Exploiting rodent cell blocks 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, Tats interactions with CCNT1 are inefficient, mapped to a single species-specific residue Y261 instead of C261 in human. 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 Tat and Rev function in humans, herein we recoded ("mousified") each in human CD4+ T cells using precision CRISPR/Cas9-facilitated gene editing. Both edits yielded cells refractory to Rev or Tat activity, respectively, with isolated, isogenic CCNT1.C261Y cell lines remarkable in their capacity to exhibit near total inactivation of viral gene expression for all X4 and R5-tropic HIV-1 strains tested, and even the more distantly related lentiviruses including HIV-2 and SIVagm. These studies validate minor and naturally-occurring, species-specific differences in otherwise conserved human host factors as compelling targets for achieving broad-acting cell-intrinsic resistance to HIVs post-integration phases. ImportanceUnlike 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 editing to engineer "mousified" versions of two of these proteins, CCNT1 and XPO1, in human T cells. CCNT1 and XPO1 are essential for efficient HIV-1 transcription and viral RNA transport, respectively, making them intriguing targets for gene-based inactivation of virus replication. Targeting either gene yielded antiviral phenotypes, with isogenic CCNT1-modified cell lines confirmed to exhibit potent, durable, and broad-spectrum resistance to HIV-1 and other pathogenic lentiviruses, and with no discernible impact on host cells. These results provide proof of concept for targeting CCNT1 (and potentially XPO1) in the context of one or more functional HIV-1 cure strategies.