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

Abstract

ABSTRACT 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 agm . 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 CCNT1 in the context of one or more functional HIV-1 cure strategies.