Endocytic pathway followed by dengue virus to infect the mosquito cell line C6/36 HT.
The endocytic pathway followed by dengue virus to infect the mosquito cells C6/36 HT was analyzed. Using DIL-labeled virions and real-time imaging it was determined that viral entry into C6/36 HT takes approximately 5 to 7 min. Pretreatment of C6/36 HT cells with sucrose and bafilomycin A, but not filipin, inhibited dengue virus infection up to 80%. Furthermore, the overexpression of dominant-negative mutants of Eps15, a molecule required for the formation of clathrin-coated vesicles, reduced dengue infection up to 50%, indicating that dengue virus entry is through clathrin-mediated endocytosis and is pH-dependent. By double-immunofluorescence assays, DIL-labeled particles were colocalized with early endosomes at 5 min and with lysosomes mainly at 30 min post-infection. Finally, disruption of the microtubule and microfilaments by nocodazole and by cytochalasin D reduced viral infection by more than 80%. Taken together these results indicate that dengue virions enter into C6/36 HT cells by clathrin-mediated endocytosis, using the endosomal pathway from early endosomes to acidic lysosomes before viral RNA is released into the cytoplasm.
Mosso
Clemente
C
Departamento de Infectómica y Patogénesis Molecular, Centro de Investigación y de Estudios Avanzados del IPN, México City 07360, Mexico.
Galván-Mendoza
Ivan J
IJ
Ludert
Juan Ernesto
JE
del Angel
Rosa M
RM
D000330:Aedes / Q000145:classification / Q000166:cytology* / Q000821:virology*; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D006224:Cricetinae; D003716:Dengue Virus / Q000235:genetics / Q000378:metabolism / Q000472:pathogenicity*; D004705:Endocytosis / Q000502:physiology*; D011992:Endosomes / Q000821:virology; D005455:Fluorescent Antibody Technique; D008247:Lysosomes / Q000821:virology; D018613:Microscopy, Confocal; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000378:metabolism; D014771:Virion / Q000472:pathogenicity; D053586:Virus Internalization
D002966:Clathrin; D017361:Viral Nonstructural Proteins