Identification of a dengue 2 virus envelope protein receptor in Aedes aegypti critical for viral midgut infection

Abstract

The establishment of a productive dengue virus (DENV) infection in the midgut epithelial cells of Aedes aegypti is critical for the viral transmission cycle. The hypothesis that DENV virions interact directly with specific mosquito midgut proteins was explored. We found that DENV serotype 2 (DENV2) pretreated with trypsin interacted with a single 31 kDa protein, identified as AAEL011180 by protein mass spectrometry. This putative receptor is a highly conserved protein and has orthologs in culicine and anopheline mosquitoes. We confirmed that impairing the expression of AAEL011180 in the midgut of Ae. aegypti females abolished the interaction with DENV2, and the virus also bound to immobilized recombinant purified receptor. Furthermore, recombinant DENV2 surface E glycoprotein bound to recombinant AAEL011180 with high affinity (38.2 nM) in binding kinetic analysis using surface plasmon resonance. The gene for this DENV2 E protein receptor (EPrRec) was disrupted, but since the gene is essential in Ae. aegypti, only heterozygote knockout (ΔEPrRec^+/-) females could be recovered. Further reducing EPrRec mRNA expression in the midgut of ΔEPrRec^+/- females by systemic dsRNA injection significantly reduced the prevalence of DENV2 midgut infection. EPrRec also interacts with heat shock protein 70 cognate 3 (Hsc70-3), and silencing Hsc70-3 expression in ΔEPrRec females also reduced the prevalence of DENV2 midgut infection.

Keywords: E protein Receptor; aedes aegypti; dengue Virus; midgut; mosquito.

Fig. 1.

Characterization of DENV2 interactions with specific proteins in Ae. aegypti midgut homogenates. (A) Left image: Coomassie-stained gel (Left) of cellular membrane and cytoskeletal midgut extract (MG ext) fraction. Next three images: Binding of DENV2 (DV), DENV2 pretreated with trypsin (DV-T), and a no bait control to membrane-immobilized midgut extracts in far-western blots. Midguts were collected at 48 h postfeeding of mosquitoes on human serum and the homogenate fraction was separated by SDS/PAGE. (B) Left: 2D Coomassie-stained gel of cellular membrane and cytoskeletal midgut extract fraction. Right: Binding of DENV2 to membrane-immobilized midgut extracts in a 2D far-western blot. The protein spot excised from the SDS-gel corresponding to the spot where DV-T bound to the membrane is indicated by the red box. (C) Left: RT-qPCR quantification of the putative receptor (Rec) mRNA transcripts in midguts following intrathoracic injection of mosquitoes with dsRNA derived from the putative receptor mRNA (dsRec) or a LacZ dsRNA (nontarget) control (dsLZ). Next three images: Coomassie-stained gel of cellular membrane and cytoskeletal midgut extract fraction following dsRec injection. Effect of dsRNA silencing on DV-T binding to midgut protein extracts in a far-western blot. The membrane was stripped and then reprobed with anti-actin-HRP antibodies in a western blot using actin as a sample loading control. (D) Left: Coomassie-stained SDS/PAGE gel showing purified recombinant Rec. Center: DV-T binds to immobilized recombinant Rec in a far-western blot and (Right) in the ELISA; t test, * P < 0.05. (E) Binding kinetics of different concentrations (500, 250, 125, 62.5, 31.5, 15.6, and 7.8 nM) of recombinant Rec protein to immobilized DENV2 E protein by surface plasmon resonance (K_d = 38 nM).

Fig. 2.

Subcellular localization of EPrRec in midgut epithelial cells of Ae. aegypti. (A) EPrRec (green) localizes to the apical section of the midgut epithelium at 24 h postfeeding of mosquitoes on serum (Left) and in cell-to-cell junctions (Right). (B) Side view of epithelial cells with EPrRec (green) localized to the submicrovillar region just below actin-rich microvilli (red). (Scale bars, 10 μm.) (C) EPrRec (green) localizes to the apical cytoplasm region in DENV2 (light blue)-infected epithelial cells at 4 d postinfection of mosquitoes via an infectious blood meal. (Scale bars, 5 μm.)

