4.5 Article

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

期刊

VIRUS RESEARCH
卷 259, 期 -, 页码 28-37

出版社

ELSEVIER
DOI: 10.1016/j.virusres.2018.10.003

关键词

Fusion inhibitors; Fusion protein; Head-stalk interface; Morbillivirus cell entry; Prefusion state stabilization

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资金

  1. University of Bern
  2. Swiss National Science Foundation [310030_173185, 31003A_162581]
  3. National Centres of Competence in Research (NCCRs) TransCure and Molecular Systems Engineering
  4. Swiss National Science Foundation (SNF) [31003A_162581, 310030_173185] Funding Source: Swiss National Science Foundation (SNF)

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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.

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