Latently-infected CD4+ T cells are thought to be the main barrier to HIV eradication or functional cure, and viral reactivation from these cells may contribute to the organ inflammation and damage observed on antiretroviral therapy. A major impediment to the development of more effective therapies for HIV, including those aimed at cure, is the lack of knowledge about the mechanisms that govern latent HIV infection in vivo. We have developed a new ?transcription profiling? approach that can simultaneously measure the degree to which different mechanisms inhibit HIV transcription in vivo. By applying this approach to cells from ART-suppressed patients, we found that a series of blocks to HIV transcriptional elongation, distal transcription/polyadenylation (completion), and multiple splicing are the main mechanisms that reversibly suppress virus production in the blood. In contrast, HIV-infected cells in the rectum showed a strong and paradoxical block to initiation of HIV transcription. A critical unanswered question is whether the same or different mechanisms operate in the small subset of cells with infectious proviruses, which constitute the true latent reservoir.
In aims 1 -3 of this proposal, we will determine the degree to which different mechanisms reversibly block virus production in individual patient cells with intact or defective proviruses by using a novel approach that combines both single genome ?whole provirus? sequencing and single genome HIV transcription profiling.
In aim 1, we will apply these methods to blood CD4+ T cells from ART-suppressed patients to determine the degree to which different mechanisms constitutively inhibit HIV transcription in blood CD4+ T cells with intact or defective proviruses, the degree to which they are reversed by short term T cell activation ex vivo, and the degree to which they differ in activated cells that do and do not produce supernatant virus.
In aim 2, we will extend these studies to PD-1+ and PD-1- CD4+ T cells from blood, lymph node, and gut to determine whether there are differences by tissue or PD-1 expression in proviral sequences and the mechanisms that reversibly suppress virus production in individual cells with defective and intact proviruses.
Aim 3 will further extend these findings by using a new method that combines single genome ?whole provirus? and integration site sequencing, in combination with ATAC-seq, to determine how integration site and chromatin accessibility affect constitutive HIV transcription and reactivation potential in cells with intact and defective proviruses. These 3 aims will help definitively answer the question of what mechanisms govern HIV latency in vivo in the blood and the tissues, which has been a major goal of HIV research for the last 20 years and should help inform new therapies aimed at eradication, functional cure, or reducing the sequelae of treated HIV infection.
The main barrier to cure of HIV is the ability of the virus to establish a silent infection (called ?latent infection?) in which production of infectious virus is reversibly turned off in some infected cells, allowing HIV to escape detection by the body?s defenses and persist in long-lived cells that can also propagate the virus during cell division. Despite over 20 years of research, it is uncertain what mechanisms allow HIV to establish this silent, latent infection. In this proposal, we will help answer this question by using a series of novel methods to determine the mechanisms that reversibly inhibit virus production in individual HIV-infected cells from both blood and tissues of infected patients, and whether they differ between cells with infectious and defective forms of the virus.