title: Functional entry of dengue virus into Aedes albopictus mosquito cells is dependent on clathrin-mediated endocytosis.
abstract: Entry of dengue virus 2 (DENV-2) into Aedes albopictus mosquito C6/36 cells was analysed using biochemical and molecular inhibitors, together with confocal and electron microscopy observations. Treatment with monodansylcadaverine, chlorpromazine, sucrose and ammonium chloride inhibited DENV-2 virus yield and protein expression, whereas nystatin, a blocker of caveolae-mediated endocytosis, did not have any effect. Using confocal microscopy, co-localization of DENV-2 E glycoprotein and the marker protein transferrin was observed at the periphery of the cytoplasm. To support the requirement of clathrin function for DENV-2 entry, overexpression of a dominant-negative mutant of Eps15 in C6/36 cells was shown to impair virus entry. The disruption of actin microfilaments by cytochalasin D also significantly affected DENV-2 replication. In contrast, microtubule disruption by colchicine treatment did not impair DENV-2 infectivity, suggesting that DENV-2 does not require transport from early to late endosomes for successful infection of mosquito cells. Furthermore, using transmission electron microscopy, DENV-2 particles of approximately 44-52 nm were found attached within electron-dense invaginations of the plasma membrane and in coated vesicles that resembled those of clathrin-coated pits and vesicles, respectively. Together, these results demonstrate for the first time that DENV-2 enters insect cells by receptor-mediated, clathrin-dependent endocytosis, requiring traffic through an acidic pH compartment for subsequent uncoating and completion of a productive infection.
authors.affiliation: Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.
authors.forename: Eliana G
authors.initials: EG
authors.lastname: Acosta
authors.affiliation: Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.
authors.forename: Viviana
authors.initials: V
authors.lastname: Castilla
authors.affiliation: Laboratorio de Virología, Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, 1428 Buenos Aires, Argentina.
authors.forename: Elsa B
authors.initials: EB
authors.lastname: Damonte
mesh_terms: D008841:Actin Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000821:virology; D000199:Actins / Q000378:metabolism; D000330:Aedes / Q000821:virology*; D000818:Animals; D001692:Biological Transport; D002522:Chlorocebus aethiops; D002966:Clathrin / Q000235:genetics / Q000502:physiology*; D015638:Cytochalasin D / Q000494:pharmacology; D003716:Dengue Virus / Q000502:physiology*; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000821:virology; D018613:Microscopy, Confocal; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism; D014168:Transferrin / Q000648:ultrastructure; D014709:Vero Cells; D014759:Viral Envelope Proteins / Q000648:ultrastructure; D053586:Virus Internalization / Q000187:drug effects
chemical_list: D000199:Actins; D002966:Clathrin; D014168:Transferrin; D014759:Viral Envelope Proteins; D015638:Cytochalasin D