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The viral capsid is essential for successfully delivering the HIV-1 genome into the nucleus. Nucleoporin 153 (NUP153) interacts with the HIV-1 capsid via a C-terminal capsid-binding motif (hereafter named CbM.1) to licence HIV-1 nuclear ingress. Deletion or mutation of CbM.1 in NUP153 causes a reduction in capsid interaction but does not prevent HIV-1 nuclear ingress or completely block capsid interaction. This paper combines molecular modelling with biochemical and HIV infection assays to identify capsid-binding motif 2 (CbM.2) in the C-terminus of NUP153 that is similar in sequence to CbM.1. CbM.2 has an FG dipeptide motif predicted to interact with a hydrophobic pocket in capsid protein (CA) hexamers similar to CbM.1. CA hexamers can interact with CbM.2, and the deletion of both CbM.1 and CbM.2 results in a lower capsid interaction than a single CbM.1 deletion. The loss of CbM.1 is complemented by CbM.2, an interaction dependent on the FG motif. In the context of the nuclear pore complex, a loss-of-function mutation in CbM.1 reduces HIV nuclear ingress as measured by transduction and 2-LTR circles, whereas the mutation of CbM.2 causes a large increase in 2-LTR circles. Our results highlighted a previously unidentified FG dipeptide-containing motif (CbM.2) in NUP153 that binds the HIV-1 capsid at the common hydrophobic pocket on CA hexamers.<\/jats:p>","DOI":"10.1099\/jgv.0.002104","type":"journal-article","created":{"date-parts":[[2025,5,14]],"date-time":"2025-05-14T15:16:07Z","timestamp":1747235767000},"source":"Crossref","is-referenced-by-count":0,"title":["The identification of a novel interaction site for the human immunodeficiency virus capsid on nucleoporin 153"],"prefix":"10.1099","volume":"106","author":[{"given":"Shunji","family":"Li","sequence":"first","affiliation":[{"name":"Department of Biological Sciences, University of Idaho, Moscow, ID 83844, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Peik","family":"Lund-Andersen","sequence":"additional","affiliation":[{"name":"Institute for Modeling Collaboration and Innovation, University of Idaho, Moscow, ID 83844, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Szu-Huan","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912, USA"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"F. 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