Engineering T cells to encode natural post-integration barriers to HIV-1 replication

HIV-1 virion production is inefficient in cells derived from mice and other rodents. Inefficient production maps, in part, to a species-specific polymorphism in the cellular gene, CCNT1. In human cells, the HIV-1 Tat protein hijacks the positive transcription elongation factor (p-TEFb) complex, composed of CCNT1 and the cellular kinase CDK9, to promote viral transcript elongation and thereby increasing net viral gene expression and virion production. In mouse cells, Tat-dependent viral transcription is poor, reflecting the inability of Tat to engage p-TEFb because of a species-specific tyrosine residue at position 261 in the mouse ortholog of CCNT1. Humans encode a cysteine at the equivalent position which Tat requires for efficient p-TEFb usage. Here, we used CRISPR/Cas9 genome engineering to substitute the native cysteine with the rodent-encoded tyrosine (C261Y) in the human Jurkat T cell line (E6-1 clone). We derived two independent Jurkat T cell clones with all endogenous CCNT1 encoding the C261Y substitution. In an effort to understand net impacts to cellular and viral gene expression, we compared the transcriptomes of uninfected parental Jurkat T cells (E6-1 clone) to the two derived clones (C1 and C2, CCNT1.C261Y). Transcriptome comparison (polyA-enriched RNA-seq) of human Jurkat T cells (E6-1 clone) to two derivative clones (C1 and C2) encoding CCNT1 with an engineered C261Y substitution via CRISPR knock-in. Total RNA was extracted from three replicate cell cultures.