An Aedes aegypti-Derived Ago2 Knockout Cell Line to Investigate Arbovirus Infections.

Christina Scherer,Jack Knowles,Janina Fuss,Anthony C Fredericks,Esther Schnettler,Kevin Maringer,Alain Kohl,Ana Fernandez-Sesma,Vattipally B Sreenu,Andres Merits,Margus Varjak

doi:10.3390/v13061066

Abstract

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.

Highlights

Arboviruses are transmitted by biting arthropods such as ticks, midges, sandflies and mosquitoes
Previous studies of RNA interference (RNAi) key proteins Argonaute 2 (Ago2) and Dicer 2 (Dcr2) in the context of arboviral infections were based on transient silencing approaches in mosquito-derived cell lines
Our findings indicate that acute infection of Semliki Forest virus (SFV) and Bunyamwera orthobunyavirus (BUNV) replication is targeted and modulated by the exo-siRNA pathway, with contributions from both Dcr2 and Ago2

Summary

Introduction

Arboviruses are transmitted by biting arthropods such as ticks, midges, sandflies and mosquitoes. Two RNAi pathways are triggered by viral infection resulting in the production of different virusspecific small RNAs: the exogenous 21 nt long small interfering (vsi)RNAs and the 24–30 nt long Piwi-interacting (vpi)RNAs. Typically, (v)piRNAs are produced by the so-called pingpong production pathway, involving PIWI proteins and Argonaute 3, which results in the characteristic composition of vpiRNAs: U1 (anti-sense), A10 (sense) bias and 10 nt overlap of the sense and antisense piRNAs. The exogenous siRNA (exo-siRNA) pathway has been proposed to be the main host response to control virus infections in Aedes aegypti mosquitoes and derived cell lines [7]. The exogenous siRNA (exo-siRNA) pathway has been proposed to be the main host response to control virus infections in Aedes aegypti mosquitoes and derived cell lines [7] This pathway is induced by viral replication-generated doublestranded RNA (dsRNA), which activates the response and involves the critical exo-siRNA effector proteins Dicer 2 (Dcr2) and Argonaute 2 (Ago). To the best of our current knowledge—based mostly on the model organism Drosophila melanogaster—siRNAs bound by Ago are methylated at their 3 end and one strand of the siRNA duplex is degraded, while the remaining ‘guide strand’ is used to target and in turn cleave complementary viral RNA, resulting in reduced or impaired viral replication and translation

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