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.