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. 2004 Dec 7;101(49):17240-5.
doi: 10.1073/pnas.0406983101. Epub 2004 Dec 6.

RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae

Kimberly M Keene  1 , Brian D Foy, Irma Sanchez-Vargas, Barry J Beaty, Carol D Blair, Ken E Olson
Affiliations

RNA interference acts as a natural antiviral response to O'nyong-nyong virus (Alphavirus; Togaviridae) infection of Anopheles gambiae

Kimberly M Keene et al. Proc Natl Acad Sci U S A. .

Abstract

RNA interference (RNAi) is triggered in eukaryotic organisms by double-stranded RNA (dsRNA), and it destroys any mRNA that has sequence identity with the dsRNA trigger. The RNAi pathway in Anopheles gambiae can be silenced by transfecting cells with dsRNA derived from exon sequence of the A. gambiae Argonaute2 (AgAgo2) gene. We hypothesized that RNAi may also act as an antagonist to alphavirus replication in A. gambiae because RNA viruses form dsRNA during replication. Silencing AgAgo2 expression would make A. gambiae mosquitoes more permissive to virus infection. To determine whether RNAi conditions the vector competence of A. gambiae for O'nyong-nyong virus (ONNV), we engineered a genetically modified ONNV that expresses enhanced GFP (eGFP) as a marker. After intrathoracic injection, ONNV-eGFP slowly spread to other A. gambiae tissues over a 9-day incubation period. Mosquitoes were then coinjected with virus and either control beta-galactosidase dsRNA (dsbetagal; note that "ds" is used as a prefix to indicate the dsRNA derived from a given gene throughout) or ONNV dsnsP3. Treatment with dsnsP3 inhibited virus spread significantly, as determined by eGFP expression patterns. ONNV-eGFP titers from mosquitoes coinjected with dsnsP3 were significantly lower at 3 and 6 days after injection than in mosquitoes coinjected with dsbetagal. Mosquitoes were then coinjected with ONNV-eGFP and dsAgAgo2. Mosquitoes coinjected with virus and AgAgo2 dsRNA displayed widespread eGFP expression and virus titers 16-fold higher than dsbetagal controls after 3 or 6 days after injection. These observations provide direct evidence that RNAi is an antagonist of ONNV replication in A. gambiae, and they suggest that the innate immune response conditions vector competence.

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Figures

Fig. 1.
Fig. 1.
Design and characterization of recombinant ONNV-eGFP. (A) p5′dsONNVic/Foy contains a full-length cDNA of the ONNV genome with a second subgenomic promoter inserted 3′ of the original subgenomic promoter. eGFP was cloned into the MCS and is transcribed under control of the first subgenomic promoter. (B) Characterization of viral transcripts of ONNV after injection into adult female A. gambiae. Lanes 1 and 2 show the ONNV-eGFP transcript profile at 48 and 72 h after infection, respectively. The blot was hybridized by using a radiolabeled ONNV E2 gene as probe, and it shows the production of the full-length genomic and two subgenomic transcripts. (C) eGFP expression at 9 dpi occurs throughout the body.
Fig. 4.
Fig. 4.
Northern blot analysis of AgAgo2 mRNA after injection of mosquitoes with ONNV-eGFP, dsRNA, or dsRNA and ONNV-eGFP. Injection of dsAgAgo2 results in the reduction of Ago2 transcript levels. Mosquitoes injected with dsAgAgo2 and virus showed partial recovery of the Ago2 mRNA accumulation. (Upper) Lane 1, mock injected; lane 2, dsβgal at 3 dpi; lane 3, dsAgAgo2 at 3 dpi; lane 4, dsAgAgo2 + ONNV-eGFP 3 at dpi; lane 5, ONNV-eGFP 2 dpi; and lane 6, ONNV-eGFP 3 dpi. (Lower) Ethidium bromide stain of Northern blot showing ribosomal RNA in each lane and verifying that equivalent amounts of total RNA were added to each lane.
Fig. 2.
Fig. 2.
eGFP expression at 3 dpi in mosquitoes coinjected with dsRNAs and ONNV-eGFP. (A) Coinjection of ONNV-eGFP and dsnsP3. (B) Coinjection of ONNV-eGFP and dsβgal. (C) Coinjection of ONNV-eGFP and dsAgAgo2. Mosquitoes injected with dsnsP3 show a dramatic reduction in eGFP expression when compared with dsβgal-injected controls, whereas mosquitoes injected with dsAgAgo2 show an increase in eGFP expression over controls.
Fig. 3.
Fig. 3.
Viral titers of ONNV-eGFP in mosquitoes coinjected with dsRNA homologous to ONNV nsP3 and AgAgo2. Compared with the nonspecific dsβgal, mosquitoes coinjected with virus and dsnsP3 had statistically significant decreases in infection at both 3 and 6 dpi (P < 0.0001). Viral titers of ONNV-eGFP increased significantly in mosquitoes at 3 and 6 dpi after coinjection with dsRNAs homologous with AgAgo2 (P < 0.0001 and P = 0.0006 at 3 and 6 dpi, respectively).
Fig. 5.
Fig. 5.
Viral titers of ONNV-eGFP in mosquitoes at 3 and 6 dpi after coinjection of dsRNA homologous to AgAgo1, AgAgo3, AgAgo4, and AgAgo5. Compared with the nonspecific dsβgal, only mosquitoes coinjected with virus and dsAgAgo3 had statistically significant increases at 3 and 6 dpi (P ≤ 0.0141).
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