Mosquito Cellular Factors and Functions in Mediating the Infectious entry of Chikungunya Virus

Abstract

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.

Dynamics of Chikungunya Virus Cell Entry Unraveled by Single-Virus Tracking in Living Cells

“…Our results are in line with an earlier study of Bernard et al, who showed that CHIKV infection of HEK-293T cells is dependent on the integrity of early endosomes, but not on late endosomes (28). In mosquito cells, both early and late endosomes are required for CHIKV infection, suggesting that in these cells, CHIKV is trafficked to maturing/late endosomes before fusion (32). This discrepancy may be related to the different virus strains used but could also be related to potential differences in the endosomal pH between cells (64).…”

“…The observation that CHIKV enters cells via CME is in line with two earlier reports on CHIKV cell entry (31,32). In these studies, significant inhibition of viral infectivity was seen in cells treated with drugs or siRNAs that have been proposed to interfere with CME.…”

“…One study showed that small interfering RNAs (siRNAs) against the clathrin heavy chain did not interfere with CHIKV infection in HEK239T cells (28), suggesting that CHIKV infects cells via a clathrin-independent pathway. However, siRNAs against clathrin did inhibit CHIKV infection of U-2 OS cells, primary human umbilical vein endothelial cells (31), and the mosquito cell line C6/36 (32). The majority of the results thus suggest that CHIKV cell entry occurs mainly via CME.…”

“…For example, Bernard et al found that integrity of Rab7-positive endosomes is not required for CHIKV infection of HEK293T cells (28), suggesting that CHIKV fuses from within early endosomes. In the mosquito cell line C6/36, however, CHIKV infection was dependent on both Rab5-and Rab7-positive endosomes (32), indicating that fusion might also occur from Rab5/Rab7-positive maturing endosomes and Rab7-positive late endosomes. Furthermore, and in line with the findings for other alphaviruses, low-pH-dependent CHIKV fusion is strongly promoted by target membrane cholesterol and sphingomyelin (33,34).…”

Chikungunya and Its Interaction With the Host Cell

“…A detailed entry mechanism description is not available so far. Some authors implicate a clathrin-dependent pathway [64,66], whereas other authors have proposed that CHIKV enters by a clathrin-independent, but Eps15-dependent pathway [65]. These later authors also have found evidence that multiple pathways maybe employed by CHIKV to enter into cells, as already proposed with dengue virus (DENV) [67,68].…”