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. 2008 Jan;82(2):974-86.
doi: 10.1128/JVI.01664-07. Epub 2007 Nov 7.

Protease activity, self interaction, and small interfering RNA binding of the silencing suppressor p1b from cucumber vein yellowing ipomovirus

Adrian Valli  1 , Gabriela Dujovny, Juan Antonio García
Affiliations

Protease activity, self interaction, and small interfering RNA binding of the silencing suppressor p1b from cucumber vein yellowing ipomovirus

Adrian Valli et al. J Virol. 2008 Jan.

Abstract

The RNA silencing pathway mediated by small interfering RNAs (siRNAs) plays an important antiviral role in eukaryotes. To counteract this defense barrier, a large number of plant viruses express proteins with RNA silencing suppression activity. Recently, it was reported that the ipomovirus Cucumber vein yellowing virus (CVYV), which lacks the typical silencing suppressor of members of the family Potyviridae, i.e., HCPro, has a duplicated P1 coding sequence and that the downstream P1 copy, named P1b, has silencing suppression activity. In this study, we provide experimental evidence that P1b is a serine protease that self-cleaves at its C terminus but that its proteolytic activity is not essential for silencing suppression. In contrast, a putative zinc finger and a conserved basic motif in the N-terminal region of the protein are required for efficient silencing suppression. In vitro gel filtration-fast protein liquid chromatography and in vivo bimolecular fluorescence complementation assays showed that P1b binds itself to form oligomeric structures and that the zinc finger-like motif is essential for the self interaction. Moreover, we observed that CVYV P1b forms complexes with synthetic siRNAs, and this ability correlated with both silencing suppression activity and enhancement of Potato virus X pathogenicity in a mutational analysis. Together, these results suggest that CVYV P1b resembles potyviral HCPro and other viral proteins in interfering RNA silencing by preventing siRNA loading into the RNA-induced silencing complex.

