Limited transferrin receptor clustering allows rapid 1 diffusion of canine parvovirus into clathrin endocytic 2 structures 3 4 5

word count: 156 21 Text word count: 6291 22 Copyright © 2012, American Society for Microbiology. All Rights Reserved. J. Virol. doi:10.1128/JVI.07194-11 JVI Accepts, published online ahead of print on 22 February 2012 on S etem er 3, 2017 by gest http/jvi.asm .rg/ D ow nladed fom ABSTRACT 23 Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet 24 the mechanisms of virus incorporation into these structures remain incompletely understood. 25 Here, we used fluorescence microscopy to directly visualize the association of single canine 26 parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surface of live feline 27 cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that 28 most capsids associated with fewer than five TfRs, and that ~25% of TfR-bound capsids laterally 29 diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not 3023 Viral pathogens usurp cell surface receptors to access clathrin endocytic structures, yet 24 the mechanisms of virus incorporation into these structures remain incompletely understood. 25 Here, we used fluorescence microscopy to directly visualize the association of single canine 26 parvovirus (CPV) capsids with cellular transferrin receptors (TfR) on the surface of live feline 27 cells and to monitor how these CPV-TfR complexes access endocytic structures. We found that 28 most capsids associated with fewer than five TfRs, and that ~25% of TfR-bound capsids laterally 29 diffused into assembling clathrin-coated pits less than 30 s after attachment. Capsids that did not 30 encounter a coated pit dissociated from the cell surface with a half-life of ~30 s. Together, our 31 results show how CPV exploits the natural mechanism of TfR endocytosis to engage the clathrin 32 endocytic pathway and reveal that the low affinity of capsids for feline TfRs limits the residence 33 time of capsids on the cell surface and thus the efficiency of virus internalization. 34