Structural basis of nanobody recognition of grapevine fanleaf and of virus resistance loss

Igor Orlova,b,c,d,1, Caroline Hemmere,f,1, Léa Ackerere,f,g, Bernard Lorberh, Ahmed Ghanname, Vianney Poignavente, Kamal Hleibiehe, Claude Sauterh, Corinne Schmitt-Keichingere,f, Lorène Belvalf, Jean-Michel Hilyf,g, Aurélie Marmonierf, Véronique Komarf, Sophie Gerschf, Pascale Schellenbergere,f,2, Patrick Broni, Emmanuelle Vignef, Serge Muyldermansj, Olivier Lemairef, Gérard Demangeatf, Christophe Ritzenthalere,3, and Bruno P. Klaholza,b,c,d,3

aCentre for Integrative Biology (CBI), Department of Integrated Structural Biology, IGBMC, Université de Strasbourg, 67404 Illkirch, France; bInstitute of Genetics and of Molecular and Cellular Biology, Department of Integrated Structural Biology, Université de Strasbourg, 67404 Illkirch, France; cCentre National de la Recherche Scientifique, UMR 7104, 67404 Illkirch, France; dInstitut National de la Santé et de la Recherche Médicale, U964, 67404 Illkirch, France; eInstitut de Biologie Moléculaire des Plantes, UPR2357 du Centre National de la Recherche Scientifique, Université de Strasbourg, F-67084 Strasbourg, France; fUniversité de Strasbourg, Institut National de Recherche pour L’Agriculture, L’Alimentation et L’Environnement, Santé de la Vigne et Qualité du Vin, UMR-A 1131, 68000 Colmar, France; gInstitut Français de la Vigne et du Vin, 30240 Le Grau du Roi, France; hUniversité de Strasbourg, Architecture et Réactivité de l’ARN, UPR 9002, Centre National de la Recherche Scientifique, Institut de Biologie Moléculaire et Cellulaire, F-67084 Strasbourg, France; iCentre de Biochimie Structurale Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, 34090 Montpellier, France; and jCellular and Molecular Immunology, Vrije Universiteit Brussel, 1050 Brussels, Belgium

