Multimodal profiling of microglia during HIV infection and substance use disorder

University Of Pennsylvania, Philadelphia PA

Investigators

Abstract

Project Summary Whether cocaine exposure supports long-term HIV expression in immune cells and whether inflammation mediates this effect are major knowledge gaps. A clear understanding of cocaine's impact on HIV expression in complex multicellular contexts is a prerequisite for the identification of novel targets for effective ART regimens, adjunctive neuroprotective therapies, and latency reversal strategies. Our overarching hypothesis is that long­ term HIV expression in macrophages and microglia contributes to neuropsychiatric damage, which is exacerbated by cocaine. During the second year of this award, we made tremendous progress in developing HID-Seq (HIV integrated proviral DNA [HID]/single-nuclear RNASeq) (Fig. 1). In brief, HID-Seq is a new approach that combines single-nuclear RNASeq (snRNASeq) with dCas9-Tn5 tagmentation. This is a scalable method that permits increased read depth that can simultaneously detect integrated HIV proviral DNA, HIV RNA species, and host transcriptome within the same cell, achieved through a multi-index barcoding strategy (Fig. 3, 4). HID-Seq will enable us to (A) determine the identity of discreet populations of productively infected (HIV DNA+/HIV RNA+), latenUnonproductively infected (HIV DNA+/HIV RNA, and uninfected (HIV DNA/HIV RNA) cells, and (B) to simultaneously link the presence of HIV DNA and RNA to functional cellular outcomes, such as inflammation, via the transcriptome in the same cell. We have achieved nearly all objectives in all 3 milestones for the R61 phase, as detailed in the Progress to Milestones section. The primary focus within the second period of R61 period was to complete the individual steps of HID-Seq, including the detection of HIV DNA and RNA (Milestone 1), the quantification of cells harboring HIV DNA and HIV RNA (Milestone 2), and the confirmation of read depth (Milestone 3). We have overcome the technical challenge in the detection of HIV DNA by revising our original design (version 1), where we would use dCas9-Tn5 fusion protein to cut and tag DNA, to version 2, where we use dCas9-Tn5 fusion protein together with free Tn5. This enabled us to increase the rate of tagmentation of the region of interest, i.e., where HIV proviral DNA is integrated into the cellular genome. • Milestones 1 and 2: In our experiment where we bulk sequenced over 5000 cells that were processed using dCas9-Tn5, we were able to detect that 2430 cells had HIV DNA, leading to a successful HIV DNA detection rate of 48.6%. We also optimized the single-nucleus RNASeq (snRNASeq) protocol, which we term HIV-snRNASeq, which enabled us to significantly increase the sensitivity of HIV RNA detection by 40-67-fold compared with the traditional methods. • Milestone 3:_We successfully detected HIV DNA in ~50% of the J-Lat cells with bulk sequencing 5000 cells, indicating a good read depth, by using this novel approach. We also developed the computational pipeline to facilitate the analysis of joint-profiling of the host-cell transcriptome and HIV genome at the single-cell level, i.e., the junction site including both the HIV and host-cell genome. We will be able to determine the identity of discreet populations of productively infected (HIV DNA+/HIV RNA+), latenUnonproductively infected (HIV DNA+/HIV RNA, and uninfected (HIV DNA/HIV RNA) cells and determine their transcriptomic profiles. With our data showing that we can detect HIV DNA and RNA with increased sensitivity and good read depth, we are confident that the ongoing analyses of the HID-Seq of J-Lat cells will reveal that we can detect the different states of cells based on the presence of HIV DNA and RNA