Complement-Related Proteins Control the Flavivirus Infection of Aedes aegypti by Inducing Antimicrobial Peptides
Figure 5
AaMCR and AaSR-C function in a pathway that opposes DENV-2 infection.
(A) The interaction between 3 AaMCR fragments and AaSR-C in co-IP assays. Three AaMCR gene fragments were cloned into the pMT/BiP/V5-His A vector. The recombinant plasmids were transiently transfected into S2 cells. The cell supernatant was used for investigation of the AaMCR/AaSR-C interaction. The protein complex was pulled down with a rabbit anti-V5 antibody and probed using a mouse anti-HA antibody. The experiment was reproduced 3 times. (B) Expression and purification of AaMCR-a in Drosophila S2 cells. The purified AaMCR-a was separated through SDS-PAGE (Left Panel) and detected with an anti-HA antibody via western blotting (Right Panel). The supernatant from empty vector-transfected S2 cells was used as the mock control. (C) AaSR-C-Ex acted as an adaptor in the interaction between the AaMCR-a and DENV-2 E proteins. The purified AaSR-C-Ex, AaMCR-a and DENV-2 E proteins were mixed and pulled down with a mouse anti-HA antibody (AaMCR-a) and detected using a rabbit anti-V5 antibody (AaSR-C) and anti-FLAG-HRP antibody (DENV-2 E). The experiment was repeated 3 times with similar results. (D) AaSR-C-Ex connected AaMCR-a to DENV-2 virions. Purified AaMCR-a or BSA was pre-coated into the ELISA plate wells. DENV-2 virions either mixed with AaSR-C-Ex or without AaSR-C-Ex were added to the protein-coated wells. The signal was detected using the flavivirus E mAb 4G2. The data are expressed as the mean ± standard error. The experiment was reproduced by 3 times with similar results. (E) Double knockdown of AaMCR and AaSR-C showed similar effects on DENV-2 infection to individual knockdown. Both AaSR-C (i) and AaMCR (ii) were knocked down using a dsRNA mixture in the AaSR-C/AaMCR co-silenced group. DENV-2 replication and the numbers of infectious DENV-2 virions in the mosquitoes were measured via qPCR (iii) and plaque assays (iv). Statistical analysis was performed using the non-parametric Mann-Whitney test. The data on gene silencing (i, ii) and from plaque assays (iv) are expressed as the mean ± standard error. The horizontal line depicts the median (iii). Each dot represents an individual mosquito. The result was representative of 3 independent experiments. (F) Immunostaining of AaMCR, AaSR-C and DENV-2 in A. aegypti hemocytes. The hemocytes were dissected from uninfected mosquitoes, AaMCR and/or AaSR-C silenced infected mosquitoes and GFP dsRNA treated infected mosquitoes at 6 days post-infection. AaSR-C was stained with anti-rabbit IgG Alexa-488 (Green); AaMCR was probed using anti-mouse IgG Alexa-546 (Red); the DENV-2 E protein was probed with DENV-2 human antiserum (purified IgG) and anti-human IgG Alexa-633 (Blue). Images were examined using a Zeiss LSM 780 meta confocal 63×objective lens.