Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells.

HIV-1 virion production is inefficient in cells derived from mice and other rodents reflecting cell-intrinsic defects to interactions between the HIV-1 auxiliary proteins Tat and Rev and host dependency factors CCNT1 (Cyclin T1) and XPO1 (exportin-1, also known as CRM1), respectively. In human cells, Tat binds CCNT1 to enhance viral RNA transcription and Rev recruits XPO1 to mediate the nuclear export of intron-containing viral RNA. In mouse cells, Tat's interactions with CCNT1 are inefficient, mapped to a single species-specific residue Y261 instead of C261 in humans. Rev interacts poorly with murine XPO1, mapped to a trio of amino acids T411/V412/S414 instead of P411/M412/F414 in humans. To determine if these discrete species-specific regions of otherwise conserved housekeeping proteins represent viable targets for inhibiting HIV-1 replication in humans, herein, we employed CRISPR/Cas9 to recode the relevant regions of CCNT1 and XPO1 in human CD4+ T cells. While efforts to modify XPO1 were inconclusive, we generated isogenic CCNT1.C261Y cell lines exhibiting remarkable resistance to HIV-1 Tat, exhibiting near total inactivation of viral gene expression for all X4- and R5-tropic HIV-1 strains tested, as well as the more distantly related primate lentiviruses HIV-2 and SIV. Induction of viral reactivation using latency reversal agents (LRAs) was also restricted in CCNT1.C261Y cells. These studies validate a minor and naturally occurring, species-specific difference in a conserved human host factor as a compelling potential target for achieving broad-acting cell-intrinsic resistance to HIV's post-integration phases. IMPORTANCE Unlike humans, mice are unable to support HIV-1 infection. This is due, in part, to a constellation of defined minor, species-specific differences in conserved host proteins needed for viral gene expression. Here, we used precision CRISPR/Cas9 gene editing to engineer a "mousified" version of one such host protein, cyclin T1 (CCNT1), in human T cells. CCNT1 is essential for efficient HIV-1 transcription, making it an intriguing target for gene-based inactivation of virus replication. We show that isogenic cell lines engineered to encode CCNT1 bearing a single mouse-informed amino acid change (tyrosine in place of cysteine at position 261) exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cell biology. These results provide proof of concept for targeting in the context of one or more functional HIV-1 cure strategies.