The RNA helicase eIF4A as a novel target in insect cells to combat arboviral infections

Abstract

Arboviruses transmitted by mosquitoes cause major global health burdens, yet few vaccines or antivirals exist. Targeting host factors required for viral replication offers a promising approach. The DEAD-box RNA helicase eIF4A, a core component of the translation initiation complex eIF4F, unwinds structured 5' UTRs and is critical for many viral RNAs. We tested whether the natural products rocaglates and pateamines, potent eIF4A inhibitors in mammalian cells, also suppress arboviral replication and translation in insect vectors. Silvestrol strongly inhibited Rift Valley fever virus (RVFV) replication in human A549 cells without cytotoxicity, expanding the list of arboviruses dependent on eIF4A. Sequence analysis showed conservation of the rocaglate-binding motif between the arboviral vector Aedes aegypti and the fruit flies Anastrepha suspensa and Drosophila melanogaster. Dual-luciferase assays in insect cells of all three species confirmed silvestrol selectively inhibited translation from purine-rich reporters below cytotoxic thresholds. Purified eIF4A variants retained helicase activity, allowing direct testing of inhibitor interactions. Thermal shift assays demonstrated robust stabilization of eIF4A-RNA complexes by both compound classes in the wildtype proteins, with unexpected rocaglate sensitivity of the putatively insensitive Ae. aegypti H161L mutant, indicating a unique binding pocket geometry of the mosquito protein. Our results identify eIF4A as a conserved and druggable host factor in insects, highlighting its potential as a novel target for transmission-blocking antivirals and insect-specific inhibitors. Author SummaryNatural compounds from the classes of rocaglates and pateamines have shown their potential to inhibit tumor cell growth and to effectively suppress replication of different viruses, including Ebola, corona, picorna, and arboviruses by inhibiting translation initiation through blocking of one of the initiation actors, the helicase eIF4A. eIF4A is needed to unwind structured 5UTRs of selected mRNAs to allow binding of the 43S preinitiation complex. The compounds bind in a pocket of eIF4A, thereby clamping the RNA onto the eIF4A surface. Although eIF4A is highly conserved in eukaryotes not all eIF4A are sensitive to rocaglates. Mainly one key amino acid in the binding pocket determines sensitivity or insensitivity. In this study we expanded the list of arboviruses as potential targets of rocaglate-based drugs. Moreover, we approached the inhibition of arboviral infections from the vector side by studying the interaction between rocaglates and pateamines with eIF4A from the yellow fever mosquito Aedes aegypti, one of the most important arboviral vectors. Our study shows that also the mosquito eIF4A can be targeted by both compound classes. Moreover, our results reveal a unique architecture of the binding pocket that retains sensitivity even upon mutation of the key amino acid.