Mutational analysis of Aedes aegypti Dicer 2 provides insights into the biogenesis of antiviral exogenous small interfering RNAs

Abstract

The exogenous small interfering RNA (exo-siRNA) pathway is a key antiviral mechanism in the Aedes aegypti mosquito, a widely distributed vector of human-pathogenic arboviruses. This pathway is induced by virus-derived double-stranded RNAs (dsRNA) that are cleaved by the ribonuclease Dicer 2 (Dcr2) into predominantly 21 nucleotide (nt) virus-derived small interfering RNAs (vsiRNAs). These vsiRNAs are used by the effector protein Argonaute 2 within the RNA-induced silencing complex to cleave target viral RNA. Dcr2 contains several domains crucial for its activities, including helicase and RNase III domains. In Drosophila melanogaster Dcr2, the helicase domain has been associated with binding to dsRNA with blunt-ended termini and a processive siRNA production mechanism, while the platform-PAZ domains bind dsRNA with 3' overhangs and subsequent distributive siRNA production. Here we analyzed the contributions of the helicase and RNase III domains in Ae. aegypti Dcr2 to antiviral activity and to the exo-siRNA pathway. Conserved amino acids in the helicase and RNase III domains were identified to investigate Dcr2 antiviral activity in an Ae. aegypti-derived Dcr2 knockout cell line by reporter assays and infection with mosquito-borne Semliki Forest virus (Togaviridae, Alphavirus). Functionally relevant amino acids were found to be conserved in haplotype Dcr2 sequences from field-derived Ae. aegypti across different continents. The helicase and RNase III domains were critical for silencing activity and 21 nt vsiRNA production, with RNase III domain activity alone determined to be insufficient for antiviral activity. Analysis of 21 nt vsiRNA sequences (produced by functional Dcr2) to assess the distribution and phasing along the viral genome revealed diverse yet highly consistent vsiRNA pools, with predominantly short or long sequence overlaps including 19 nt overlaps (the latter representing most likely true Dcr2 cleavage products). Combined with the importance of the Dcr2 helicase domain, this suggests that the majority of 21 nt vsiRNAs originate by processive cleavage. This study sheds new light on Ae. aegypti Dcr2 functions and properties in this important arbovirus vector species.

Fig 1. Loss-of-function mutations in Aaeg Dcr2 domains affect the exo-siRNA pathway and antiviral activity.

(A) Left panel, schematic diagram of Dmel and Aaeg Dcr2 showing functional domains. Right panel, table showing loss-of-function mutations performed on Aaeg Dcr2 helicase and RNase III domains based on Dmel Dcr2. Abbreviations: DUF = domain of unknown function; PAZ = PIWI-Argonaute-Zwille domain, and dsRBD = dsRNA binding domain. (B) Top panel, multiple sequence alignment of human Dcr1 and insect Dcr2 showing conserved motifs/residues across species with mutagenesis performed on the helicase domain Motif I, IV, and VI that map to Aaeg Dcr2 at K39N, Y232G, G488R, respectively, and the RNase III domain mutations mapping to D1198A, E1341A, D1444A, and E1548A (collectively, mtR3). Bottom panel, immunoblot showing expression of myc-tagged Dcr2 in AF319 cells transfected with pPUb plasmids encoding WT or mutant Dcr2 with pPUb-myc-eGFP (peGFP) as control using anti-myc antibody with γ-tubulin as loading control. Representative blot of n = 2 independent repeats is shown. (C) Silencing activity of mutant Dcr2 assessed by RNAi reporter assay using AF319 cells co-transfected with mutant or WT Dcr2 plasmid (peGFP as control) and FFLuc and RLuc reporter plasmids with dsRNA targeting FFLuc (dsFLuc) or dsLacZ (non-targeting control). At 24 hpt, luciferase levels measured and presented as mean±SEM relative light units (FFLuc/RLuc) versus dsLacZ set to 1 from n = 3 independent repeats with * = p<0.05, ** = p<0.01 versus controls according to two-way ANOVA. (D) Antiviral activity of mutant Dcr2 in AF319 cells (peGFP as control). Cells were infected at 24 hpt with SFV-FFLuc (MOI = 0.1) for 48 h. Measured FFLuc readings shown as mean±SEM relative light units compared to eGFP control set to 1 from n = 3 independent repeats with * = p<0.05, ** = p<0.01 according to Student’s t-test.

Fig 2. Naturally occurring Dcr2 variants among field-derived mosquito populations.