Fig. 3.

Effect of EPrRec silencing on DENV2 infection of Ae. aegypti. (A) Left: RT-qPCR quantification of EPrRec mRNA in midguts 4 d post-intrathoracic injection of dsRNA targeting EPrRec (dsRec) or LacZ (dsLacZ; nontarget control), t test P < 0.01. Center: Effect of EPrRec silencing on DENV2 infection prevalence and (Right) on virus titers (intensity of infection) in midguts. Mosquitoes were offered a DENV2 containing artificial bloodmeal 4 d postinjection of dsRNA. Midguts were collected at 7 d postinfection with DENV2. Virus titers were determined by the plaque assay, and horizontal lines represent median DENV2 titers. (B) Schematic of the gene structure of AAEL011180 (pERec) and the design of the insertion construct to facilitate CRISPR/Cas9-mediated gene disruption. Using sgRNA #3, the genomic DNA would be cleaved at nucleotide position 80 of the coding sequence of AAEL011180 (located at the 5’ end of the 2. exon). LHA: left homology arm; RHA: right homology arm; 3xP3: promoter to drive eye tissue–specific gene expression of the mCherry marker; SV40: transcription terminator. (C) Genotyping of individual female ΔEPrRec mosquitoes using a multiplex PCR assay. The 375 bp band signal is indicative of an allele harboring the insertion transgene that disrupts pERec (ko), while the 130 bp band signal is indicative of the wild-type (WT) allele. Hemizygous individual (+/−); WT individuals (+/+). (D) Left: RT-qPCR quantification of EPrRec mRNA transcripts in midguts of ΔEPrRec^+/−, and WT Ae. aegypti; t test, P < 0.01. Center: Infection prevalence and (Right) virus titers in individual midguts of ΔEPrRec^+/−, and WT Ae. aegypti at 5 d postinfection of mosquitoes with DENV2. Virus titers were determined by the plaque assay, and horizontal lines represent median DENV2 titers. (E) Left: RT-qPCR quantification of EPrRec mRNA transcripts in midguts of ΔEPrRec^+/− mosquitoes following silencing via dsRNA injection targeting EPrRec (dsRec) or control LacZ (dsLacZ); t test, P < 0.001. Center: Infection prevalence and (Right) virus titers in individual midguts of ΔEPrRec^+/− and WT Ae. aegypti at 5 d postinfection of mosquitoes with DENV2. Mosquitoes had been intrathoracically injected either with dsRec to silence EPrRec or with dsLacZ as a nontarget control. Fisher’s exact test, P = 0.0003. Virus titers were determined by the plaque assay, and horizontal lines represent median DENV2 titers.

Fig. 4.

EPrRec interacts with Hsc70 to establish DENV2 infection of midguts in Ae. aegypti. (A) Recombinant EPrRec binds to immobilized recombinant Hsc70 in the ELISA. t test, P<0.01. (B) Left: Schematic representation of recombinant EPrRec binding to immobilized recombinant Hsc70 in the ELISA. Right: Binding of increasing concentrations of recombinant EPrRec to immobilized recombinant Hsc70 in the ELISA. (C) Left: RT-qPCR quantification of Hsc70 mRNA in midguts of ΔEPrRec^+/− females following intrathoracic injection of dsHsc70 RNA into ΔEPrRec^+/− females to silence Hsc70 (dsHsc70) or LacZ dsRNA as a nontarget control (dsLacZ). t test, P < 0.01. Center: Effect of Hsc70 silencing on DENV2 infection prevalence and (Right) virus titers (intensity of infection) in individual midguts of ΔEPrRec^+/− females. Fisher’s exact test, P < 0.001. Midguts were assayed at 5 d postinfection of mosquitoes with DENV2 via infectious blood meals. Virus titers were determined by the plaque assay, and horizontal lines represent median DENV2 titers.