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Figures

FIG. 1.
FIG. 1.
Conserved domains present in P1b-like proteins. (A) Genome maps of monopartite viruses from the family Potyviridae. The arrows represent protease activities. The question mark indicates that this proteolytic activity had not been demonstrated experimentally prior to this work. (B) Partial amino acid alignment of P1b of the ipomovirus CVYV (considered to start at amino acid 526 of the polyprotein) and P1s of the ipomovirus SPMMV and the tritimoviruses Brome streak mosaic virus (BStMV), Oat necrotic mottle virus (ONMV), and WSMV. Boxed amino acids are identical or chemically similar between the two ipomoviral sequences (green boxes), between the three tritimoviral sequences (yellow boxes), or between at least four of the aligned sequences (black boxes). Dashes represent gaps. The conserved domains are indicated below the sequence alignment. The position of the first residue of each aligned segment is indicated on the left side of the sequence. Cys and His residues predicted to form a zinc finger domain are marked with asterisks. The arrow indicates the predicted autocatalytic cleavage site. The residues that were mutated in this work and their positions in the P1b sequence are indicated above the alignment. (C) Schematic representation of CVYV P1b showing the locations of conserved domains and a plot of charge density along the protein.
FIG. 2.
FIG. 2.
CVYV P1b cleaves at its C terminus and accumulates in CVYV-infected cucumber plants. (A) Schematic representation of the N terminus of the CVYV polyprotein and the C-terminally TAP-tagged P1b reporter. Letters inside the boxes represent either conserved amino acids or conserved domains. LXKA, positively charged conserved motif; Zn-F, putative zinc finger; H D S, H221, D229, and S264 predicted to constitute the protease catalytic triad; and YC, amino acids at the predicted P1b-P3 junction. (B) Western blot analysis with PAP complex of extracts of leaf patches of N. benthamiana plants infiltrated with agrobacteria carrying empty pBin19 (lane 6), p35S-P1b-CTAP (lane 5), or derivatives of this plasmid with the indicated mutations (lanes 1 to 4), collected at 3 dpi. (C) Western blot analysis with anti-P1b serum of extracts of cucumber leaves systemically infected with CVYV (lane 1) or equivalent leaves of mock-inoculated cucumber plants (lane 2) and extracts of leaf patches of N. benthamiana plants infiltrated with agrobacteria carrying p35S-P1b (lane 3) or empty pBin19 (lane 4). The positions of prestained molecular mass markers (in kilodaltons; New England Biolabs) run in the same gels are indicated to the right of the panels. Blots stained with Ponceau red are shown at the bottom as a loading control.
FIG. 3.
FIG. 3.
Effects of mutations in CVYV P1b conserved domains on silencing suppression activity. N. benthamiana plants were coinfiltrated with agrobacteria carrying p35S:GFP and p35S:GF-IR plus empty pBin19 (vector), p35S-NTAP-P1b (wild type), or derivatives of this plasmid with the indicated mutations. (A) GFP fluorescence pictures taken under a UV lamp at 7 dpi. (B) Western blot analysis with PAP complex and anti-GFP antibodies (upper panels) and Northern blot analysis of GFP mRNA and siRNA (middle and bottom panels) of infiltrated leaves harvested at 7 dpi. A protein blot stained with Ponceau red and RNA agarose (25S rRNA) and polyacrylamide (5S + tRNA) gels stained with ethidium bromide are shown as loading controls.
FIG. 4.
FIG. 4.
Analysis of CVYV P1b self interaction by BiFC assay. N. benthamiana plants were coinfiltrated with agrobacteria carrying pBin61:P19 plus p35S-NYFP-P1b (wild type or the indicated mutants) plus p35S-CYFP-P1b (wild type or the indicated mutants). Plants coinfiltrated with agrobacteria carrying pBin61:P19 plus p35S-CYFP-P1b were used as a negative control (control). (A) Schematic representation of plasmids used in the assay. (B) YFP fluorescence pictures taken under a fluorescence microscope at 6 dpi. Bars, 50 μm.
FIG. 5.
FIG. 5.
Oligomerization of CVYV P1b in solution. N-terminally tagged CVYV P1b proteins purified by affinity chromatography were analyzed by gel filtration-FPLC. (A) Elution fractions of NTAP-P1b, intact or digested with acTEV protease (NCBP-P1b), were subjected to Western blot analysis with PAP complex or biotinylated calmodulin, respectively. (B) Elution fractions of NTAP-P1b mutants were subjected to Western blot analysis with PAP complex. Arrows indicate the elution positions of the following molecular mass markers: aldolase (158 kDa), serum albumin (68 kDa), and ovalbumin (50 kDa).
FIG. 6.
FIG. 6.
CVYV P1b binds siRNA in vitro. (A) Crude protein extract (2 μl) from agroinfiltrated leaves expressing NTAP-P1b (lanes 1 to 3) or TBSV P19 (lanes 2 to 5) and harvested at 3 dpi was incubated with 32P-labeled double-stranded siRNAs. The PAP complex was included in the binding mixtures loaded in lanes 2 and 5 (0.1 μl) and 3 and 6 (0.2 μl). (B) NTAP-P1b purified by affinity chromatography (250 nM) (lanes 3 to 6) or control bovine serum albumin (BSA; 250 nM) (lanes 1 and 2) was incubated with siRNAs. The PAP complex was included in the binding mixtures loaded in lanes 4 (0.02 μl), 5 (0.1 μl), and 2 and 6 (0.2 μl). Protein-siRNA complexes were resolved in polyacrylamide gels and revealed by autoradiography.
FIG. 7.
FIG. 7.
Null silencing suppression CVYV P1b mutants are unable to bind siRNAs. Crude protein extract (1 or 4 μl) from leaves infiltrated with agrobacteria carrying p35S-NTAP-P1b (wild type or the indicated mutants) or empty pBin19 (vector) and harvested at 3 dpi was incubated with 32P-labeled double-stranded siRNAs. Protein-siRNA complexes were resolved in polyacrylamide gels and revealed by autoradiography. The amount of NTAP-P1b protein present in the crude extracts was estimated by Western blot analysis with PAP complex (top panel). The silencing suppression activity of each NTAP-P1b mutant is indicated at the bottom.
FIG. 8.
FIG. 8.
CVYV P1b enhances PVX pathogenicity. (A) Schematic representation of Gateway-adapted T-DNA of pGWC-PVX with the CVYV P1b coding sequence inserted in its cloning site. (B) Symptoms of infection with wild-type PVX (empty) or PVX expressing CVYV P1b (wild type or the indicated mutants) in N. clevelandii and N. benthamiana plants. Photographs were taken at 21 dpi. (C) Western blot analysis with anti-CVYV P1b and anti-PVX CP sera of extracts of N. benthamiana leaves systemically infected with wild-type PVX (empty) or PVX expressing CVYV P1b (wild type or the indicated mutants). Healthy N. benthamiana leaves were also analyzed. The leaves were harvested at 10 dpi. A blot stained with Ponceau red is shown at the bottom as a loading control.
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