Biology subjects

Haecker, I.

Publications and source records attributed to Haecker, I..

4 recordsLinked to original sources

bioRxiv2025

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

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.

molecular biology↗

bioRxiv2025

Mosquito Sex Separation using Complementation of Selectable Traits and Engineered Neo-Sex Chromosomes

Effective and scalable sex separation remains a critical challenge for mosquito genetic control strategies. Genetic sexing strains (GSS) address this by genetically linking maleness with selectable traits, enabling efficient removal of females before release. Here, we describe a robust platform for the development of GSSs in the invasive Aedes albopictus mosquito by integrating a CRISPR-engineered selectable phenotype with sex conversion via nix, the male-determining factor. As a proof-of-concept, we disrupt the yellow gene to generate a vivid pigmentation marker, then rescue its function in males using nix-containing transgenes, creating a stable strain where all females are yellow and all engineered males are dark. The resulting GSS males are fertile, robust, and despite lacking the ancestral M locus, exhibit gene expression profiles closely resembling wild-type males. We benchmark sex separation based on pigmentation and discover that yellow mutant females exhibit slower larval development, enhancing protandry-based sorting. The GSS strain is compatible with existing size-based sex sorting systems, allowing for improved separation accuracy through the integration of natural and engineered sexually dimorphic traits. Additionally, we find that GSS females lay desiccation-sensitive eggs, reducing the risk of accidental female releases. Our approach is the first to engineer a sex-linked selectable trait by precisely targeting an endogenous gene and restoring its function in males, establishing a versatile platform for GSS development in Aedes mosquitoes.

synthetic biology↗

bioRxiv2020

White pupae genes in the Tephritids Ceratitis capitata, Bactrocera dorsalis and Zeugodacus cucurbitae: a story of parallel mutations

Mass releases of sterilized male insects, in the frame of sterile insect technique programs, have helped suppress insect pest populations since the 1950s. In the major horticultural pests Bactrocera dorsalis, Ceratitis capitata, and Zeugodacus cucurbitae, a key phenotype white pupae (wp) has been used for decades to selectively remove females before releases, yet the gene responsible remained unknown. Here we use classical and modern genetic approaches to identify and functionally characterize causal wp− mutations in these distantly related fruit fly species. We find that the wp phenotype is produced by parallel mutations in a single, conserved gene. CRISPR/Cas9-mediated knockout of the wp gene leads to the rapid generation of novel white pupae strains in C. capitata and B. tryoni. The conserved phenotype and independent nature of the wp− mutations suggest that this technique can provide a generic approach to produce sexing strains in other major medical and agricultural insect pests.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genetics↗

bioRxiv2018

Highly efficient homology-directed repair using Cas9 protein in Ceratitis capitata

BackgroundThe Mediterranean fruit fly Ceratitis capitata is a highly polyphagous and invasive insect pest, causing vast economical damage in horticultural systems. A currently used control strategy is the sterile insect technique (SIT) that reduces pest populations through infertile matings with mass-released, sterilized insects. Transgenic approaches hold great promise to improve key aspects of a successful SIT program. However, there is strict or even prohibitive legislation regarding the release of genetically modified organisms (GMO), while novel CRISPR-Cas technologies might allow to develop genetically enhanced strains for SIT programs classified as non-transgenic.\n\nResultsHere we describe highly efficient homology-directed repair genome editing in C. capitata by injecting pre-assembled CRISPR-Cas9 ribonucleoprotein complexes using different guide RNAs and a short single-stranded oligodeoxynucleotide donor to convert an enhanced green fluorescent protein in C. capitata into a blue fluorescent protein. Six out of seven fertile and individually backcrossed G0 individuals generated 57-90% knock-in rate within their total offspring and 70-96% knock-in rate within their phenotypically mutant offspring.\n\nConclusionConsidering the possibility that CRISPR-induced alterations in organisms could be classified as a non-GMO in the US and Europe, our approach to homology-directed repair genome editing can be used to genetically improve strains for pest control systems like SIT without the need to struggle with GMO directives. Furthermore, it can be used to recreate and use mutations, found in classical mutagenesis screens, for pest control systems.

genetics↗