blocks to global transcription. Drug candidates for reactivation, called latency reversing agents (LRA), target different steps in the transcription process (e.g. histone tail modification, polymerase elongation) but are designed to avoid causing general activation of T cells, which would result in massive immune dysfunction. To date, clinical trials with the most promising LRA drugs have failed to reduce the latent HIV-1 reservoir. Curiously, LRA treatment can robustly induce viral RNA expression in primary HIV-1 latency models, however a comparable increase in viral proteins and replication is not observed, which is required for successful clearance. These findings have led to the proposal of an additional post-transcriptional block to viral protein expression in resting T cells. The ~10kb HIV-1 RNA genome folds into a complex three dimensional structure and specific features of this structure are critically involved in regulating multiple post-transcriptional steps including transport of the viral RNA out of the nucleus, translation of viral protein, and packaging of the RNA genome into the viral particle. The proposed research will test the hypothesis that the post-transcriptional block, which is not bypassed by LRA stimulation, is due to specific aspects of HIV-1 RNA structure, which is an innovative idea for the HIV cure field. We will employ a novel technique (DMS-MaPseq) developed recently in the Rouskin lab, which allows for targeted RNA structure probing in living cells. Importantly, the simplicity of the DMS-MaPseq approach will enable RNA structure probing of the entire HIV-1 genome in several different cellular conditions. This work will be significant because it will provide critical information on the native HIV-1 RNA structure, which has never been examined in its physiological context within T-cells where host factors can dramatically influence RNA folding. In addition, this high-risk, high-reward project can directly benefit the prominent ?shock and kill strategy? for latently infected T cell clearance by providing insight into the post- transcriptional block to LRA activity.
Despite over 30 years of intensive research, HIV-1 infection remains an incurable global threat to human health. Although the replication of HIV-1, a single-stranded RNA virus, can be successfully suppressed with highly active antiretroviral therapy (HAART), those who are infected must receive treatment for their lifetime. The proposed research will test the hypothesis that a major block to current strategies for HIV-1 cure is caused by a defect in the HIV-1 genomic RNA structure.
