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. 2020 Apr 8;5(2):e00081-20.
doi: 10.1128/mSphere.00081-20.

The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and ago-2 Expression

Melanie McFarlane #  1 , Floriane Almire #  1 , Joy Kean  1 , Claire L Donald  1 , Alma McDonald  1 , Bryan Wee  2 , Mathilde Lauréti  1 , Margus Varjak  1 , Sandra Terry  1 , Marie Vazeille  3 , Rommel J Gestuveo  1   4 , Isabelle Dietrich  1 , Colin Loney  1 , Anna-Bella Failloux  3 , Esther Schnettler  1 , Emilie Pondeville #  5 , Alain Kohl #  5
Affiliations

The Aedes aegypti Domino Ortholog p400 Regulates Antiviral Exogenous Small Interfering RNA Pathway Activity and ago-2 Expression

Melanie McFarlane et al. mSphere. .

Abstract

Arboviruses are pathogens of humans and animals. A better understanding of the interactions between these pathogens and the arthropod vectors, such as mosquitoes, that transmit them is necessary to develop novel control measures. A major antiviral pathway in the mosquito vector is the exogenous small interfering RNA (exo-siRNA) pathway, which is induced by arbovirus-derived double-stranded RNA in infected cells. Although recent work has shown the key role played by Argonaute-2 (Ago-2) and Dicer-2 (Dcr-2) in this pathway, the regulatory mechanisms that govern these pathways have not been studied in mosquitoes. Here, we show that the Domino ortholog p400 has antiviral activity against the alphavirus Semliki Forest virus (Togaviridae) both in Aedes aegypti-derived cells and in vivo Antiviral activity of p400 was also demonstrated against chikungunya virus (Togaviridae) and Bunyamwera virus (Peribunyaviridae) but not Zika virus (Flaviviridae). p400 was found to be expressed across mosquito tissues and regulated ago-2 but not dcr-2 transcript levels in A. aegypti mosquitoes. These findings provide novel insights into the regulation of an important aedine exo-siRNA pathway effector protein, Ago-2, by the Domino ortholog p400. They add functional insights to previous observations of this protein's antiviral and RNA interference regulatory activities in Drosophila melanogasterIMPORTANCE Female Aedes aegypti mosquitoes are vectors of human-infecting arthropod-borne viruses (arboviruses). In recent decades, the incidence of arthropod-borne viral infections has grown dramatically. Vector competence is influenced by many factors, including the mosquito's antiviral defenses. The exogenous small interfering RNA (siRNA) pathway is a major antiviral response restricting arboviruses in mosquitoes. While the roles of the effectors of this pathway, Argonaute-2 and Dicer-2 are well characterized, nothing is known about its regulation in mosquitoes. In this study, we demonstrate that A. aegypti p400, whose ortholog Domino in Drosophila melanogaster is a chromatin-remodeling ATPase member of the Tip60 complex, regulates siRNA pathway activity and controls ago-2 expression levels. In addition, we found p400 to have antiviral activity against different arboviruses. Therefore, our study provides new insights into the regulation of the antiviral response in A. aegypti mosquitoes.

Keywords: RNA interference; ago-2; arbovirus; innate immunity; mosquito; p400.

