An Aedes aegypti-derived Ago2 knockout cell line to investigate arbovirus infections

Scherer, C. et al. (2021) An Aedes aegypti-derived Ago2 knockout cell line to investigate arbovirus infections. Viruses, 13(6), 1066. (doi: 10.3390/v13061066) (PMID:34205194) (PMCID:PMC8227176)

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Mosquitoes are known as important vectors of many arthropod-borne (arbo)viruses causing disease in humans. These include dengue (DENV) and Zika (ZIKV) viruses. The exogenous small interfering (si)RNA (exo-siRNA) pathway is believed to be the main antiviral defense in arthropods, including mosquitoes. During infection, double-stranded RNAs that form during viral replication and infection are cleaved by the enzyme Dicer 2 (Dcr2) into virus-specific 21 nt vsiRNAs, which are subsequently loaded into Argonaute 2 (Ago2). Ago2 then targets and subsequently cleaves complementary RNA sequences, resulting in degradation of the target viral RNA. Although various studies using silencing approaches have supported the antiviral activity of the exo-siRNA pathway in mosquitoes, and despite strong similarities between the siRNA pathway in the Drosophila melanogaster model and mosquitoes, important questions remain unanswered. The antiviral activity of Ago2 against different arboviruses has been previously demonstrated. However, silencing of Ago2 had no effect on ZIKV replication, whereas Dcr2 knockout enhanced its replication. These findings raise the question as to the role of Ago2 and Dcr2 in the control of arboviruses from different viral families in mosquitoes. Using a newly established Ago2 knockout cell line, alongside the previously reported Dcr2 knockout cell line, we investigated the impact these proteins have on the modulation of different arboviral infections. Infection of Ago2 knockout cell line with alpha- and bunyaviruses resulted in an increase of viral replication, but not in the case of ZIKV. Analysis of small RNA sequencing data in the Ago2 knockout cells revealed a lack of methylated siRNAs from different sources, such as acute and persistently infecting viruses-, TE- and transcriptome-derived RNAs. The results confirmed the importance of the exo-siRNA pathway in the defense against arboviruses, but highlights variability in its response to different viruses and the impact the siRNA pathway proteins have in controlling viral replication. Moreover, this established Ago2 knockout cell line can be used for functional Ago2 studies, as well as research on the interplay between the RNAi pathways.

Item Type:Articles
Additional Information:This research was supported by the European Union’s Horizon 2020 research and innovation program (, accessed on 19 May 2021) under ZIKAlliance grant agreementNo 734548 (AK, ES), the UK Medical Research Council (MC_UU_12014/8) (AK) and (MR/R010315/1) (KM), and the Wellcome Trust (096062) (KM). KM receives salary support related to this work from UK Research and Innovation (UKRI) through Biotechnology and Biological Sciences Research Council (BBSRC) grants BBS/E/I/00007032 and BBS/E/I/00007033.
Glasgow Author(s) Enlighten ID:Vattipally, Dr Sreenu and Knowles, Mr Jack and Schnettler, Dr Esther and Varjak, Dr Margus and Kohl, Professor Alain
Creator Roles:
Knowles, J.Investigation
Vattipally, S.Software, Data curation
Varjak, M.Resources
Kohl, A.Conceptualization, Methodology, Writing – review and editing, Supervision, Project administration, Funding acquisition
Schnettler, E.Conceptualization, Methodology, Validation, Investigation, Resources, Writing – review and editing, Supervision, Project administration, Funding acquisition
Authors: Scherer, C., Knowles, J., Sreenu, V. B., Fredericks, A. C., Fuss, J., Maringer, K., Fernandez-Sesma, A., Merits, A., Varjak, M., Kohl, A., and Schnettler, E.
College/School:College of Medical Veterinary and Life Sciences
College of Medical Veterinary and Life Sciences > School of Infection & Immunity
College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
Journal Name:Viruses
ISSN (Online):1999-4915
Copyright Holders:Copyright © 2021 The Authors
First Published:First published in Viruses 13(6):1066
Publisher Policy:Reproduced under a Creative Commons License

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Project CodeAward NoProject NamePrincipal InvestigatorFunder's NameFunder RefLead Dept
Medical Research Council (MRC)MC_UU_12014/8