The CD225/Dispanins superfamily consists of integral membrane proteins that regulate vesicular transport and membrane fusion events driving critical cellular functions, including glucose transport, neurotransmission, and antiviral immunity. However, how the conserved CD225 domain contributes to the diverse roles played by CD225 proteins during membrane trafficking was unknown. Here, we reveal that the CD225 domain contains a SNARE-like motif that enables interaction with cellular SNARE fusogens and modulation of SNARE complex formation. We show that CD225 member proline rich transmembrane protein 2 (PRRT2) encodes an R-SNARE-like motif that enables interaction with neuronal Q-SNARE proteins. Importantly, mutations in PRRT2 linked to neurological disease in humans reside within key structural features of the R-SNARE-like motif and cause loss of Q-SNARE binding, revealing that SNARE binding via this motif in PRRT2 is critical for its role in regulating neurotransmission in vivo. Another CD225 member, interferon-induced transmembrane protein 3 (IFITM3), provides cell-intrinsic protection against Influenza A virus infection and has been shown to accelerate the trafficking of incoming virions to lysosomes for degradation. Here we demonstrate that an R-SNARE-like motif in IFITM3 is predicted to adopt a VAMP-like fold and to enable interaction with endosomal Q-SNARE proteins involved in homotypic late endosome fusion. Specifically, IFITM3 binds to Q-SNARE syntaxin 7 in cells and in vitro, and mutations in IFITM3 that abrogate syntaxin 7 binding exhibit a loss of antiviral activity against Influenza A virus, indicating that this previously uncharacterized activity is central to the antiviral mechanism of IFITM3. IFITM3 deficiency in human cells resulted in enhanced co-immunoprecipitation between Q-SNARE syntaxin 7/8 and R-SNARE VAMP8, while IFITM3 overexpression inhibited this interaction. Furthermore, overexpression of IFITM3 WT, but not a mutant of IFITM3 defective for syntaxin 7/8 binding, disrupted assembly of the SNARE complex controlling homotypic late endosome fusion. Our findings invoke a mechanistic model whereby IFITM3 diverts incoming virus towards lysosomes by preventing homotypic late endosome fusion. Our identification of an R-SNARE-like motif in many members of the CD225 family suggests that SNARE modulation plays a previously unrecognized role in the diverse membrane trafficking functions performed by these proteins and provides fresh insight into how their loss of function results in disease or susceptibility to infection.
