The VP3 factor from viruses of Birnaviridae family suppresses RNA silencing by binding both long and small RNA duplexes

Abstract

RNA silencing is directly involved in antiviral defense in a wide variety of eukaryotic organisms, including plants, fungi, invertebrates, and presumably vertebrate animals. The study of RNA silencing-mediated antiviral defences in vertebrates is hampered by the overlap with other antiviral mechanisms; thus, heterologous systems are often used to study the interplay between RNA silencing and vertebrate-infecting viruses. In this report we show that the VP3 protein of the avian birnavirus Infectious bursal disease virus (IBDV) displays, in addition to its capacity to bind long double-stranded RNA, the ability to interact with double-stranded small RNA molecules. We also demonstrate that IBDV VP3 prevents the silencing mediated degradation of a reporter mRNA, and that this silencing suppression activity depends on its RNA binding ability. Furthermore, we find that the anti-silencing activity of IBDV VP3 is shared with the homologous proteins expressed by both insect- and fish-infecting birnaviruses. Finally, we show that IBDV VP3 can functionally replace the well-characterized HCPro silencing suppressor of Plum pox virus, a potyvirus that is unable to infect plants in the absence of an active silencing suppressor. Altogether, our results support the idea that VP3 protects the viral genome from host sentinels, including those of the RNA silencing machinery.

Figure 1. VP3 protein from IBDV suppresses the RNA silencing.

ssRNA-triggered (A) and dsRNA-triggered (B) silencing assays in N. benthamiana plants. The figure shows GFP fluorescence pictures taken under an epifluorescence microscope using the same exposure time (left panels), and Northern blot analyses of GFP mRNA and GFP-derived siRNAs (right panels) in agroinfiltrated leaf patches from two plants expressing the indicated constructs, and collected at 6 days post agroinfiltration. Two different probes were used for detection of GFP siRNAs: the GF probe (for primary plus secondary siRNAs) corresponds to the GFP fragment included in the inverted repeat RNA expressed from the silencing trigger plasmid p35S:GF-IR; and the P probe (specific for secondary siRNAs) corresponds to the 3′ terminal region of the GFP gene, which is not included in p35S:GF-IR. EtBr-stained rRNA and 5S+tRNA are shown as loading controls.

Figure 2. IBDV VP3 binds long and short dsRNAs, and the positively charged domain Patch 1 is involved in this interaction.

(A) Schematic representation of wild type IBDV VP3 and derivative mutants used in this assay (red bars indicate mutated patches). The distribution of electrostatic potential on VP3 surface (adapted from [56]) is shown at the right. Both Patch 1 and Patch 2 positively charged regions are indicated in blue, whereas negatively charged regions of the protein are shown in red. (B) (Left panel) Purified IBDV VP3 WT protein (final concentration of 10, 20, 40 and 80 nM) was incubated with the IBDV genomic dsRNAs. (Central and right panels) Purified IBDV VP3 protein (final concentration of 80, 160, 320, 640 and 1200 nM) was incubated with ³²P-labelled 21-nt or 26-nt ds small RNAs. (C) (Upper panels) Purified VP3 ΔC (final concentration of 12, 24, 48 and 96 nM) and Patch 1 or Patch 2 mutant VP3 (final concentration of 10, 20, 40 and 80 nM) were incubated with IBDV genomic dsRNAs. (Lower panels) Purified VP3 ΔC (final concentration of 90, 180, 360, 720 and 1440 nM), and Patch 1 or Patch 2 mutant VP3 (final concentration of 80, 160, 320, 640 and 1200 nM) were incubated with ³²P-labelled 21-nt small RNAs. In both B and C, protein-IBDV genomic dsRNA complexes were resolved in agarose gels, stained with EtBr, and photographed under UV light, and protein-small RNA complexes were resolved in polyacrylamide gels and revealed by autoradiography.

Figure 3. Positive correlation between dsRNA binding and RNA silencing suppression activity revealed by a dsRNA-triggered silencing suppression test.

GFP fluorescence pictures taken under an epifluorescence microscope (A) and Northern blot analysis of GFP mRNA accumulation (B) of leaves of two N. benthamiana plants infiltrated with agrobacteria carrying the plasmid indicated above each lane (red bars indicate mutated patches). EtBr-stained rRNA is shown as loading control.

Figure 4. VP3 proteins from fish- and insect-infecting birnaviruses suppress RNA silencing in a dsRNA-triggered test.

(A) (Upper panels) purified DXV VP3 (final concentration of 8.5, 17, 34 and 68 nM) and IPNV VP3 (final concentration of 10, 20, 40 and 80 nM) were incubated with IBDV genomic dsRNAs. Protein-dsRNA complexes were resolved in agarose gels, stained with EtBr, and photographed under UV light. (Lower panels) purified DXV VP3 (final concentration of 70, 140, 280, 560 and 1120 nM) and IPNV VP3 (final concentration of 80, 160, 320, 640 and 1280 nM) were incubated with ³²P-labelled 21-nt ds small RNAs. Protein-small RNA complexes were resolved in polyacrylamide gels and revealed by autoradiography. (B) GFP fluorescence pictures taken under an epifluorescence microscope using the same exposure time, and (C) Northern blot analysis of GFP mRNA in agroinfiltrated leaf patches from two N. benthamiana plants expressing the indicated constructs, and collected at 6 days post agroinfiltration. EtBr-stained rRNA is shown as loading control.

Figure 5. IBDV VP3 is able to functionally replace the HCPro silencing suppressor in a PPV infection.

(A) Schematic representation of full-length cDNA clones derived from PPV and employed to infect N. benthamiana biolistically. The coding sequence of the GFP protein inserted between the NIb and CP cistrons is represented with a green rectangle. Black arrows indicate self-cleavages by the corresponding viral proteases, whereas the grey arrow indicates a cleavage in trans by the action of NIaPro. (B) GFP expression pattern of plants infected with the indicated viruses. Pictures of inoculated leaves collected at 7 days post inoculation (dpi), and the forth (+4) and fifth (+5) leaves above the inoculated one and the most apical leaves collected at 22 dpi were taken under an epifluorescence microscope. (C) Western blot analyses of plant tissue showing GFP foci collected at 9 dpi from inoculated leaves (upper panel) and at 22 dpi from upper non-inoculated leaves (+4 and +5 leaves) (lower panel) of two independent infected plants. (D) Western blot analysis of old (O) and young (Y) leaves collected at 38 dpi from two independent infected N. benthamiana plants. A polyclonal serum specific for PPV CP was used for assessment of virus accumulation, whereas immunoreaction with polyclonal sera specific for IBDV VP3 confirmed the identity of the infecting chimera. The membranes stained with Ponceau red showing the Rubisco are included as loading controls.