(A) Dcr2 haplotypes (in grey) were identified from early generation colonies of Ae. aegypti initially collected from Cameroon (Ben), French Guiana (Cay), Guadeloupe (Guad), Cambodia (KC and Rat), and Gabon (Lope) and aligned to a reference WT Dcr2 sequence (GenBank ID: AY713296; thick black line) to determine SNPs. Black bars show disagreements to WT Dcr2 with silent and non-synonymous SNPs highlighted by cyan and magenta bars, respectively. (B) Phylogenetic tree of putative amino acid sequences of haplotypes highlighting the amino acid net charge at positions 246/253 (+: positive, -: negative, =: neutral) and an H or N at position 1514. Numbers on branches indicate % consensus support with the substitution rate measured by the scale bar. (C) Alignment of haplotypes highlighting amino acid variants N246D and Q253K in the helicase, and H1514N in the RNase IIIB domain. (D) Mutagenesis of WT Dcr2 to generate amino acid mutations at position 246/253 (aa 246/253) and a theoretical "D/Q" variant (N246D; negative charge) not observed among the surveyed mosquito populations. To check for Dcr2 expression, AF319 cells transfected with variant or WT Dcr2 (peGFP as control) were lysed and probed for myc-Dcr2 by immunoblot using anti-myc tag and γ-tubulin (loading control) antibodies. Representative blot of n = 2 independent repeats is shown. (E) RNAi reporter assay was performed to determine the effect of the variants on silencing activity. AF319 cells co-transfected with WT or variant Dcr2 plasmids (peGFP as control) and FFLuc and RLuc reporter plasmids with dsRNA targeting FFLuc (dsFLuc) or dsLacZ (non-targeting control). At 24 hpt, luciferase levels were measured and shown as mean±SEM relative light units (FFLuc/RLuc) versus dsLacZ set to 1 compared to eGFP control from n = 3 independent repeats with * = p<0.05 according to two-way ANOVA. (F) Antiviral activity of Dcr2 variants against SFV-FFLuc (MOI = 0.1) was determined in AF319 cells transiently expressing variant or WT Dcr2 (peGFP as control). Cells were infected 24 hpt. At 48 hpi, FFLuc readings were measured and presented as mean±SEM relative light units compared to eGFP control set to 1 from n = 3 independent repeats with * = p<0.05 versus control according to two-way Student’s t-test.

Fig 3. Locations of conserved amino acids critical for Aaeg Dcr2 function can be predicted and are distributed between surface and buried residues.

Center, AlphaFold prediction (mean pLDDT score = 81.4) for the structure of WT Aaeg Dcr2 (GenBank ID: AAW48725), colored by predicted domains from annotations and Interproscan. Residues representing conserved amino acids involved in Dcr2 activity and natural haplotype variants are highlighted in red and purple, respectively. Inset panels show enlarged representations of conserved amino acid residues targeted in mutagenesis experiments, with dashed lines showing contacts in red, hydrogen bonds in cyan, and distances in yellow. Clockwise from upper right: loss-of-function mutations in the RNase III domains line a predicted cleft in the core of the protein; G488 and K39 are forecast to be buried near the surface of the helicase domain; Y232 appears to form hydrogen bonds and extensive contacts with a neighbouring α-helix (residues 360–374) and β-strand (residues 496–502); natural haplotype variant residues N246 and Q253 (distance = 10.71Å) are likely positioned on the exposed surface of the helicase domain; H1514 is seen at the surface exposed tip of an α-helix.

Fig 4. Domain loss-of-function mutations in Aaeg Dcr2 affect vsiRNA production.

(A) Distribution of 18–35 nt vsRNAs mapped to the SFV (GenBank ID: KP699763) genome (sense; magenta) or anti-genome (anti-sense; cyan) shown as mean % mapped reads in bars with replicates shown as circles from n = 2 independent repeats from AF319 cells transiently expressing mutant or WT Dcr2 and infected with SFV (MOI = 1) at 48 hpi. (B) SFV-derived 21 nt vsiRNAs from (A), mapped to SFV genome (magenta) or anti-genome (cyan) with the mean number of mapped reads shown from n = 2 independent repeats. The SFV genome organization (top panel) is shown for reference.

Fig 5. Characterising 21 nt vsiRNAs with vpiRNA signature: vsi-piRNAs.

(A) Heat maps showing mean overlap probabilities of z-scores of 18–30 nt SFV-derived vsRNAs from AF319 transiently expressing mutant or WT Dcr2 from n = 2 independent repeats. vsRNA lengths are shown horizontally, and the number of nucleotide overlaps listed vertically. Red arrow labelled Dcr2 indicates expected 2 nt overlap from dsRNA cleavage with cells boxed in black. Black arrow labeled pp (ping-pong) shows expected 10 nt overlap from potential ping-pong amplification. (B) Representative sequence logos of n = 2 independent repeats (other replicate in S5 Fig) of 21 nt vsRNAs mapping to the genome (left panel) or anti-genome (right panel) of SFV from AF319 transiently expressing mutant or WT Dcr2. Overall height of the stack indicates conservation presented as bits, with the height of each symbol reflecting the relative frequency of the corresponding nucleotide at a given position. Error bars indicate an approximate Bayesian 95% credible interval.

Fig 6. Viral dsRNA cleavage pattern of Aaeg Dcr2 during SFV infection.

Number of overlapping pairs (top panel) and probability z-scores (bottom panel) of 21 nt virus-derived small RNAs from AF319 transiently expressing mutant or WT Dcr2 infected with SFV. Mean of n = 2 independent repeats is presented as bars or red line (grey region indicates the range).

Fig 7. Functional Aaeg Dcr2 generates diverse but highly consistent true 21 nt vsiRNAs.

(A) Multi-dimensional scaling (MDS) plot of vsRNAs (16–46 nt) and (B) 21 nt vsRNAs from AF319 cells transiently expressing functional Dcr2 (WT or Y232G; mainly true 21 nt vsiRNAs) or mutant Dcr2 (G488R, K39N, mtR3; mainly vsi-piRNAs) per replicate with axes presented as log fold change (LogFC). (C) Heatmap showing the relationship of 21 nt vsRNAs generated from AF319 cells transiently expressing WT or mutant Dcr2 (as indicated above) during SFV infection. Hierarchical clustering of treatment replicates (columns) and 21 nt vsRNAs (rows) performed by first calculating a distance matrix using Pearson correlation distance and clustered using Ward’s minimum variance method. Each library is shown, and the scale corresponds to log-transformed TMM normalized counts per million.