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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\u2019s 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\n                    <jats:sub>agm<\/jats:sub>\n                    . 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\u2019s post-integration phases.\n                  <\/jats:p>\n                  <jats:sec>\n                    <jats:title>Importance<\/jats:title>\n                    <jats:p>\n                      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 \u201cmousified\u201d 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\n                      <jats:italic>CCNT1<\/jats:italic>\n                      in the context of one or more functional HIV-1 cure strategies.\n                    <\/jats:p>\n                  <\/jats:sec>","DOI":"10.1128\/mbio.00420-23","type":"journal-article","created":{"date-parts":[[2023,9,7]],"date-time":"2023-09-07T09:01:35Z","timestamp":1694077295000},"update-policy":"https:\/\/doi.org\/10.1128\/asmj-crossmark-policy-page","source":"Crossref","is-referenced-by-count":6,"title":["Exploiting a rodent cell block for intrinsic resistance to HIV-1 gene expression in human T cells"],"prefix":"10.1128","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0001-5761-9056","authenticated-orcid":false,"given":"Ryan T.","family":"Behrens","sequence":"first","affiliation":[{"name":"McArdle Laboratory for Cancer Research and Carbone Cancer Center, University of Wisconsin-Madison , Madison, Wisconsin, USA"},{"name":"Institute for Molecular Virology, University of Wisconsin-Madison , Madison, Wisconsin, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jyothi Krishnaswamy","family":"Rajashekar","sequence":"additional","affiliation":[{"name":"Department of Internal Medicine, Section of Infectious Diseases, Yale University School of Medicine , New Haven, Connecticut, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"James W.","family":"Bruce","sequence":"additional","affiliation":[{"name":"McArdle Laboratory for Cancer Research and Carbone Cancer Center, University of Wisconsin-Madison , Madison, Wisconsin, USA"},{"name":"Institute for Molecular Virology, University of Wisconsin-Madison , Madison, Wisconsin, USA"},{"name":"Morgridge Institute for Research, University of Wisconsin-Madison , Madison, Wisconsin, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"suffix":"III","given":"Edward L.","family":"Evans","sequence":"additional","affiliation":[{"name":"McArdle 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