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Imaging multiple intermediates of single‐virus membrane fusion mediated by distinct fusion proteins
Author(s) -
Joo KyeIl,
Tai April,
Lee ChiLin,
Wong Clement,
Wang Pin
Publication year - 2010
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.20841
Subject(s) - lipid bilayer fusion , fusion mechanism , fusion , microbiology and biotechnology , endosome , viral entry , fusion protein , endocytosis , viral envelope , virus , sindbis virus , viral membrane , cell fusion , chemistry , biology , biophysics , virology , biochemistry , cell , viral replication , gene , recombinant dna , linguistics , philosophy , rna , intracellular
Membrane fusion plays an essential role in the entry of enveloped viruses into target cells. The merging of viral and target cell membranes is catalyzed by viral fusion proteins, which involves multiple sequential steps in the fusion process. However, the fusion mechanisms mediated by different fusion proteins involve multiple transient intermediates that have not been well characterized. Here, we report a synthetic virus platform that allows us to better understand the different fusion mechanisms driven by the diverse types fusion proteins. The platform consists of lentiviral particles coenveloped with a surface antibody, which serves as the binding protein, along with a fusion protein derived from either influenza virus (HAmu) or Sindbis virus (SINmu). By using a single virus tracking technique, we demonstrated that both HAmu‐ and SINmu‐bearing viruses enter cells through clathrin‐dependent endocytosis, but they required different endosomal trafficking routes to initiate viral fusion. Direct observation of single viral fusion events clearly showed that hemifusion mediated by SINmu upon exposure to low pH occurs faster than that mediated by HAmu. Monitoring sequential fusion processes by dual labeling the outer and inner leaflets of viral membranes also revealed that the SINmu‐mediated hemifusion intermediate is relatively long‐lived as compared with that mediated by HAmu. Taken together, we have demonstrated that the combination of this versatile viral platform with the techniques of single virus tracking can be a powerful tool for revealing molecular details of fusion mediated by various fusion proteins. Microsc. Res. Tech. 73:886–900, 2010. © 2010 Wiley‐Liss, Inc.