Transcriptional programs of HIV silencing and cell survival in HIV-infected memory CD4 T cells under antiretroviral therapy


BACKGROUND: Rare memory CD4 T cells harboring HIV under antiretroviral therapy (ART) represent an important barrier to HIV cure, but the infeasibility of isolating and characterizing these cells in their 'natural' state has bred uncertainty about whether they possess distinctive attributes that HIV cure-directed therapies might exploit.
METHODS: We developed a custom microfluidic process termed Focused Interrogation of cells by Nucleic acid Detection and Sequencing (FIND-Seq), which captures polyadenylated RNA and genomic DNA from millions of single cells within water-in-oil droplets and then sorts single-cell transcriptomes based solely on HIV DNA detection. Using blood cells from people with HIV (PWH) who had initiated ART during chronic infection and experienced >1 year of virologic suppression (n = 6), memory CD4 T cell transcriptomes were sorted by FIND-Seq into HIV DNA+ and uninfected cell fractions and sequenced. Host cell transcriptomic profiles of HIV DNA+ and uninfected memory CD4 T cells were compared by differential gene expression, co-expression network analysis, and gene ontology.
RESULTS: HIV DNA+ memory CD4 T cells from ART-treated PWH consistently showed inhibition of six transcriptomic pathways including death receptor signaling, necroptosis signaling, and Ga12/13 signaling. Gene co-expression network analysis revealed two small gene clusters associated with HIV DNA+ cells. Gene ontology revealed significant enrichment of these clusters for factors related to epigenetic gene regulation, RNA processing, and the regulation of cell death signaling. Individual genes in these clusters included HIV transcriptional activators that were lower in HIV DNA+ cells and HIV silencing factors affecting both transcriptional and post-transcriptional steps in HIV gene expression that were higher in HIV DNA+ cells. Remaining genes in these clusters not previously associated with HIV gene expression had roles in chromatin modification, RNA processing, and the survival and proliferation of CD4 T cells.
CONCLUSIONS: Whole transcriptome sequencing of unmanipulated HIV DNA+ memory CD4 T cells under ART reveals these cells as a highly selected population with distinctive gene expression patterns that can promote HIV persistence through HIV silencing, cell survival, and cell proliferation. These findings help reconcile previous observations made in ex vivo and in vivo studies, and suggest important next steps in research towards an HIV cure.