Role of clathrin-mediated endocytosis during vesicular stomatitis virus entry into host cells.
Vesicular stomatitis virus (VSV) is well established to enter cells by pH-dependent endocytosis, but mechanistic aspects of its internalization have remained unclear. Here, we examined the functional role of clathrin in VSV entry by expression of a dominant-negative mutant of Eps15 (GFP-Eps15Delta95/295), a protein essential for clathrin-mediated endocytosis. Whereas expression of GFP alone had no effect on VSV infection, expression of GFP-Eps15Delta95/295 severely limited infection. As independent ways to examine clathrin function, we also examined cells that had been treated with chlorpromazine and utilized small interfering RNA (siRNA) techniques. Inhibition of clathrin-mediated endocytosis by chlorpromazine treatment, as well as clathrin knock-down using siRNA duplexes directed against the clathrin heavy chain, also prevented VSV infection. In combination with previous morphological approaches, these experiments establish clathrin as an essential component needed for endocytosis of VSV.
Sun
Xiangjie
X
Department of Microbiology and Immunology, Cornell University, C4127 Veterinary Medical Center, Ithaca, NY 14853, USA.
Yau
Vivian K
VK
Briggs
Benjamin J
BJ
Whittaker
Gary R
GR
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000502:physiology; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D033922:Clathrin Heavy Chains / Q000235:genetics; D006224:Cricetinae; D004705:Endocytosis / Q000502:physiology*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins / Q000235:genetics / Q000502:physiology; D034741:RNA, Small Interfering / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014168:Transferrin / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000472:pathogenicity* / Q000502:physiology
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D002966:Clathrin; C486206:EPS15 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins; D034741:RNA, Small Interfering; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D033922:Clathrin Heavy Chains; D049452:Green Fluorescent Proteins; D002746:Chlorpromazine