PMID:15367621

Citation	Chu, JJ and Ng, ML (2004) Infectious entry of West Nile virus occurs through a clathrin-mediated endocytic pathway. J. Virol. 78:10543-55
Abstract	The pathway of West Nile flavivirus early internalization events was mapped in detail in this study. Overexpression of dominant-negative mutants of Eps15 strongly inhibits West Nile virus (WNV) internalization, and pharmacological drugs that blocks clathrin also caused a marked reduction in virus entry but not caveola-dependent endocytosis inhibitory agent, filipin. Using immunocryoelectron microscopy, WNV particles were seen within clathrin-coated pits after 2 min postinfection. Double-labeling immunofluorescence assays and immunoelectron microscopy performed with anti-WNV envelope or capsid proteins and cellular markers (EEA1 and LAMP1) revealed the trafficking pathway of internalized virus particles from early endosomes to lysosomes and finally the uncoating of the virus particles. Disruption of host cell cytoskeleton (actin filaments and microtubules) with cytochalasin D and nocodazole showed significant reduction in virus infectivity. Actin filaments are shown to be essential during the initial penetration of the virus across the plasma membrane, whereas microtubules are involved in the trafficking of internalized virus from early endosomes to lysosomes for uncoating. Cells treated with lysosomotropic agents were largely resistant to infection, indicating that a low-pH-dependent step is required for WNV infection. In situ hybridization of DNA probes specific for viral RNA demonstrated the trafficking of uncoated viral RNA genomes to the endoplasmic reticulum.
Links	PubMed PMC516396 Online version:10.1128/JVI.78.19.10543-10555.2004
Keywords	Actin Cytoskeleton/virology; Animals; Antigens, CD/analysis; Biological Transport; Capsid Proteins/analysis; Cercopithecus aethiops; Clathrin-Coated Vesicles/ultrastructure; Clathrin-Coated Vesicles/virology; Cryoelectron Microscopy; Cytochalasin D/pharmacology; Cytoskeleton/drug effects; Cytoskeleton/virology; Endocytosis; Endoplasmic Reticulum/virology; Endosomes/virology; Hydrogen-Ion Concentration; Lysosomal-Associated Membrane Protein 1; Lysosome-Associated Membrane Glycoproteins; Lysosomes/virology; Membrane Proteins/analysis; Microscopy, Fluorescence; Microscopy, Immunoelectron; Microtubules/virology; Nocodazole/pharmacology; RNA, Viral/analysis; Vero Cells; Vesicular Transport Proteins; Viral Envelope Proteins/analysis; Virion/metabolism; Virus Replication; West Nile virus/pathogenicity; West Nile virus/physiology
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
WNV:POLG	involved_in	GO:0075512: clathrin-dependent endocytosis of virus by host cell	ECO:0000314: direct assay evidence used in manual assertion		P		Seeded From UniProt	complete

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