The catalytic tetrad of Aedes aegypti argonaute 2 is critical for the antiviral activity of the exogenous siRNA pathway

Abstract

Viruses transmitted by biting arthropods, arboviruses, pose a significant global health and economic threat. Climate change is exacerbating this issue by expanding the range of disease-carrying vectors. Effective control of arbovirus transmission often relies on targeting the vectors, making it crucial to understand the interactions between the virus and its vector. The exogenous siRNA (exo-siRNA) pathway is a key antiviral defense mechanism in mosquitoes such as Aedes aegypti. Argonaute 2 (Ago2) is a central protein in this pathway, responsible for antiviral activity. While the PIWI domain of Ago proteins is known to mediate slicing activity, not all Ago proteins possess this slicing function. To understand the antiviral mechanism of Ago2 in Ae. aegypti, we aimed to confirm the presence of the catalytic tetrad, a group of amino acids known to be crucial for slicing activity. Here, we confirmed the tetrad (D740, E780, D812, and H950) in Ae. aegypti Ago2 and demonstrated its essential role in antiviral and siRNA pathway activity. Our findings show that the catalytic tetrad is necessary for the degradation of siRNA passenger strands. When the tetrad is absent, siRNA duplexes accumulate, leading to a loss of siRNA pathway function. This underscores the critical role of the tetrad in the antiviral defense mechanism of Ae. aegypti.

Keywords: Argonaute; RNA interference (RNAi); RNA virus; alphavirus; antiviral; mosquito; small-interfering RNA (siRNA).

Figure 1

Multiple sequence alignment of DEDH domain across vector mosquitoes.A, schematic diagram of the domain structure of Ago2. B, Ago2 sequences from D. melanogaster (UniProt ID: Q9VUQ5), An. gambiae (UniProt ID: A0A453Z118), Ae. aegypti (UniProt ID: C5J0H4) and Ae. albopictus (UniProt ID: I3VLA6) were aligned using Benchling (https://www.benchling.com/), and conserved amino acids D740, E780, D812 and H950 were identified relative to D. melanogaster Ago2 sequence.

Figure 2

Growth curve and stability analysis of the AF525-V5-eGFP, AF525-V5-Ago2, and AF525-V5-Ago2mut cell lines.A, Western blot analysis of cell lysates generated from the stable cell lines after initial generation (passage 0) and after long-term passage (passage 20). V5-tagged proteins are present in corresponding lanes confirming stable protein expression when probed with an anti-V5 antibody. Anti-tubulin was used as a loading control. Both cell line clones are indicated on the Western blot image. Relative protein expression levels are indicated below image. Protein expression levels were measured by calculating the density of each band normalised to the corresponding tubulin band and calculated relative to cell line 1. B and C, a total of 8 × 10⁴ cells were seeded into 24 well plates, and the number of cells present in the monolayer enumerated at the time points indicated for both clone one (B) and clone two (C) of each stable cell line. Points represent the average ± SD of three independent experiments.

Figure 3

Effect of catalytic domain mutant on the antiviral activity of Ago2. AF525 cells stably expressing eGFP (AF525-V5-eGFP; black), Ago2 (AF525-V5-Ago2; pink) and Ago2mut (AF525-V5-Ago2mut; teal) were (A) infected with SFV4(3H)-FFLuc at differing MOIs (0.1, 1, and 10 PFU/cell) and virus replication assessed 24 h post-infection (h.p.i.) by measuring FFLuc activity in infected cell monolayers. B, infected with wt SFV at a MOI of 1 PFU/cell and relative virus RNA level was assessed at 24 h.p.i. by RT-qPCR (C) infected with wt SFV at a MOI of 1 PFU/cell and virus titer was assessed at 24 h.p.i. by plaque assay. Bars show the average ± SEM of three independent experiments performed in triplicate. Significance was determined by one-way ANOVA. Here, ns: non-significant, ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, ∗∗∗∗p ≤ 0.0001.

Figure 4

Effect of the Ago2 catalytic tetrad on exo-siRNA pathway activity. AF525 cells stably expressing eGFP (AF525-V5-eGFP), Ago2 (AF525-V5-Ago2), and Ago2mut (AF525-V5-Ago2) were transfected with plasmids expressing FFLuc and Renilla luciferases and either dsRNA to FFLuc (dsFFLuc; pink) or eGFP (dseGFP; black). Luciferase activities in each cell line were determined by calculating activity relative to the dseGFP-transfected control. Cells were lysed at 24 h post-transfection (p.t.). Bars show the average of three independent experiments ±SEM performed in triplicate. Significance was assessed by a 2-way ANOVA, ns: non-significant, ∗∗∗∗p ≤ 0.0001.

Figure 5

Mutation in the catalytic tetrad of Ago2 in AF525 cells has a limited effect on the magnitude of 21 nt vsiRNA reads produced in response to SFV infection. Small RNA analysis of AF525 cells stably expressing eGFP, Ago2, and Ago2mut, and infected with SFV (MOI 5 PFU/cell), analyzed at 24 h.p.i. A, histogram of 21 nt vsiRNAs that mapped to the SFV genome (positive) or antigenome (negative) with colors indicating the nt prevalence of the first base for each read length. Shown as mean % mapped reads (Y axis, percentage reads) from three independent experiments. Error bars show the standard error of the mean. B, coverage of SFV-derived 21 nt vsiRNAs over the viral genome, genomic (magenta) or antigenomic (cyan) sense RNAs are shown (Y axis, vsiRNA reads per million) from three independent experiments. Error bars show the standard error of the mean. C, mean number of 21 nt viral RNAs per million reads mapping to the SFV genome (magenta, +) and antigenome (cyan, −) of all treatments. The graph indicates a mean value of three (n = 3) independent repeats with the range of values given. Ordinary one-way ANOVA with Tukey's multiple comparisons test was used to compare groups. Here, ns: insignificant ∗p ≤ 0.05, and ∗∗∗p ≤ 0.0001. GenBank ID for SFV: KP699763.

Figure 6

Mutation of the catalytic tetrad in Ago2 increases the absolute number of SFV-derived vsiRNA duplex pairs. Samples as described in Figure 5 were reanalysed for bioinformatic analysis. A, number of overlapping 21 nt vsiRNA pairs, normalized per million mapped SFV reads, with the most abundant vsiRNA duplex pairs indicated by the orange background as 21 nt reads overlapping by 18-21 nt. B, probability z-score of overlapping 21 nt vsiRNA reads across conditions, e.g., eGFP, Ago2 or Ago2mut samples. C, number of most abundant 21 nt vsiRNA duplexes overlapping by 18-21 nt across conditions, e.g., eGFP, Ago2, or Ago2mut samples, normalized to number of million mapped reads. All graphs indicate a mean value of three (n = 3) independent repeats with the range of values indicated. Ordinary one-way ANOVA with Tukey's multiple comparisons test was used to compare groups in (C) with ∗p ≤ 0.05 and ∗∗∗p ≤ 0.0001.