Abstract
Flaviviruses deliver their genome into the cell by fusing the viral lipid membrane to an endosomal membrane. The sequence and kinetics of the steps required for nucleocapsid delivery into the cytoplasm remain unclear. Here we dissect the cell entry pathway of virions and virus-like particles from two flaviviruses using single-particle tracking in live cells, a biochemical membrane fusion assay and virus infectivity assays. We show that the virus particles fuse with a small endosomal compartment in which the nucleocapsid remains trapped for several minutes. Endosomal maturation inhibitors inhibit infectivity but not membrane fusion. We propose a flavivirus cell entry mechanism in which the virus particles fuse preferentially with small endosomal carrier vesicles and depend on back-fusion of the vesicles with the late endosomal membrane to deliver the nucleocapsid into the cytoplasm. Virus entry modulates intracellular calcium release and phosphatidylinositol-3-phosphate kinase signaling. Moreover, the broadly cross-reactive therapeutic antibody scFv11 binds to virus-like particles and inhibits fusion.
Highlights
Many enveloped RNA viruses utilize the endocytic pathway to enter host cells [1,2]
In order to deliver their genome into the hostcell cytoplasm, where it can be replicated, viruses must fuse their envelope with a cellular lipid membrane
The viral genome remains trapped in these compartments for several minutes until the compartments fuse with the surrounding endosomal membrane
Summary
Many enveloped RNA viruses utilize the endocytic pathway to enter host cells [1,2]. Endocytosis begins at the cell membrane, where these viruses bind to their cellular receptors and ends at the lysosome, the ‘‘stomach’’ of the cell. Enveloped viruses and bacterial toxins enter the endocytic pathway by binding receptors on the cell surface that are coupled to the endocytic machinery, in particular clathrin adaptors. These microbial cargoes undergo sorting at two different checkpoints [4,5,6]. The level of cholesterol decreases along the endocytic pathway and is replaced with ceramide in late endosomes and lysosomes, where it maintains membrane fluidity [10]. Unlike cholesterol and PS, the anionic lipid BMP (bis(monoacylglycero)phosphate), known as LBPA (lysobisphosphatidic acid), is abundant in internal membranes of lysosomes and late endosomes, and depleted in the EEs [7]. Autoantibodies against this lipid result in human disorders such as Niemann-Pick type C (NPC) syndrome, characterized by dysfunctional sorting and trafficking in late endosomes [11]
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