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

The establishment of a productive dengue virus (DENV) infection in the midgut epithelial cells of <i>Aedes aegypti</i> 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 <i>Ae. aegypti</i> 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 <i>Ae. aegypti,</i> 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.