Relevance of potential endocytosis motifs in Cedar virus glycoprotein G for its biological activity

The glycoprotein (G) and fusion protein (F) of henipaviruses play crucial roles in receptor binding and entry into host cells, also enabling virus spread from cell to cell without the release of infectious particles. For Cedar virus (CedV), the proteolytic activation of the F protein precursor into F1 and F2-and thus, its biological activity-depends on clathrin-mediated endocytosis driven by classical endocytosis motifs YXXΦ and YY in the cytoplasmic tail of the F protein. Similar motifs are present in the cytoplasmic tail of CedV G protein. In this study, we investigated whether these motifs influence CedV G protein expression and transport, endocytosis from the plasma membrane, and overall the biological activity-more specifically receptor binding and mediation of fusion together with the fusion protein. Our data show that the expression of CedV G mutants is comparable to parental G in MDCK cells. Endocytosis can be detected for both parental G and its mutants. However, some G protein mutants show reduced biological activity, as indicated by a decrease in fusion when G mutants are co-expressed with CedV F protein. Interestingly, neither co-expression of CedV F and G mutants on the cell surface nor the binding of G mutants to EFNB2 receptors appears to be compromised. Consequently, the putative endocytosis motifs are not relevant for biological activity of CedV G.IMPORTANCEThe glycoprotein (G) and fusion protein (F) of henipaviruses mediate host cell entry and direct cell-to-cell virus spread. For the non-pathogenic Cedar virus (CedV), activation of F depends on clathrin-mediated endocytosis mediated by specific YXXΦ and YY motifs in its cytoplasmic tail. The presence of similar motifs in the cytoplasmic domain of CedV G suggested a potential role in G protein trafficking and function. Here, we show that mutation of these motifs does not impair CedV G surface expression, internalization, or receptor binding. However, some mutants showed reduced ability to mediate fusion with CedV F, despite unaltered surface expression and receptor binding. This suggests that these putative endocytosis motifs are not critical for the biological activity of CedV G. These insights will help dissect mechanisms of viral entry and fusion in CedV and contribute to the comparison of these processes with those of the highly pathogenic henipaviruses HeV and NiV.