Dicer-2 mutations in Aedes aegypti cells lead to a diminished antiviral function against Rift Valley fever virus and Bunyamwera virus infection

Abstract

Mosquitoes are known to transmit different arthropod-borne viruses belonging to various virus families. The exogenous small interfering RNA pathway plays an important role in the mosquito defence against such virus infections, with Dicer-2 (Dcr2) as one of the key proteins that initiates the cleavage of viral dsRNAs into 21 nt long virus-derived small interfering RNAs. Previous data identified the importance of various motifs in Dcr2 for its small interfering RNA (siRNA)-mediated antiviral activity. However, all these data focus on positive-strand RNA viruses, although negative-strand RNA viruses, like Bunyaviricetes, include several important mosquito-borne viruses. Here, we aim to investigate the importance of different domains of Dcr2 for antiviral activity against viruses of the Bunyaviricetes. For this, we used the Aedes aegypti-derived Dcr2 knock-out cell line Aag2-AF319 to study the importance of the helicase, RNase III and PIWI-Argonaute-Zwille domains of Dcr2 on the antiviral activity of two viruses belonging to different families of the Bunyaviricetes: the Rift Valley fever virus (RVFV) vaccine strain MP12 (Phenuiviridae, Phlebovirus) and the Bunyamwera orthobunyavirus (BUNV; Peribunyaviridae, Orthobunyavirus). All three domains were determined to be critical for the antiviral activity against both RVFV and BUNV. Interestingly, one specific mutation in the helicase domain (KN) did not result in a loss of antiviral activity for RVFV, but for BUNV, despite losing the ability to produce 21 nt siRNAs.

Keywords: Bunyaviricetes; Dcr2; RVFV; antiviral RNAi; arbovirus; mosquito.

Fig. 1.. Antiviral activity of different mutant Ae. aegypti Dcr2 against RVFV and BUNV. (a) Table of Dcr2 mutants with corresponding mutations and their domain location. (b) Effect of mutant Dcr2 on BUNV and (c) RVFV vRNA. Aag2-AF319 cells were transfected with pPUb-plasmids expressing WT or mutant Dcr2s, with pPUb-myc-EGFP as control. The cells were infected at 24-h post-transfection (hpt) with BUNV or RVFV (both MOI 0.1), respectively. At 72-h post-infection (hpi), total RNA was isolated and analysed using qRT-PCR. Relative quantification (RQ) was calculated using S7 rRNA as a housekeeper gene for qRT-PCR and EGFP as control. The results are shown as sem of at least three independent biological replicates. The dots represent different biological replicates, consisting of two technical replicates each with * = P < 0.05 and ** = P < 0.01 according to Student’s t-test.

Fig. 2.. Mutations in Dcr2 in Aag-AF319 cells reduce the vsiRNA production against RVFV M segment. Small RNA sequencing of Aag2-AF319 cells transiently expressing WT Dcr2, EGFP, Dcr2 KN and Dcr2 GR mutations and infected with RVFV (MOI 1) at 48 hpi. Histogram of small RNA reads 18–30 nt in length, mapped to the RVFV antigenome (positive) and genome (negative) with colours indicating first base nt prevalence for each read length, shown as counts per million (CPM) from two independent experiments ± sd.

Fig. 3.. Differences in the mapping of 21 nt vsiRNAs in KN and GR Dcr2 mutants in RVFV infection. Coverage of RVFV-derived 21 nt vsiRNAs over the antigenome (magenta) or genome (cyan) (Y-axis, vsiRNA reads per million) for the L, M and S segments of RVFV from two independent experiments ± sd.

Fig. 4.. Limited differences in vpiRNA generation of Aag2-AF319 cells with Dcr2 WT, KN and GR mutants and EGFP control during RVFV infection. Probability of overlapping pairs z-scores of 25–30 nt virus-derived small RNAs. Mean of n=2 independent repeats is presented with the grey region indicating the range.