Edited by Wah Chiu, Stanford University, Stanford, CA, and approved March 23, 2020 (received for review August 8, 2019) Grapevine fanleaf virus (GFLV) is a picorna-like plant virus trans- outstanding interest as therapeutics against human diseases and mitted by that affects vineyards worldwide. Nanobody pathogens (9–11), including (12–14). Recent reports also (Nb)-mediated resistance against GFLV has been created recently, revealed their effectiveness in conferring resistance against plant and shown to be highly effective in plants, including grapevine, viruses. Thus, transient expression of Nbs against broad bean but the underlying mechanism is unknown. Here we present the mottle virus attenuated the spreading of the cognate virus in high-resolution cryo electron microscopy structure of the GFLV– Vicia faba (15). Recently, we showed that the constitutive ex- Nb23 complex, which provides the basis for molecular recognition pression of a single Nb (Nb23) that is specific to GFLV confers by the Nb. The structure reveals a composite binding site bridging monogenic resistance to a wide range of GFLV isolates in both over three domains of one capsid protein (CP) monomer. The struc- the model plant Nicotiana benthamiana and grapevine (16), but ture provides a precise mapping of the Nb23 epitope on the GFLV the molecular basis of GFLV recognition and the mechanism of capsid in which the antigen loop is accommodated through an resistance induced by Nb23 are unknown. Moreover, while one induced-fit mechanism. Moreover, we uncover and characterize of the homozygous N. benthamiana lines tested was fully resistant several resistance-breaking GFLV isolates with amino acids map- ping within this epitope, including C-terminal extensions of the CP, Significance which would sterically interfere with Nb binding. Escape variants with such extended CP fail to be transmitted by nematodes linking Grapevine fanleaf virus (GFLV), a picorna-like plant virus, se- Nb-mediated resistance to vector transmission. Together, these verely affects vineyards worldwide. While nanobodies (Nbs) data provide insights into the molecular mechanism of Nb23- confer resistance to GFLV in plants, the underlying molecular mediated recognition of GFLV and of virus resistance loss. mechanism of action is unknown. Here we present the high- resolution cryo electron microscopy structure of the GFLV–Nb structural biology | virus | GFLV | nanobody complex. It uncovers the epitope on the capsid surface, which is a composite binding site into which the antigen loop is ac- ineyards under production for wine grapes, table grapes, or commodated through an induced fit mechanism. Furthermore, Vdried grapes account for a total of 7.4 million hectares and we describe several resistance-breaking isolates of GFLV with more than 77.8 million metric tons worldwide, for a global trade reduced Nb binding capacity. Those escape variants that carry a value of 31 billion Euros (1). This positions grapevine ( vi- C-terminal extension in the capsid protein also fail to be nifera) as a highly valuable crop. However, grapevine is suscep- transmitted by nematodes. Together, these data provide tible to a wide range of pathogens, including viruses. With more structure–function insights into Nb–GFLV recognition and the than 70 viral species belonging to 27 genera identified so far, molecular mechanism leading to loss of resistance. grapevine represents the cultivated crop with the highest number of infecting viruses (2, 3). While the pathogenicity of all these Author contributions: I.O., J.-M.H., S.M., O.L., G.D., C.R., and B.P.K. designed research; I.O., viruses is not established, a number of them cause severe dis- C.H., L.A., B.L., A.G., V.P., K.H., C.S., C.S.-K., L.B., A.M., V.K., and S.G. performed research; I.O., C.H., L.A., B.L., A.G., V.P., K.H., C.S., C.S.-K., L.B., J.-M.H., A.M., V.K., S.G., P.S., P.B., eases, such as the viruses responsible for fanleaf, leafroll, and E.V., S.M., O.L., G.D., C.R., and B.P.K. analyzed data; and C.R. and B.P.K. wrote the paper. rugose wood diseases that have been reported in nearly all vine- The authors declare no competing interest. growing areas. Grapevine fanleaf virus (GFLV) is a - This article is a PNAS Direct Submission. transmitted virus (4) and principal causal agent of grapevine Published under the PNAS license. fanleaf degeneration. It belongs to the genus of the Picornavirales Data deposition: Cryo electron microscopy map and coordinates of the atomic model can family in the order and possesses a be found in the Protein Data Bank (accession no. 5FOJ) and the Electron Microscopy Data bipartite single-stranded positive-sense RNA genome (5). The Bank (accession no. 3246). structure of the icosahedral capsid is known from our previous 1I.O. and C.H. contributed equally to this work. T = crystal structure analysis (6) and follows a pseudo 3 tri- 2Present address: University of Sussex, Falmer Brighton, BN1 9RH, United Kingdom. angulation. It is composed of 60 copies of the capsid protein 3To whom correspondence may be addressed. Email: [email protected] or klaholz@ (CP), which folds into three jelly roll β sandwiches (6). igbmc.fr. Since their discovery (7), single-domain antigen-binding frag- This article contains supporting information online at https://www.pnas.org/lookup/suppl/ ments of camelid-derived heavy chain-only antibodies, also doi:10.1073/pnas.1913681117/-/DCSupplemental. known as nanobodies (Nbs) (8), have proven to be of First published May 5, 2020.

10848–10855 | PNAS | May 19, 2020 | vol. 117 | no. 20 www.pnas.org/cgi/doi/10.1073/pnas.1913681117 Downloaded by guest on October 2, 2021 to GFLV, another line showed infection at low frequency reveals that Nb23 bridges over 3 domains of the CP, and it (3.2%), suggesting the existence of escape variants (EVs) con- provides unprecedented insights into the epitope and the resi- taining resistance-breaking (RB) mutations susceptible to in- dues involved in the interface, including the mechanism of mo- terfering with Nb23–GFLV interaction (16). lecular recognition of the antigen-binding loops. We find a To address the molecular basis of Nb23–GFLV recognition, perfect correlation between mutations detected in RB variants we determined the cryo electron microscopy (cryo-EM) structure and the Nb23 epitope observed in the structure, which explains of the GFLV–Nb23 complex at high resolution. The structure the resistance loss in EVs. In agreement with the fact that the BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 1. Structural analysis of the GFLV–Nb23 complex by cryo-EM. (A) Typical cryo-EM micrographs of the GFLV–Nb23 complex; most particles are intact and comprise the genome inside (an empty particle is indicated at the bottom with an arrow). (Scale bar, 500 Å.) (B) Local resolution estimation, section through Nb23 and the capsid. (C) Plot showing FSC versus spatial frequency of the icosahedral averaged reconstruction. Average resolution of the reconstructions is given where the FSC curve crosses below a correlation value of 0.143. (D) Overall structure of the GFLV–Nb23 complex with Nb23 shown in cyan and the capsid in radial coloring (SI Appendix, Figs. S1 and S2), including a segmentation of the 3D reconstruction for the Nb23 and the GFLV capsid parts, and a cross section through the icosahedral reconstruction (left to right); the icosahedron is shown as an overlaid polygon. (E) Sections through the refined 3D reconstruction (from top to bottom of the 3D map). (F) Sections through the 3D reconstruction while under refinement; at lower resolution, the overall parts are clearly visible: Nb, capsid (secondary structures of the CP are visible) and genome inside the capsid (averaged out due to the icosahedral symmetry that is imposed during 3D reconstruction while the genome might have a different structural organization).