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Figures

FIG 1
FIG 1
Detection of p400 transcripts in tissues of NBF A. aegypti females. Presence of p400 transcripts was determined in hemocytes, salivary glands, digestive tracts, ovaries, and carcasses of NBF females by RT-qPCR using the 2−ΔΔCT method and expression in hemocytes as the reference sample. Bars represent the fold change in gene expression from 3 independent biological replicates (pools of 25 digestive tracts or ovaries, pools of 60 salivary glands, and pools of perfused hemocytes from 70 females per replicate). Error bars show the minimum and maximum fold change.
FIG 2
FIG 2
Detection of p400 protein in tissues of NBF A. aegypti females. Expression of p400 was analyzed in perfused hemocytes (A to C), salivary glands (D), digestive tracts (E to I), and ovaries (J to P) by immunofluorescence assay using an anti-p400 antibody. The signal was determined using an Alexa Fluor 568 goat anti-mouse IgG (H+L) (red). Nuclei are stained by DAPI (blue signal), and F-actin is stained by phalloidin 488 (green signal). Images were acquired on a Zeiss LSM 710 inverted confocal microscope with 40×, 63×, and 100× oil immersion objectives. Scale bars are 5 μm (A to C, F to H, and N to P) and 40 μm (D, E, and I to M). (A) Perfused prohemocyte. (B and C) Perfused differentiated hemocytes. (D) Salivary glands. (E) Crop. (F to H) Enlargement of panel E. (I) Midgut. (J) Oviduct. (K and L) Ovarioles showing the germarium and primary follicle (pf). (M) Ovarioles surrounded by the ovarian sheath. (N to P) Enlargement of panel M. Yellow star, developing cyst in the germarium; yellow arrowhead, germ line stem cells. The cystoblast is located between the germ line stem cells and the developing cyst. Images shown are representative of a minimum of 10 tissues per experiment from 3 independent experiments.
FIG 3
FIG 3
p400 knockdown significantly enhances replication of SFV in A. aegypti females. p400 and SFV expression in whole dsLacZ- or dsp400-injected females 3 days after an SFV4-infected blood meal were analyzed by RT-qPCR. Normalized expression-per-sample values were calculated as described by Taylor et al. (78) in order to obtain normalized expression values, relative to the ribosomal S7 transcript as reference, with a geomean of 1 for the dsLacZ control group. Bars represent the geomean and 95% confidence intervals from n = 73 dsLacZ and n = 66 dsp400. The results were analyzed using a Mann-Whitney test using log2-normalized expression values. This experiment is representative of two independent biological replicates (second replicate shown in Fig. S1). (A) p400 transcript levels are significantly reduced in dsp400-injected females compared to that in dsLacZ-injected females (mean fold change, 1.7; geomean fold change, 1.6; ****, P < 0.0001). (B) SFV4 titers are significantly higher in dsp400-injected females than in dsLacZ-injected ones (mean fold change, 5.3; geomean fold change, 10.1; ****, P < 0.0001). A base 10 log scale is used for the y axis.
FIG 4
FIG 4
p400 knockdown significantly enhances replication of SFV, CHIKV, and BUNV but not ZIKV. (A) p400 knockdown efficiency in Aag2 cells was analyzed by RT-qPCR using the 2−ΔΔCT method and p400 expression calculated relative to eGFP dsRNA transfection. Bars show the fold change in gene expression from 3 independent experiments (3 wells in each independent experiment, with an average of three wells per condition/experiment used for statistical analysis). Error bars represent the minimum and maximum fold change. The results were analyzed by a one-sample t test. p400 transcript levels are significantly reduced after dsp400 transfection (P = 0.039; *, P < 0.05). The effect of p400 knockdown on SFV-FFLuc (B), CHIKV-2SG-FFLuc (C), BUNV-NLuc (D), and ZIKV-NLuc (E) was assessed by a luciferase assay. Bars show the means of the results from 3 independent experiments (3 wells in each independent experiment, with the average of three replicates per condition/experiment used for statistical analysis). Values were calculated relative to the control eGFP dsRNA-transfected sample, which was set to 1. Statistical significance was determined by performing a one-sample t test (SFV P = 0.0129, CHIKV P = 0.0286, and BUNV P = 0.0257; *, P < 0.05). Error bars show the standard error of mean.
FIG 5
FIG 5
p400 knockdown leads to reduced silencing efficiency. RNA silencing activity in the presence of p400 knockdown was determined using a sensor assay. Aag2 cells were transfected with plasmids constitutively expressing firefly (FFLuc) or Renilla luciferase and dsRNA targeting either p400 or eGFP as a control. At 24 h post-initial transfection, dsRNA against FFLuc or eGFP was transfected. Silencing activity was assessed by measuring the relative levels of FFLuc 24 h post-second transfection after normalization to RLuc (internal transfection control) levels. The level of silencing was calculated relative to respective control eGFP dsRNA-transfected samples (dseGFP+dseGFP as a control for condition dseGFP+dsFFLuc, and dsp400+dseGFP as a control for condition dsp400+dsFFluc), which were set to 1. Bars show the means from 3 independent experiments (3 wells in each independent experiment, average of three wells per condition/experiment used for statistical analysis). Error bars show the standard error of mean. Significance was determined by an unpaired t test to determine the P value (P = 0.034; *, P < 0.05).
FIG 6
FIG 6
Effect of p400 knockdown on ago-2 and dcr-2 transcript levels in A. aegypti females. (A) ago-2 transcript levels in whole dsLacZ- or dsp400-injected females 3 days after an SFV4-infected blood meal was analyzed by RT-qPCR. Normalized expression-per-sample values were obtained as described by Taylor et al. (78) in order to obtain normalized expression values, relative to the ribosomal S7 transcript as a reference, with a geomean of 1 for the dsLacZ control group. Bars show the geomean and 95% confidence intervals from n = 73 dsLacZ and n = 66 dsp400 females of a single experiment, also used in Fig. 3. This experiment is representative of two independent experiments (second replicate shown in Fig. S1). The results were analyzed using a Mann-Whitney test using log2-normalized expression values. ago-2 transcript levels are significantly reduced in dsp400-injected females compared to those in dsLacZ-injected ones (mean fold change, 1.2; geomean fold change, 1.1; P = 0.0197; *, P < 0.05). (B) Transcript levels of p400, ago-2, and dcr-2 in whole non-blood-fed (NBF) females 4 days after injection with dsLacZ or dsp400 were analyzed by RT-qPCR using the 2−ΔΔCT method and expression in dsLacZ females as the reference sample. Bars represent the fold change in gene expression (3 independent biological replicates, with pools of 10 females per replicate). Error bars show the minimum and maximum fold change. The results were analyzed using a one-sample t test. p400 and ago-2 transcript levels are significantly reduced in dsp400-injected females compared to those in dsLacZ-injected females (70% reduction, P = 0.0047; and 42% reduction, P = 0.0067; **, P < 0.01).
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