Summary The cellular and viral determinants required for HIV-1 infection of nondividing cells have been a subject of intense scrutiny. Here we identify the 68 kDa subunit of cleavage factor Im, CPSF6, as an inhibitor of HIV-1 infection. When enriched in the cytoplasm by high level expression or mutation, CPSF6 prevents nuclear entry of the virus. Similar to TRIM5 and Fv1 type restrictions, CPSF6 targets the viral capsid (CA). N74D mutation of the HIV-1 CA leads to a loss of interaction with CPSF6 and evasion of the nuclear import restriction. Interestingly, N74D mutation of CA changes HIV-1 nucleoporin (NUP) requirements. Whereas wild-type HIV-1 requires NUP153, N74D HIV-1 mimics the NUP requirements of feline immunodeficiency virus (FIV) and is more sensitive to NUP155 depletion. These findings reveal a remarkable flexibility in HIV-1 nuclear transport and highlight a single residue in CA as essential in regulating interactions with NUPs.

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Transportin 3 (Tnpo3, Transportin-SR2) is implicated in nuclear import of splicing factors and HIV-1 replication. Herein, we show that the majority of cellular Tnpo3 binding partners contain arginineserine (RS) repeat domains and present crystal structures of human Tnpo3 in its free as well as GTPase Ran-and alternative splicing factor/splicing factor 2 (ASF/SF2)-bound forms. The flexible β-karyopherin fold of Tnpo3 embraces the RNA recognition motif and RS domains of the cargo. A constellation of charged residues on and around the arginine-rich helix of Tnpo3 HEAT repeat 15 engage the phosphorylated RS domain and are critical for the recognition and nuclear import of ASF/SF2. Mutations in the same region of Tnpo3 impair its interaction with the cleavage and polyadenylation specificity factor 6 (CPSF6) and its ability to support HIV-1 replication. Steric incompatibility of the RS domain and RanGTP engagement by Tnpo3 provides the mechanism for cargo release in the nucleus. Our results elucidate the structural bases for nuclear import of splicing factors and the Tnpo3-CPSF6 nexus in HIV-1 biology.T he transport of macromolecules between cytoplasm and nucleus is orchestrated by a family of nuclear import and export receptors (1). Referred to as importins and exportins, these proteins bind their specific cargoes and translocate them across the nuclear pore complex. The process is regulated by the small GTPase Ran that partitions between cytoplasm and nucleus in the predominantly GDP-and GTP-bound form, respectively. Importins associate with their cargoes in the cytoplasm, and the competitive binding of RanGTP induces them to release their cargoes in the nucleus (2). Most nuclear import/export receptors belong to the β-karyopherin family of proteins, with 22 members encoded in the human genome (3). The majority of β-karyopherins bind their cargoes directly, recognizing a linear nuclear localization or export signal and/or a specific tertiary/quaternary structural feature (4, 5).A fundamentally important type of a nuclear localization signal (NLS), comprising sequences rich in Arg-Ser and/or ArgAsp/Glu/Gly dipeptides (referred to as RS or RS-like domains), belongs to the family of Ser/Arg-rich (SR) proteins. These nuclear proteins also contain RNA recognition motif (RRM) domains and play essential roles in pre-mRNA splicing and 3′ processing and participate in transcription regulation, mRNA transport, translation, and nonsense-mediated mRNA decay (6). The splicing factors alternative splicing factor/splicing factor 2 (ASF/SF2) and SC35 along with the cleavage and polyadenylation specificity factor 6 (CPSF6, also known as CF-Im-68) are among the best-characterized metazoan SR proteins (7-10). The SR protein family can be further extended by inclusion of a structurally and functionally diverse group of nuclear proteins that possess RS repeats but lack RRM domains (11). The RS domains are processively phosphorylated on their Ser residues by a set of dedicated kinases (12-16). Phosphorylation of SR proteins is thought to...

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