Single cell transcriptomic analysis of acute and early ART-treated HIV-1-infected CD4+T cells from a reservoir-marking humanized mouse model
Human immunodeficiency virus (HIV-1) infects and depletes CD4+ T helper cells and can persist within these cells in a latent state during treatment with effective antiretroviral therapy (ART).  A latent viral reservoir represents a major barrier to a cure for HIV-1 infection.  A longstanding obstacle to understanding the reservoir has been the inability to identify and characterize latent cells without activating them and disrupting their resting state.  To address this challenge, we have developed an HIV-1-induced lineage tracing (HILT) humanized mouse model that irreversibly marks HIV-infected cells.  The system employs a genetically encoded Cre-lox switch transduced into hematopoietic stem cells which are transplanted into immunodeficient mice.  Infection of CD4+ T cells in HILT mice with a Cre-expressing HIV clone irreversibly marks infected cells by switching dsRedExpress2 to EGFP expression, a phenotype that is maintained in cell harboring proviruses that are transcriptionally silent during ART.  Single cell RNA sequencing (scRNAseq) of infected T cells in the presence or absence of ART reveals HIV infection in diverse CD4+ T cell transcriptional lineages indicating that infected cells with active or suppressed transcription exist in many different T cell lineages.  We examined genetic pathways that are more frequently altered in infected cells when compared with uninfected bystander cells.   A comparison of pathways that are regulated in opposite directions in acute versus early ART-treated (persistent) infection identified EIF2a-, sirtuin-, and ILK-signaling pathways which contain known transcriptional regulators of HIV transcription.  Lastly, we find that resveratrol, a drug that impact sirtuin pathways can influence latency establishment in an acute infection setting. Overall design: Utilizing the HIV-induced-lineage-tracing (HILT) system within humanized mice. Humanized mouse model was based on NOD-NSG Il2rgNULL?(NOD.Cg-Prkdcscid?Il2rgtm1Wjl/SzJ, NSG). 0-3 days old neonates of NSG mice were non-lethally irradiated (100 cGy) and injected intra-hepatically with 1 x 105 human cord blood-derived CD34+ cells (Stemcell Technologies, Cat# 70008) that had been transduced with pHR_SIN CS(RG)W. Mice were injected with NLCreI (RHPA) virus intravenously into the retro-orbital sinus. At 12 days post infection, some infected mice were placed under ART treatment to study latent population. Spleens were harvested and processed. The remaining cells were stained with antibodies described in engraftment panel and sorted using Fluorescence activated cell sorting (FACS). Cells were sorted to enrich for GFP+ infected cells. In mouse A1 GFP+ and dsRed+ cells were pooled and processed together as a mixed population. In acutely infected mice A2/A3, GFP+ HIV infected cells were pooled with unmarked cells, dsRed+ sample processing. Within T1 and T2, GFP+ latently infected cells were sorted into carrier wild type mouse B cells and processed. In T3 GFP+ and dsRed+ cells were pooled and processed together as a mixed population. Within, U1 and U2, Unmarked and dsRed+ bystander cells were sorted separately and sequenced separately. Processing was done using 10x Genomics Chromium Next GEM Single Cell 3' Reagent Kits v.3.1 (CG000204 Rev D). For sequencing, batched libraries were run on an Illumina NovaSeq S4 flowcell (2 x 100 nt paired-end read length) with a target of 50,000-100,000 reads per cell.