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However, the regulatory mechanisms controlling Vpx stability remain unclear. In this study, we identify the von Hippel\u2013Lindau (VHL) tumor suppressor as a novel E3 ubiquitin ligase that specifically targets Vpx for proteasomal degradation. Mechanistically, we demonstrate that VHL-mediated degradation depends on the oxygen-dependent hydroxylation of Vpx at proline residue 41 (Pro41), a modification catalyzed by prolyl hydroxylase domain-containing protein 3 (PHD3). Using an integrated approach combining crosslinking mass spectrometry and molecular modeling analyses, we elucidate the structural architecture of the PHD3-Vpx complex, revealing the spatial orientation of the catalytic domain of PHD3 required for Pro41 hydroxylation. Furthermore, we establish the physiological significance of this pathway in human macrophages, where pharmacological inhibition or genetic ablation of VHL or PHD3 enhances HIV-2 infection by facilitating Vpx-mediated SAMHD1 degradation. Collectively, our findings unveil a previously unrecognized oxygen-sensitive regulatory mechanism influencing HIV-2 infection and suggest novel therapeutic strategies targeting Vpx stability through modulation of its prolyl hydroxylation status.<\/jats:p>","DOI":"10.1371\/journal.ppat.1013241","type":"journal-article","created":{"date-parts":[[2025,6,16]],"date-time":"2025-06-16T17:39:59Z","timestamp":1750095599000},"page":"e1013241","update-policy":"https:\/\/doi.org\/10.1371\/journal.ppat.corrections_policy","source":"Crossref","is-referenced-by-count":1,"title":["PHD3-VHL axis controls HIV-2 infection through oxygen-dependent hydroxylation and degradation of 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