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1.
J Mol Biol ; 434(16): 167715, 2022 08 30.
Article in English | MEDLINE | ID: mdl-35798161

ABSTRACT

Viruses of the sobemovirus genus are plant viruses, most of which generate very important agricultural and financial losses. Among them, the rice yellow mottle virus (RYMV) is one of the most damaging pathogens devastating rice fields in Africa. RYMV infectivity and propagation rely on its protein P1, identified as a key movement and potential long-distance RNA silencing suppressor. Here we describe P1's complete 3D structure and dynamics obtained by an integrative approach combining X-Ray crystallography and NMR spectroscopy. We show that P1 is organized in two semi-independent and topologically unrelated domains, each harboring an original zinc finger. The two domains exhibit different affinities for zinc and sensitivities to oxidoreduction conditions, making the C-terminal P1 region a potential labile sensor of the plant redox status. An additional level of regulation resides on the capacity of P1 to oligomerize through its N-terminal domain. Coupling P1 structure information with site-directed mutagenesis and plant functional assays, we identified key residues in each zinc domain essential for infectivity and spread in rice tissues. Altogether, our results provide the first complete structure of a sobemoviral P1 movement protein and highlight structural and dynamical properties that may serve RYMV functions to infect and invade its host plant.


Subject(s)
Oryza , Plant Viruses , Viral Proteins , Zinc Fingers , Crystallography, X-Ray , Nuclear Magnetic Resonance, Biomolecular , Oryza/virology , Plant Viruses/pathogenicity , Protein Domains , Viral Proteins/chemistry , Viral Proteins/genetics , Zinc/metabolism
2.
Plant Cell ; 33(11): 3402-3420, 2021 11 04.
Article in English | MEDLINE | ID: mdl-34436604

ABSTRACT

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.


Subject(s)
Arabidopsis Proteins/genetics , Arabidopsis/genetics , Host-Pathogen Interactions , Plant Defense Against Herbivory/genetics , Plant Viruses/physiology , RNA, Double-Stranded/genetics , RNA, Plant/genetics , RNA-Binding Proteins/genetics , Arabidopsis/virology , Arabidopsis Proteins/metabolism , RNA, Double-Stranded/metabolism , RNA, Plant/metabolism , RNA-Binding Proteins/metabolism , Virus Replication
3.
Proc Natl Acad Sci U S A ; 117(20): 10848-10855, 2020 05 19.
Article in English | MEDLINE | ID: mdl-32371486

ABSTRACT

Grapevine fanleaf virus (GFLV) is a picorna-like plant virus transmitted by nematodes that affects vineyards worldwide. Nanobody (Nb)-mediated resistance against GFLV has been created recently, and shown to be highly effective in plants, including grapevine, but the underlying mechanism is unknown. Here we present the high-resolution cryo electron microscopy structure of the GFLV-Nb23 complex, which provides the basis for molecular recognition by the Nb. The structure reveals a composite binding site bridging over three domains of one capsid protein (CP) monomer. The structure provides a precise mapping of the Nb23 epitope on the GFLV capsid in which the antigen loop is accommodated through an induced-fit mechanism. Moreover, we uncover and characterize several resistance-breaking GFLV isolates with amino acids mapping within this epitope, including C-terminal extensions of the CP, which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Nb-mediated resistance to vector transmission. Together, these data provide insights into the molecular mechanism of Nb23-mediated recognition of GFLV and of virus resistance loss.


Subject(s)
Nepovirus/drug effects , Plant Diseases/immunology , Single-Chain Antibodies/chemistry , Single-Chain Antibodies/pharmacology , Animals , Antibodies, Viral/immunology , Capsid/chemistry , Capsid Proteins/chemistry , Capsid Proteins/drug effects , Cryoelectron Microscopy , Epitopes/chemistry , Models, Molecular , Nematoda/virology , Nepovirus/ultrastructure , Plant Diseases/virology , Plant Leaves/virology , Plant Viruses/immunology , Plant Viruses/physiology , Protein Conformation , Vitis
4.
Biomol NMR Assign ; 13(2): 345-348, 2019 10.
Article in English | MEDLINE | ID: mdl-31346897

ABSTRACT

RNA silencing describes a pan-eukaryotic pathway of gene regulation where doubled stranded RNA are processed by the RNAse III enzyme Dicer or homologs. In particular, plants use it as a way to defend themselves against pathogen invasions. In turn, to evade the plant immune response, viruses have developed anti-RNA silencing mechanisms. They may indeed code for proteins called "viral suppressor of RNA silencing" which block the degrading of viral genomic or messenger RNA by the plant. The Rice Mottle Virus is an African virus of the sobemovirus family, which attacks the most productive rice varieties cultivated on this continent. It encodes P1, a cysteine-rich protein described as a potential RNA silencing suppressor. P1 is a 157 amino-acid long protein, characterized by a high propensity to aggregate concomitant with a limited stability with time in the conditions used in structural studies. To overcome this problem, shorter fragments were also studied. This strategy enabled the assignment of more than 90% backbone resonances of P1. This assignment should set the base of future NMR investigation of the protein structure and of its interactions with rice cellular partners.


