Endocytosis of chikungunya virus into mammalian cells: role of clathrin and early endosomal compartments.
BACKGROUND
The replicative cycle of chikungunya virus (CHIKV), an alphavirus that recently re-emerged in India and in Indian Ocean area, remains mostly unknown. The aim of the present study was to investigate the intracellular trafficking pathway(s) hijacked by CHIKV to enter mammalian cells.


RESULTS
Entry pathways were investigated using a variety of pharmacological inhibitors or overexpression of dominant negative forms of proteins perturbating cellular endocytosis. We found that CHIKV infection of HEK293T mammalian cells is independent of clathrin heavy chain and- dependent of functional Eps15, and requires integrity of Rab5-, but not Rab7-positive endosomal compartment. Cytoskeleton integrity is crucial as cytochalasin D and nocodazole significantly reduced infection of the cells. Finally, both methyl beta-cyclodextrin and lysomotropic agents impaired CHIKV infection, supporting that a cholesterol-, pH-dependent step is required to achieve productive infection. Interestingly, differential sensitivity to lysomotropic agents was observed between the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in Reunion Island.


CONCLUSIONS
Together our data indicate that CHIKV entry in its target cells is essentially mediated by clathrin-independent, Eps15-dependent endocytosis. Despite that this property is shared by the prototypal 37997 African strain of CHIKV and the LR-OPY1 virus isolated from the recent outbreak in La Réunion Island, differential sensitivity to lysomotropic agents may support that the LR-OPY1 strain has acquired specific entry mechanisms.
Bernard
Eric
E
Centre d'études d'agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS-UMR5236, Université Montpellier 1,2, Montpellier, France.
Solignat
Maxime
M
Gay
Bernard
B
Chazal
Nathalie
N
Higgs
Stephen
S
Devaux
Christian
C
Briant
Laurence
L
D002460:Cell Line; D002646:Chikungunya virus / Q000378:metabolism*; D002784:Cholesterol; D002966:Clathrin / Q000378:metabolism*; D003599:Cytoskeleton / Q000235:genetics / Q000378:metabolism; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology*; D011992:Endosomes / Q000378:metabolism*; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D018613:Microscopy, Confocal; D034622:RNA Interference; D047392:beta-Cyclodextrins / Q000494:pharmacology; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D020696:rab5 GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism; D000090802:rab7 GTP-Binding Proteins
D002966:Clathrin; D047392:beta-Cyclodextrins; C108732:methyl-beta-cyclodextrin; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D002784:Cholesterol; D020691:rab GTP-Binding Proteins; D020696:rab5 GTP-Binding Proteins