The advent of antiviral therapy (ART) revealed a treatment-resistant reservoir in CD4+ T cells capable of refueling HIV viremia when treatment is stopped. This reservoir is a major barrier to achieving a cure for HIV infection. Recent work suggests reservoir decay on ART is slower in some individuals due to proliferation of T cells containing intact proviruses since identical intact sequences are predominant after many years on ART in roughly half of subjects. These identical sequences likely represent clones of cells that have proliferated. The driving forces behind proviral clonal expansion remain mysterious, but integration into introns of oncogenes likely plays a role. To study this question, we combine proviral sequencing with integration site sequencing. Our innovation is recognizing that to properly assign proviruses we need longer stretches of HIV DNA than typically provided by current methodologies. We propose a new method to clone integration sites that is based on long-range PCR techniques previously utilized by our group.
In Aim 1 we propose to develop a method that will capture unique junctions that are created in proviruses with large deletions. These deleted proviruses retain splicing ability and fall into two broad categories, those with strong and those with weak potential to express HIV proteins.
In Aim 2, we propose a method to clone the integration site of intact proviruses which requires amplifying longer stretches of HIV DNA that are contiguous with the human DNA. With this in mind, we describe a systematic approach to identify the chromosomal address of the intact proviruses including intact proviral clones. We hypothesize that intact proviral clones are generally in introns and that this placement within the intron plays an important role in clonal expansion as it permits splicing of HIV to downstream exons of oncogenes. This in turn provides a new target for HIV eradication strategies. The premise of our proposal is largely based on preliminary data from our group showing that there are two counterbalancing forces that cause (1) proviral contraction through immune clearance and (2) proviral expansion through clonal proliferation after splicing to a downstream oncogene. We ask in this proposal if the same two forces act on both defective and intact replication-competent proviruses. The significance of our proposal include that it may contribute to growing evidence that the reservoir is more visible than previously realized. Our work suggests that perturbing these two forces by either enhancing immune clearance or targeting splicing or downstream exons of splicing may reduce reservoir size. We envision this work could lead to a larger study to understand if dysfunctional cytotoxic T cells have a role in intact proviral clonal expansion.
Our proposal studies why HIV reservoirs decay at different rates in different treated infected individuals. Slower decay may occur when HIV induces infected cells to aberrantly proliferate. Identifying how HIV activates cell division may provide new targets for HIV cure.
