Chikungunya virus (CHIKV) is an arthropod-borne virus responsible for recent epidemics in the Asia Pacific regions. A customized gene expression microarray of 18,760 transcripts known to target Aedes mosquito genome was used to identify host genes that are differentially regulated during the infectious entry process of CHIKV
infection on C6/36 mosquito cells. Several genes such as
epsin I (EPN1),
epidermal growth factor receptor pathway substrate 15 (EPS15) and Huntingtin interacting
protein I (HIP1) were identified to be differentially expressed during CHIKV
infection and known to be involved in
clathrin-mediated endocytosis (CME). Transmission electron microscopy analyses further revealed the presence of CHIKV particles within invaginations of the plasma membrane, resembling
clathrin-coated pits. Characterization of vesicles involved in the endocytic trafficking processes of CHIKV revealed the translocation of the virus particles to the early endosomes and subsequently to the late endosomes and lysosomes. Treatment with receptor-mediated endocytosis inhibitor,
monodansylcadaverine and
clathrin-associated drug inhibitors,
chlorpromazine and
dynasore inhibited CHIKV entry, whereas no inhibition was observed with
caveolin-related drug inhibitors. Inhibition of CHIKV entry upon treatment with low-endosomal pH inhibitors indicated that low pH is essential for viral entry processes. CHIKV entry by
clathrin-mediated endocytosis was validated via overexpression of a dominant-negative mutant of Eps15, in which infectious entry was reduced, while
siRNA-based knockdown of genes associated with CME, low endosomal pH and RAB trafficking
proteins exhibited significant levels of CHIKV inhibition. This study revealed, for the first time, that the infectious entry of CHIKV into mosquito cells is mediated by the
clathrin-dependent endocytic pathway.