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Primary resistance mechanism of the canine distemper virus fusion protein against a small-molecule membrane fusion inhibitor

Kalbermatter, David, Shrestha, Neeta, Ader-Ebert, Nadine, Herren, Michael, Moll, Pascal, Plemper, Richard K., Altmann, Karl-Heinz, Langedijk, Johannes P., Gall, Flavio, Lindenmann, Urs, Riedl, Rainer, Fotiadis, Dimitrios, Plattet, Philippe
Virus research 2019 v.259 pp. 28-37
viral fusion proteins, bioactive properties, glycoproteins, membrane fusion, antibodies, Canine morbillivirus, electron microscopy, Measles morbillivirus, hydrophobicity, mutants, cysteine
Morbilliviruses (e.g. measles virus [MeV] or canine distemper virus [CDV]) employ the attachment (H) and fusion (F) envelope glycoproteins for cell entry. H protein engagement to a cognate receptor eventually leads to F-triggering. Upon activation, F proteins transit from a prefusion to a postfusion conformation; a refolding process that is associated with membrane merging. Small-molecule morbilliviral fusion inhibitors such as the compound 3G (a chemical analog in the AS-48 class) were previously generated and mechanistic studies revealed a stabilizing effect on morbilliviral prefusion F trimers. Here, we aimed at designing 3G-resistant CDV F mutants by introducing single cysteine residues at hydrophobic core positions of the helical stalk region. Covalently-linked F dimers were generated, which highlighted substantial conformational flexibility within the stalk to achieve those irregular F conformations. Our findings demonstrate that “top-stalk” CDV F cysteine mutants (F-V571C and F-L575C) remained functional and gained resistance to 3G. Conversely, although not all “bottom-stalk” F cysteine variants preserved proper bioactivity, those that remained functional exhibited 3G-sensitivity. According to the recently determined prefusion MeV F trimer/AS-48 co-crystal structure, CDV residues F-V571 and F-L575 may directly interact with 3G. A combination of conformation-specific anti-F antibodies and low-resolution electron microscopy structural analyses confirmed that 3G lost its stabilizing effect on “top-stalk” F cysteine mutants thus suggesting a primary resistance mechanism. Overall, our data suggest that the fusion inhibitor 3G stabilizes prefusion CDV F trimers by docking at the top of the stalk domain.