Search

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. Author Summary: Deciphering the much neglected aspects of cellular factors in contributing to the infectious entry of CHIKV into mosquito cells may enhance our understanding of the conservation or diversity of these host factors amongst mammalian and arthropod for successful CHIKV replication. The study revealed that the infectious entry of chikungunya virus (CHIKV) into mosquito cells is mediated by the clathrin-dependent endocytic pathway. A customized gene expression microarray known to target the Aedes mosquito genome was used to identify host genes that are differentially regulated upon CHIKV infection. A combination of bio-imaging studies and pharmacological inhibitors confirmed the involvement of clathrin-mediated endocytosis as well as the importance of low endosomal pH during CHIKV infectious entry. Furthermore, the clathrin heavy chain, Eps15, RAB5, RAB7 and vacuolar ATPase B are shown to be essential for the infectious entry process of CHIKV. This study aims to underline the importance of cellular factors, particularly those associated with clathrin-dependent endocytosis, in mediating the infectious entry of CHIKV into mosquito cells. [ABSTRACT FROM AUTHOR]