The PAZ domain of Aedes aegypti Dicer 2 is critical for accurate and high-fidelity size determination of virus-derived small interfering RNAs.

The exogenous siRNA (exo-siRNA) pathway is a critical RNA interference response involved in controlling arbovirus replication in mosquito cells. It is initiated by the detection of viral long double-stranded RNA (dsRNA) by the RNase III enzyme Dicer 2 (Dcr2), which is processed into predominantly 21 nt virus-derived small interfering RNAs (vsiRNAs) that are taken up by the Argonaute 2 (Ago2) protein to target viral single-stranded RNAs. The detailed understanding of Dicer structure, function and domains owes much to studies outside the context of viral infection and studies in model organisms, and as such how Dcr2 domains contribute to detecting viral dsRNA to mount antiviral responses in infected mosquito cells remains less well understood. Here, we used a Dcr2 reconstitution system in <i>Aedes aegypti</i> derived Dcr2 knockout (KO) cells to assess the contribution of the PIWI-Argonaute-Zwille (PAZ) domain to induction of the exo-siRNA pathway following infection with Semliki Forest virus (SFV; <i>Togaviridae</i>, <i>Alphavirus</i>). Amino acids critical for PAZ activity were identified, and loss of PAZ function affected the production of 21 nt vsiRNAs-with enrichment of 22 nt SFV-derived small RNAs observed-and silencing activity. This study establishes PAZ domain's functional contribution to Dcr2 processing of viral dsRNA to 21 nt vsiRNAs.