Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing

Abstract

Homology-dependent RNA silencing occurs in many eukaryotic cells. We reported recently that nodaviral infection triggers an RNA silencing-based antiviral response (RSAR) in Drosophila, which is capable of a rapid virus clearance in the absence of expression of a virus-encoded suppressor. Here, we present further evidence to show that the Drosophila RSAR is mediated by the RNA interference (RNAi) pathway, as the viral suppressor of RSAR inhibits experimental RNAi initiated by exogenous double-stranded RNA and RSAR requires the RNAi machinery. We demonstrate that RNAi also functions as a natural antiviral immunity in mosquito cells. We further show that vaccinia virus and human influenza A, B, and C viruses each encode an essential protein that suppresses RSAR in Drosophila. The vaccinia and influenza viral suppressors, E3L and NS1, are distinct double-stranded RNA-binding proteins and essential for pathogenesis by inhibiting the mammalian IFN-regulated innate antiviral response. We found that the double-stranded RNA-binding domain of NS1, implicated in innate immunity suppression, is both essential and sufficient for RSAR suppression. These findings provide evidence that mammalian virus proteins can inhibit RNA silencing, implicating this mechanism as a nucleic acid-based antiviral immunity in mammalian cells.

Fig. 1.

B2 of FHV suppresses RNA silencing induced by a replicating virus RNA (A) or dsRNA (B) in cultured fruit fly cells. (A) Cells were transfected with buffer (Mock), pFR1, pFR1-ΔB2 alone or with pB2gfp, pfB2, or dsRNA of AGO2, as indicated at the top of each lane. Total RNA was extracted 2 days after induction for Northern blot analysis by a probe specific to the B2 coding region of FHV. (B) Cells were either cotransfected or sequentially transfected with a protein-expressing plasmid (pB2gfp or pgfp) and a dsRNA (targets GFP or lacZ mRNA as a control). Total RNA was extracted 2 days after the last transfection and analyzed by Northern blot hybridization with a probe specific to the GFP mRNA. RNA species corresponding to FHV RNAs 1 and 3, mRNA of B2-GFP, and GFP were indicated. Note that the mRNA from pB2 comigrated with RNA3 (A, lane 5). Equal RNA loading was shown by methylene blue staining of rRNA (Lower).

Fig. 2.

Induction and suppression of RNA silencing by NoV. Fruit fly cells were transfected with pNR1, pNR1-ΔB1 or pNR1-ΔB2 alone or with a B2-expressing plasmid (fB2 or nB2), and either dsRNA or siRNA specific for AGO2. An siRNA targeting lacZ mRNA was used as a control. Total RNA was extracted 2 days after induction for Northern blot analysis by a probe specific to the B2 coding region of NoV.

Fig. 5.

Suppression of the RNAi antiviral response by E3L and NS1 proteins. Fruit fly cells were transfected with a pFR1-derived plasmid plus either another plasmid expressing a viral RNAi suppressor (fB2, nB2, E3L, C/NS1, B/NS1, A/NS1, or p19) or a dsRNA/siRNA (to mRNA of AGO2 or lacZ). NS1-A1 contained the R38A/K41A mutation, NS1-A2 corresponded to the N-terminal 82 aa of WT A/NS1, and NS1-A3 contained the D92E mutation. p19fs was a frameshift mutant of p19 that terminates after the first 24 aa. RNA was extracted and analyzed as described for Fig. 2.

Fig. 3.

An RNA-based antiviral immunity in mosquito cells. A. gambiae 4a-2s4 cells were transfected with pONR1, pONR1-ΔB2 alone or with a pIZ-based plasmid coding for fB2 or nB2, plus dsRNA targeting the PAZ domain of A. gambiae AGO1 or AGO2. A lacZ dsRNA was used as a control. An identical blot was probed for the accumulation of AGO2 mRNA with DNA corresponding to the C-terminal PIWI domain of A. gambiae AGO2. RNA was extracted and analyzed as for Fig. 2.

Fig. 4.

Suppression of the RNAi antiviral response visualized by GFP expression. (A) Genome organization and expression of FHV RNA1 and R1gfp. RNA1 encodes protein A, the catalytic subunit of the viral RdRP, and directs transcription of RNA3, mRNA for protein B2. Nucleotides 2,802–3,001 of RNA1 as encoded in pFR1 were replaced with the coding sequence for enhanced GFP (eGFP) to give pFR1gfp so that eGFP was translated from a chimeric RNA3 as a fusion protein with the N terminus of B2. (B) Detection of GFP expression in fruit fly cells from a FHV RNA1 mutant defective in silencing suppression. Cells were transfected with pFR1gfp alone or with a plasmid expressing fB2, E3L, or NS1A and photographed 2 days after induction.

Fig. 6.

Binding of GST-NS1 fusion protein to long and short dsRNAs in vitro. GST (0.4 μM, lanes 3 and 6), GST-NS1 (lanes 4 and 7), or GST-NS1-A1 (lanes 5 and 8) was incubated with ³²P-radiolabeled dsRNA (Right) or siRNA (Center) before the RNA–protein complexes were resolved in a nondenaturing polyacrylamide gel electrophoresis. The positions of the free 76-nt dsRNA and the 21-nt siRNA are indicated. The lower band in lanes 6–8 migrates in the same position as T7-generated single-stranded RNA.