Fig 1.
Application of RNA-affinity chromatography to identify Aedes aegypti proteins that interact with DENV2 3’UTR.
(A) Schematic of the RNA-affinity chromatography coupled with quantitative mass spectrometry (MS). (B) Identification of the DENV2 3’UTR-bound proteins. Of the 385 proteins detected, 14 interacted more than 1.5 fold (p<0.1) with DENV2 3’UTR, as compared to the control RNA. Interacting proteins are shown in red and their names are indicated with an arrow.
Table 1.
List of A. aegypti proteins that interact with DENV2 3’UTR and their corresponding human homologs.
Fig 2.
Binding affinities of AePur and AeStaufen to DENV2 3’UTR and sfRNA.
(A) Western blots of AePur immunoprecipitates from orally-infected mosquito lysates. (B) gRNA and sfRNA fold enrichments in AePur immunoprecipitates as compared to IgG control. (C) Western blots of AeStaufen-V5 (AeStau-V5) immunoprecipitates from infected C6/36 cell lysates. (D) gRNA and sfRNA fold enrichments in AeStaufen immunoprecipitates as compared to IgG control. (A, C) Three biological repeats (R1-3) are shown. (B, D) Bars show mean ± s.e.m. from three repeats. **, p-value < 0.01; ***, p-value < 0.001 as determined by unpaired T-test.
Fig 3.
Effects of the 3’UTR-bound proteins on DENV infection in mosquitoes.
Four days post-dsRNA injection to silence each of the 14 3’UTR-bound proteins, mosquitoes were orally infected with 106 pfu/ml of DENV2. Infection was quantified 7 days later using focus forming unit (FFU) assay. (A) Infection rate. Bars show percentage ± s.e. *, p-value < 0.05 as determined by Z-test. (B) FFU per infected mosquito. Bars show geometric means ± 95% C.I. *, p-value < 0.05; ***, p-value < 0.001 as determined by Dunnett’s test compared to control mosquitoes injected with dsRNA control. N, number of orally-infected mosquitoes. Data from three biological repeats, each using a specific set of dsRNA targets, were combined. DsRNA control was included in each repeat.
Fig 4.
Human PurA and PurB interactions with DENV RNA and effect on infection in human cells.
(A) Western blots of FLAG-tagged HsPurA and HA-tagged HsPurB immunoprecipitates from Huh7 cells 24 hpi with DENV2 at a MOI of 5. (B) gRNA and sfRNA fold enrichments in FLAG-tagged HsPurA and HA-tagged HsPurB immunoprecipitates as compared to IgG control. Bars show mean ± s.e.m. from two repeats. (C) Western blots of HsPurA and HsPurB from cells silenced for either of the HsPUR proteins. Three different siRNAs were used for each protein. The picture is representative of multiple repeats. GAPDH was used as a loading control. (D) Effect of HsPURA or HsPURB silencing on viral load estimated by plaque forming unit (PFU) quantities in supernatants at 24 hpi with a MOI of 0.1. Bars indicate means ± s.e.m. (E) Western blots of HsPurA and HsPurB from cells, where both proteins were genetically-depleted. Four different double knock-out lines (PURA/B KO) for each protein were tested. The picture is representative of multiple repeats. GAPDH was used as a loading control. (F) Effect of HsPurA and HsPurB genetic ablation on viral loads estimated by PFU quantities in supernatants at 24 and 48hpi, with a MOI of 0.1. Bars indicate means ± s.e.m. NT, non-transfected control; siNC, transfected with control siRNA; WT, wild-type.
Fig 5.
Effect of AeStaufen on gRNA and sfRNA copies, and viral titers in different mosquito tissues.
(A) AeStaufen relative expression in whole mosquitoes, carcass (what is left after dissection), midgut, and salivary glands at 10 days post non-infectious (Ni) or infectious blood-feeding (DENV). Bars represent means ± s.e.m. from three replicates of pools of ten tissues. (B-D) Effect of AeStaufen silencing (dsAeStau) on gRNA copies (B), sfRNA copies (C) and sfRNA:gRNA ratio (D) in the carcass, midgut and salivary glands at 10 days post-oral infection. (E-F) Effect AeStaufen silencing (dsAeStau) on viral titers as determined by focus forming unit (FFU) assay in salivary glands (E) and saliva (F) at 10 days post-oral infection. (B-F) dsLacZ was injected as control. (B, C, E, F) Bars indicate geometric means ± 95% C.I. (D) Bars indicate means ± s.e.m. N, number of mosquitoes analyzed. *, p-value < 0.05; **, p-value < 0.01; ***, p-value < 0.001 as determined by unpaired t-test or by LSD-Fisher test (A).
Fig 6.
Effect of AeStaufen on sfRNA secretion in the saliva.
(A-B) Effect of AeStaufen silencing (dsAeStau) on gRNA copies (A) and sfRNA:gRNA ratio (B) in saliva collected at 10 days post-oral infection. (C-D) Effect of AeStaufen silencing (dsAeStau) on gRNA copies (C) and sfRNA:gRNA ratio (D) in the salivary glands at 7 days post-inoculation. dsLacZ was injected as control. (A, C) Bars indicate geometric means ± 95% C.I. (B, D) Bars indicate means ± s.e.m. N, number of mosquitoes analyzed. *, p-value < 0.05; **, p-value < 0.01 as determined by unpaired t-test.