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

<div><p>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 <em>Aedes</em> 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.</p> </div

Colocalization of CHIKV with early and late endocytic vesicular markers within C6/36 cells.

<p>(a) TEM analyses reveal that CHIKV virus particles are contained within the endocytic vesicle (black arrows). Scale bar represents 200 nm. (b) Anti-EEA1 antibody (red) is used to stain the early endosomes at 10 mins p.i. Most of the virus-containing endosomes were distributed closer to the cell periphery. (c) Lysotracker (red) is used to stain late endosomes and lysosomes at 15 mins p.i. CHIKV particles are found mainly in the vesicles (green), suggesting that endocytosed CHIKV particles have been trafficked to the late endosomes and lysosomes.</p

Effects of low endosomal pH inhibitors on the entry of CHIKV into C6/36 cells.

<p>C6/36 cells were pre-treated with different drug inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. Low endosomal pH inhibitors show dose-dependent inhibition of CHIKV entry into (a) bafilomycin A-, (b) concanamycin A-treated cells, infected with CHIKV Singapore/07/2008 strain, (c) concanamycin A-treated infected with CHIKV SGEHICHD122508 strain and (d) concanamycin A-treated cells infected with CHIKV SGEHIDSD67Y2008 strain. The log virus titre is plotted against the concentrations of drug used. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates *<i>p</i> values<0.05, **<i>p</i> values of <0.01 and ***<i>p</i> values<0.0001 by Student's <i>t</i> test. Asterisks indicate statistically significant results relative to control group (▪).</p

siRNA-based knockdown on cellular genes of CLTC, RAB5, RAB7 and vacuolar-ATPase B.

<p>Scrambled siRNAs (represented by striped bars) against (a) CLTC, (b) RAB5, (c) RAB7 and (d) vacuolar ATPase B were transfected into C6/36 cells across various concentrations (0–10 nM) and subjected to CHIKV infection. No virus inhibition is observed for all tested scrambled siRNAs when compared to non-transfected cells (TC). Gene-specific siRNAs (represented by solid bars) against (a) CLTC, (b) RAB5, (c) RAB7 and (d) vacuolar ATPase B are transfected into C6/36 cells at different concentrations (0–10 nM) and subjected to CHIKV infection. Significant dose-dependent inhibition of CHIKV infection are observed from 5 nM to 10 nM, with approximately 2-log reductions seen across all genes tested. The asterisk indicates *<i>p</i> values<0.05, **<i>p</i> values of <0.01 and ***<i>p</i> values<0.0001 by Student's <i>t</i> test. Asterisks indicate statistically significant results relative to control group (▪).</p

Heatmap displaying differential regulation of genes related to clathrin-mediated endocytotic pathway.

<p>Fold changes upon CHIKV infection, relative to mock-infected samples, are as depicted by the colour key: gene upregulation is denoted in red while gene downregulation is denoted in blue.</p

Effects of clathrin-mediated endocytic inhibitors on the entry of CHIKV into C6/36 cells.

<p>C6/36 cells were pre-treated with different inhibitors for 3 hours before CHIKV infection. Supernatants were harvested 24 hours p.i for viral plaque assays. The log virus titre is plotted against the concentrations of drug used. Dose-dependent inhibition of CHIKV entry into (a) monodansylcadaverine-, (b) chlorpromazine- and (c) dynasore-treated cells is observed. In contrast, minimal inhibition of CHIKV infectious entry into (d) filipin- and (e) nystatin-treated cells is noted. Cholesterol-dependent endocytosis of CHIKV into C6/36 cells is further analysed. Dose-dependent inhibition of CHIKV infection is observed with (f) methyl-β cyclodextrin treatment of C6/36 cells. Furthermore, minimal inhibition on the infectious entry of CHIKV into (g) EIPA-treated C6/36 cells is observed. Cell viability upon drug treatments is represented by the line graphs. The asterisk indicates *<i>p</i> values<0.05, **<i>p</i> values of <0.01 and ***<i>p</i> values<0.0001 by Student's <i>t</i> test using GraphPad Prism version 5.00 for Windows, GraphPad Software. Asterisks indicate statistically significant results relative to control group (▪).</p

Bio-imaging analysis of CHIKV entry process into C6/36 cells using electron microscope (a and b) and immunofluorescene labeling.

<p>(a) CHIKV particles are negatively-stained and observed to be approximately 60–70 nm in size. (b) Attachment of CHIKV particles at the plasma membrane of C6/36 cells and uptake of CHIKV particles (arrow) by coated pits (arrowheads) (scale bar represent 200 nm). CHIKV viral particles (green) (c and d) are seen to co-localize with clathrin molecules (red) at 5 and 10 mins p.i. (arrows). Cell nuclei are stained with DAPI (blue).</p