Résumé
Interferon (IFN)-induced antiviral proteins represent a crucial first line of defence against influenza A virus (IAV) infection. Among them, we previously identified the short isoform of Nuclear Receptor Coactivator 7 (NCOA7-AS) as a key player. Specifically, knockout of NCOA7-AS partially alleviates the IFN-mediated restriction of IAV infection (Doyle et al, 2018). Conversely, ectopic expression of NCOA7-AS inhibits IAV entry by impairing the fusion efficiency between viral and endosomal membranes. This effect likely results from enhanced acidification of the endolysosomal compartment via NCOA7-AS interaction with V1 subunits of the vacuolar ATPase (V-ATPase).Here, we confirm the functional importance of this interaction by identifying NCOA7-AS glycine 91 as a critical residue. Its mutation disrupts endolysosomal pH regulation and abolishes the antiviral activity of NCOA7-AS.Additional cellular partners of NCOA7-AS were identified by mass spectrometry, notably sorting nexins (SNX) 1, 2, 5, and 6. These SNXs mediate retrograde transport of cellular cargoes from the plasma membrane and endosomes to the trans-Golgi network. All four SNXs contributed to NCOA7-AS–mediated overacidification of endolysosomes, but only SNX5 and SNX6 were required for its antiviral activity. Biochemical assays with recombinant proteins confirmed a direct interaction between the amino-terminal domain of NCOA7-AS and the Phox domains of SNX5 or SNX6.The structure of the NCOA7-AS/SNX5 complex was solved at high resolution by X-ray crystallography, revealing a conserved interaction mode shared with other SNX5 cargoes. Moreover, the hydrophobic interaction mediated by SNX5-F136 and NCOA7-AS-Y14 proved essential for both the in vitro binding and antiviral activity in cells.These findings shed new light on the molecular mechanism of action of NCOA7-AS, identifying novel essential cellular partners and key residues required for its antiviral function.