Biology subjects

Saleh, M. C.

Publications and source records attributed to Saleh, M. C..

5 recordsLinked to original sources

bioRxiv2025

Macrodomain ADP-ribose binding but not ADP-ribosylhydrolase activity is critical for chikungunya virus infection of Aedes mosquitoes

Viral macrodomains are promising antiviral targets that counteract host ADP-ribosylation-mediated antiviral responses in mammals. However, their role in dual-host viruses within the mosquito vector remains largely unknown. Here, we investigated the role of the Chikungunya virus (CHIKV) nsP3 macrodomain catalytic activity by mutating the active-site asparagine 24 (N24). N24 mutant viruses rapidly selected for compensatory second-site mutations at aspartate 31 (D31) in mammalian Vero cells, while interferon-competent human cells favored reversion at position 24 instead, suggesting adaptive responses to macrodomain functional impairment. Catalytically inactive double-mutant viruses replicated poorly in interferon-competent human cells but comparably to wild-type in mosquito cells. In Aedes mosquitoes in vivo, macrodomain mutations modulated CHIKV replication and infectivity in a mutation-specific manner: one mutant reduced them while another enhanced them. Biochemical and structural analyses confirmed that these phenotypes arise in the context of a catalytically inactive macrodomain that retains ADP-ribose binding capacity, with D31 mutations lining the substrate exit path. These findings demonstrate that macrodomain catalytic activity modulates CHIKV dissemination and transmission potential in Aedes mosquitoes, revealing fundamental differences in host-dependent evolutionary advantage for a catalytically inactive viral enzyme.

molecular biology↗

bioRxiv2024

Limited impact of the siRNA pathway on transposable element expression in Aedes aegypti

Transposable elements (TEs) are DNA sequences that can change their position within a genome. In the germline of arthropods, post-transcriptional regulation of TE expression is mainly mediated by the Piwi-interacting RNA (piRNA) pathway. piRNAs are small RNAs of 24-30 nucleotides (nt) in length produced from genomic precursor transcripts as well as through a ping-pong amplification cycle. In somatic tissues, certain insects, such as Drosophila, instead rely on the small interfering RNA (siRNA) pathway as a key regulator of TE expression. siRNAs are 21nt small RNAs produced from double-stranded RNA by the endonuclease Dicer2, which guides an RNA-induced silencing complex to degrade a complementary RNA. However, whether the siRNA pathway also regulates TE expression in the mosquito Aedes aegypti, a medically significant vector species with abundant somatic piRNAs, is unknown. To address this question, we investigated the expression of TEs and small RNAs in both somatic and gonadal tissues of a Dicer2 mutant line of Ae. aegypti and its wild-type counterpart. Our results show a modified pattern of TE expression and a decrease in TE-derived 21nt small RNAs in the Dicer2 mutant, but no major shift of TE transcript abundance. The lack of a functional siRNA pathway also causes perturbations in piRNA ping-pong signatures and the expression of certain piRNA-associated genes, but without clear evidence for compensation by increased piRNA pathway activity. We conclude that the mosquito Ae. aegypti produces siRNAs targeting TEs but these lack a critical role in the regulation of TE expression both in somatic and in gonadal tissues.

genomics↗

bioRxiv2023

Challenges in the Biotechnological Implementation of Oral RNA Interference as an Antiviral Strategy in Aedes aegypti

Mosquitoes, particularly Aedes aegypti, are critical vectors for globally significant pathogenic viruses. This study examines the limitations of oral RNA interference (RNAi) as a strategy to disrupt viral transmission by Ae. aegypti. We hypothesized that double-stranded RNA (dsRNA) targeting the Zika virus (ZIKV) or chikungunya virus (CHIKV) genomes produced by engineered bacterial symbionts could trigger an antiviral response. Mosquitoes mono-colonized with Escherichia coli producing dsZIK or dsCHIK did not display reduced viral titers following exposure to virus-contaminated bloodmeals and failed to generate dsZIK- or dsCHIK-derived small interfering RNAs. To address potential limitations of bacterial dsRNA release, we explored dsRNA inoculation via feeding and injection. While viral replication was impeded in mosquitoes injected with dsZIK or dsCHIK, no antiviral effect was observed in dsRNA-fed mosquitoes. These findings highlight complexities of implementing oral RNAi as an antiviral strategy in Ae. aegypti and warrant further exploration of local and systemic RNAi mechanisms.

microbiology↗

bioRxiv2022

Decoding rRNA Sequences for Improved Metagenomics in Sylvatic Mosquito Species

As mosquito-borne virus epidemics are often preceded by undetected spillover events, surveillance and virus discovery studies in non-urban mosquitoes informs pre-emptive and responsive public health measures. RNA-seq metagenomics is a popular methodology but it is constrained by overabundant rRNA. The lack of reference sequences for most mosquito species is a major impediment against physical and computational removal of rRNA reads. We describe a strategy to assemble novel rRNA sequences from mosquito specimens, producing an unprecedented dataset of 234 full-length 28S and 18S rRNA sequences of 33 medically important species from countries with known histories of mosquito-borne virus circulation (Cambodia, the Central African Republic, Madagascar, and French Guiana). We also evaluate the utility of rRNA sequences as molecular barcodes relative to the mitochondrial cytochrome c oxidase I (COI) gene. We show that rRNA sequences can be used for species identification when COI sequences are ambiguous or unavailable, revealing evolutionary relationships concordant with contemporary mosquito systematics. This expansion of the rRNA reference library improves mosquito RNA-seq metagenomics by permitting the optimization of species-specific rRNA depletion protocols for a broader species range and streamlined species identification by rRNA barcoding. In addition, rRNA barcodes could serve as an additional tool for mosquito taxonomy and phylogeny.

bioinformatics↗

bioRxiv2019

Tudor-SN promotes early replication of dengue virus in the Aedes aegypti midgut

Diseases caused by mosquito-borne viruses have been on the rise for the last decades, despite the implementation of vector control methods primarily based on insecticides. An alternative control method currently in development is the use of lab-engineered mosquitoes that are incapable to carry viruses. This has stimulated efforts to identify optimal target genes that are naturally involved in mosquito antiviral defenses or required for viral replication. Although several antiviral immune pathways such as RNA interference (RNAi) have been previously characterized in mosquitoes, the genes that prevent or promote early viral replication in the midgut remain elusive. Here, we investigated the role of a member of the Tudor protein family, Tudor-SN, upon dengue virus infection in the mosquito Aedes aegypti. Tudor-SN expression was upregulated early after dengue virus infection and was subsequently positively correlated with viral loads in the midgut. Using RNAi-mediated knockdown, we showed that the loss of Tudor-SN reduced dengue virus replication in the Ae. aegypti derived cell line Aag2 and in the midgut of Ae. aegypti females in vivo. Using immunofluorescence assays, we found that Tudor-SN localizes to the nucleolus in both Ae. aegypti and Aedes albopictus cells. Finally, we used a reporter assay to demonstrate that Tudor-SN was not required for RNAi function in vivo. Collectively, these results define a novel proviral role for Tudor-SN upon early dengue virus infection of the Ae. aegypti midgut.

microbiology↗