Understanding HIV-1–host interactions can identify the cellular environment supporting HIV-1 reactivation and mechanisms of clonal expansion. We developed HIV-1 SortSeq to isolate rare HIV-1–infected cells from virally suppressed, HIV-1–infected individuals upon early latency reversal. Single-cell transcriptome analysis of HIV-1 SortSeq+ cells revealed enrichment of nonsense-mediated RNA decay and viral transcription pathways. HIV-1 SortSeq+ cells up-regulated cellular factors that can support HIV-1 transcription (IMPDH1 and JAK1) or promote cellular survival (IL2 and IKBKB). HIV-1–host RNA landscape analysis at the integration site revealed that HIV-1 drives high aberrant host gene transcription downstream, but not upstream, of the integration site through HIV-1–to–host aberrant splicing, in which HIV-1 RNA splices into the host RNA and aberrantly drives host RNA transcription. HIV-1–induced aberrant transcription was driven by the HIV-1 promoter as shown by CRISPR-dCas9–mediated HIV-1–specific activation and could be suppressed by CRISPR-dCas9–mediated inhibition of HIV-1 5′ long terminal repeat. Overall, we identified cellular factors supporting HIV-1 reactivation and HIV-1–driven aberrant host gene transcription as potential therapeutic targets to disrupt HIV-1 persistence.

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HIV-1-infected cells, which can survive drug treatment and immune cell killing, prevent an HIV-1 cure. Immune recognition of infected cells requires HIV-1 protein expression; however, HIV-1 protein expression is limited in infected cells after long-term therapy.

Purpose of review CD4+ T cell loss is the hallmark of uncontrolled HIV-1 infection. Strikingly, CD4+ T cell depletion is a strong indicator for disease severity in the recently emerged coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We reviewed recent single-cell immune profiling studies in HIV-1 infection and COVID-19 to provide critical insight in virus-induced immunopathogenesis. Recent findings Cytokine dysregulation in HIV-1 leads to chronic inflammation, while severe SARS-CoV-2 infection induces cytokine release syndrome and increased mortality. HIV-1-specific CD4+ T cells are dysfunctional, while SARS-CoV-2-specific CD4+ T cells exhibit robust Th1 function and correlate with protective antibody responses. In HIV-1 infection, follicular helper T cells (TFH) are susceptible to HIV-1 infection and persist in immune-sanctuary sites in lymphoid tissues as an HIV-1 reservoir. In severe SARS-CoV-2 infection, TFH are absent in lymphoid tissues and are associated with diminished protective immunity. Advancement in HIV-1 DNA, RNA, and protein-based single-cell capture methods can overcome the rarity and heterogeneity of HIV-1-infected cells and identify mechanisms of HIV-1 persistence and clonal expansion dynamics. Summary Single-cell immune profiling identifies a high-resolution picture of immune dysregulation in HIV-1 and SARS-CoV-2 infection and informs outcome prediction and therapeutic interventions.

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Despite antiretroviral therapy (ART), HIV-1 persists in proliferating T cell clones that increase over time. To understand whether early ART affects HIV-1 persistence in vivo, we performed single-cell ECCITE-seq and profiled 89,279 CD4+ T cells in paired samples during viremia and after immediate versus delayed ART in six people in the randomized interventional Sabes study. We found that immediate ART partially reverted TNF responses while delayed ART did not. Antigen and TNF responses persisted despite immediate ART and shaped the transcriptional landscape of CD4+ T cells, HIV-1 RNA+ cells, and T cell clones harboring them (cloneHIV-1). Some HIV-1 RNA+ cells reside in the most clonally expanded cytotoxic T cell populations (GZMB and GZMK Th1 cells). CloneHIV-1+ were larger in clone size, persisted despite ART, and exhibited transcriptional signatures of antigen, cytotoxic effector, and cytokine responses. Using machine-learning algorithms, we identified markers for HIV-1 RNA+ cells and cloneHIV-1+ as potential therapeutic targets. Overall, by combining single-cell immune profiling and T cell expansion dynamics tracking, we identified drivers of HIV-1 persistence in vivo.

HIV-1 integration introduces ectopic transcription factor binding sites into host chromatin. We postulate that the integrated provirus serves as an ectopic enhancer that recruits additional transcriptional factors to the integration locus, increases chromatin accessibility, changes 3D chromatin interactions, and enhances both retroviral and host gene expression. We used 4 well-characterized HIV-1-infected cell line clones having unique integration sites and low to high levels of HIV-1 expression. Using single-cell DOGMA-seq, which captured the heterogeneity of HIV-1 expression and host chromatin accessibility, we found that HIV-1 transcription correlated with HIV-1 accessibility and host chromatin accessibility. HIV-1 integration increased local host chromatin accessibility within ~5-30 kb distance. CRISPRa and CRISPRi-mediated HIV-1 promoter activation and inhibition confirmed integration site–dependent HIV-1-driven changes of host chromatin accessibility. HIV-1 did not drive chromatin confirmation changes at the genomic level (by Hi-C) or the enhancer connectome (by H3K27ac HiChIP). Using 4C-seq to interrogate HIV-1-chromatin interactions, we found that HIV-1 interacted with host chromatin ~100–300 kb from the integration site. By identifying chromatin regions having both increased transcription factor activity (by ATAC-seq) and HIV-1-chromtain interaction (by 4C-seq), we identified enrichment of ETS, RUNT, and ZNF-family transcription factor binding that may mediate HIV-1-host chromatin interactions. Our study found that HIV-1 promoter activity increased host chromatin accessibility, increased HIV-1-host chromatin interactions in an integration site–dependent manner, within the existing chromatin boundaries.