Subject(s)
Nuclear Magnetic Resonance, Biomolecular , Plant Viruses , Viral Proteins/chemistry
5.
Front Plant Sci ; 9: 70, 2018.
Article in English | MEDLINE | ID: mdl-29449856

ABSTRACT

Double-stranded RNA (dsRNA) plays essential functions in many biological processes, including the activation of innate immune responses and RNA interference. dsRNA also represents the genetic entity of some viruses and is a hallmark of infections by positive-sense single-stranded RNA viruses. Methods for detecting dsRNA rely essentially on immunological approaches and their use is often limited to in vitro applications, although recent developments have allowed the visualization of dsRNA in vivo. Here, we report the sensitive and rapid detection of long dsRNA both in vitro and in vivo using the dsRNA binding domain of the B2 protein from Flock house virus. In vitro, we adapted the system for the detection of dsRNA either enzymatically by northwestern blotting or by direct fluorescence labeling on fixed samples. In vivo, we produced stable transgenic Nicotiana benthamiana lines allowing the visualization of dsRNA by fluorescence microscopy. Using these techniques, we were able to discriminate healthy and positive-sense single-stranded RNA virus-infected material in plants and insect cells. In N. benthamiana, our system proved to be very potent for the spatio-temporal visualization of replicative RNA intermediates of a broad range of positive-sense RNA viruses, including high- vs. low-copy number viruses.

6.
Methods Mol Biol ; 1701: 169-187, 2018.
Article in English | MEDLINE | ID: mdl-29116505

ABSTRACT

Single-domain antibodies libraries of heavy-chain only immunoglobulins from camelids or shark are enriched for high-affinity antigen-specific binders by a short in vivo immunization. Thus, potent binders are readily retrieved from relatively small-sized libraries of 107-108 individual transformants, mostly after phage display and panning on a purified target. However, the remaining drawback of this strategy arises from the need to generate a dedicated library, for nearly every envisaged target. Therefore, all the procedures that shorten and facilitate the construction of an immune library of best possible quality are definitely a step forward. In this chapter, we provide the protocol to generate a high-quality immune VHH library using the Golden Gate Cloning strategy employing an adapted phage display vector where a lethal ccdB gene has to be substituted by the VHH gene. With this procedure, the construction of the library can be shortened to less than a week starting from bleeding the animal. Our libraries exceed 108 individual transformants and close to 100% of the clones harbor a phage display vector having an insert with the length of a VHH gene. These libraries are also more economic to make than previous standard approaches using classical restriction enzymes and ligations. The quality of the Nanobodies that are retrieved from immune libraries obtained by Golden Gate Cloning is identical to those from immune libraries made according to the classical procedure.


Subject(s)
Camelus/genetics , Cloning, Molecular/methods , Gene Library , Genetic Vectors , Peptide Library , Single-Chain Antibodies/genetics , Animals , Camelus/immunology , Single-Chain Antibodies/immunology
7.
J Mol Biol ; 425(14): 2423-35, 2013 Jul 24.
Article in English | MEDLINE | ID: mdl-23542010

ABSTRACT

Viral suppressors of RNA interference (VSRs) target host gene silencing pathways, thereby operating important roles in the viral cycle and in host cells, in which they counteract host innate immune responses. However, the molecular mechanisms of VSRs are poorly understood. We provide here biochemical and biophysical features of the dual suppressor/activator VSR P1 protein encoded by the rice yellow mottle virus. In silico analyses of P1 suggested common features with zinc finger proteins and native mass spectrometry unambiguously confirmed that recombinant P1 binds reversibly two zinc atoms, each with a different strength. Additionally, we demonstrate that the reaction of P1 with H2O2 leads to zinc release, disulfide bond formation, and protein oligomerization. A reversible protein modification by redox alterations has only been described for a limited number of zinc finger proteins and has never been reported for VSRs. Those reported here for P1 might be a general feature of Cys-rich VSRs and could be a key regulatory mechanism for the control of RNA silencing.


Subject(s)
Carrier Proteins/metabolism , RNA Interference , RNA Viruses/immunology , RNA Viruses/physiology , Viral Proteins/metabolism , Virus Replication , Carrier Proteins/chemistry , Carrier Proteins/genetics , Computational Biology , Disulfides/metabolism , Host-Pathogen Interactions , Hydrogen Peroxide/metabolism , Mass Spectrometry , Oryza/immunology , Oryza/virology , Oxidation-Reduction , Protein Multimerization , Protein Processing, Post-Translational , RNA Viruses/genetics , Viral Proteins/chemistry , Viral Proteins/genetics , Zinc/metabolism
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