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 EIF2α-, 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.