The Berlin patient demonstrates that cure of HIV is possible, and the likely mechanism of cure (transplantation with CCR5?32 cells) suggests that cell-intrinsic resistance represents a potential therapeutic scenario for a full or functional cure for HIV/AIDS. For several years, we have studied natural, cell-intrinsic barriers to HIV-1 gene expression in cells derived from mice and other rodents. Two of these barriers map to genes encoding host proteins Cyclin T1 (CCNT1) and Exportin-1 (XPO1, also known as CRM1) that are essential regulators of HIV-1 gene expression and latency reversal. In human cells, CCNT1 interacts with the viral Tat protein to promote HIV transcriptional elongation, while XPO1 is bound by the viral Rev protein to mediate nuclear export of intron-containing viral mRNAs and the viral RNA genome. In mouse cells, Tat-Ccnt1 and Rev-Xpo1 interactions are inefficient due to species-specific differences between mouse and human orthologues of either protein. Why these highly conserved genes evolved differences in the rodent and primate lineages is unknown, and the rise of CRISPR/Cas9 technology provides us with the unprecedented opportunity to test the hypothesis that knocking-in mouse-specific features of CCNT1 and XPO1 into human T cells will suppress HIV-1 replication in vitro and in vivo with little net impact on host cellular biology. Indeed, our project is premised on exciting preliminary data showing that modifying a single species-specific codon of human CCNT1 in a human T cell line is sufficient to abolish HIV-1 Tat activity and viral replication, with no discernable effects on cell proliferation. Herein we request support for collaborative studies between the Sherer and Kumar labs to further our studies of mouse-informed CCNT1 and XPO1 gene modifications in human T cells, and to determine the potential of targeting these genes as a resistance/treatment strategy for HIV-1.
Mice exhibit strong cell-intrinsic barriers to HIV-1 gene expression that map to minor, species-specific differences to mouse and human versions of genes encoding the Cyclin T1 (CCNT1) and Exportin-1 (XPO1) host proteins. Herein, we propose to engineer minor (1 to 3 codon) ?mouse-informed? changes into human CCNT1 and XPO1 genes in CD4+ T cells using CRISPR/Cas9 and then to study the antiviral effects of these modifications on HIV replication both in vitro and in humanized mice engrafted with gene- modified cells. Our studies will provide new insight into the evolution of essential HIV-host interactions and may inform the development of a cell-based anti-HIV therapeutic strategy.
