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Design of antiviral AGO2-dependent short hairpin RNAs

ABSTRACT: The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interference RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibit significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA effector and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based algorithm with high predictive power. Using this algorithm, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and developed them into agshRNAs according to the rule of agshRNA design. These agshRNAs inhibited viral replication in infected cells and their efficiencies of antiviral effects displayed a consistent trend with the score ranking of siRNA sequences predicted by the algorithm. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RISC. We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develop a novel approach that combines machine learning-based algorithm and agshRNA design to custom design antiviral agshRNAs with high efficiency.

ORGANISM(S): Homo sapiens

PROVIDER: GSE261262 | GEO | 2024/09/18

REPOSITORIES: GEO

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Project description:In Drosophila, the siRNA pathway is initiated when exogenous or endogenous double stranded RNA (dsRNA) is processed into siRNAs by Dicer-2 (Dcr-2) and a dsRNA-binding protein (dsRBP) cofactor called Loquacious (Loqs). The siRNAs are then loaded onto Argonaute-2 (Ago2) protein by the action of Dcr-2 with another dsRBP cofactor called R2D2. Loaded Ago2 executes the destruction of target RNAs that have sequence complementarity to the siRNA. Dcr-2, R2D2, and Ago2 have also been shown to be required for innate antiviral defense in Drosophila. However, the biogenesis of virus-derived siRNAs (vsiRNAs) and their targets in virus-infected cells remain unclear. Here, we analyzed the antiviral response in Drosophila by monitoring the replication of different RNA viruses and deep sequencing of small RNAs in infected animals. We show that vsiRNAs are generated by Dcr-2 processing of dsRNA formed during viral genome replication and transcription. These vsiRNAs then directly target viral transcripts but not genomes, to inhibit viral replication. The biogenesis of vsiRNAs was virtually independent of Loqs and R2D2. R2D2, however, was essential for sorting and loading of vsiRNAs onto Ago2 and effective silencing of viral RNA expression. Loqs was completely dispensable for silencing of viruses in contrast to its role in silencing of endogenous targets. Our results suggest the existence of a specific siRNA pathway triggered by viral infection independent of conserved dsRBP cofactors and separate from the endogenous pathway. Inhibition of virus replication resulting from direct injection of viral RNA into Drosophila embryos was also not dependent on Loqs, suggesting the distinction of the two pathways is not related to the mode of entry but recognition of intrinsic features of viral RNA or its mode of replication. We speculate that this unique framework might be necessary for a prompt and efficient antiviral response We analyzed the small RNA reponse to viral infection by deep sequencing of small RNA libraries from wild type and RNAi mutant adult flies infected with Sindbis birus and Vesicular Stomatatis virus.

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Design of antiviral AGO2-dependent short hairpin RNAs

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