Orlov et al. PNAS | May 19, 2020 | vol. 117 | no. 20 | 10849 Downloaded by guest on October 2, 2021 conformational surface epitope recognized by the Nb23 par- comprises the two strands of the capsid β-sheet region 370 to 391 tially covers a cavity involved in vector transmission (6, 16, (domain A of the CP) in which Asp371 forms a salt bridge and 17), we show that EVs preexist in natural viral populations at Thr373 a hydrogen bond with Arg55Nb23. An additional anchor low frequency. We also uncover that the most frequently point of the antibody is provided through Thr58Nb23 and foundEVswithextendedCParedeficientintransmissionby Lys65Nb23, which interact with Asn375 located in a CP loop nematodes. (Fig. 2E). In the second β-strand of the 370 to 391 region, the backbone of Val379 interacts with Thr110Nb23,andtheSer380 Results backbone and Met381 form contacts with Ser109Nb23 and To gain molecular insights into the mechanism of GFLV rec- Trp108Nb23, respectively. CDR3 forms a long antigen-binding ognition by Nb23, precisely map the epitope, and decipher the loop with a short α-helical turn carrying Leu104Nb23,whichis interactions between Nb23 and the CP, we decided to analyze accommodated within a hydrophobic pocket formed by Tyr216, the structure of the GFLV viral particle decorated with Nb23. Phe370, Phe502, Val504, and an alanine cluster formed by The structure of the GFLV–Nb23 complex was determined by residues 387, 388, and 391 (Fig. 2D). Trp108Nb23 appears to be high-resolution single-particle cryo-EM, using angular reconsti- a key residue, in that it inserts into the binding site within a tution (18, 19), and refined to an average resolution of 2.8 Å cavity at the junction of the A and B domains of the CP to form (Fig. 1 and SI Appendix, Figs. S1 and S2). The cryo-EM map was π-stacking interactions with Phe370 (Fig. 2E). Importantly, the used to build an atomic model with proper model geometry and Phe502 and Val504 residues at the very C-terminal end of the statistics, using the Phenix software (20), which involved manual CP are in direct contact with Nb23 and form hydrophobic model building and refinement notably of the antigen-binding contacts with Leu104Nb23,Tyr107Nb23, and Trp108Nb23.Fi- loop region (Methods). The three-dimensional (3D) reconstruc- nally, Lys54Nb23 interacts with the free carboxylate moiety tion displays the 3 core parts of the complex: the virus capsid, the of Val504. Nb at the outer surface, and the genome inside (Fig. 1). The Interestingly, comparison of the CP structures in the presence structure reveals that Nb23 binds at the surface of the GFLV and absence of Nb23 (Fig. 2F) reveals that Nb23 induces a capsid in the vicinity of the 5-fold axis (Fig. 1 D–F), at the level of conformational change of the CP 370 to 375 loop region to ac- the individual CP monomers that form the pentamers of the commodate the Nb23 side-chains. This significant transition in- pseudoT = 3 icosahedral capsid. The outer isocontour surface of dicates that the molecular recognition of the CP occurs through the GFLV–Nb23 reconstruction (Fig. 1 and SI Appendix, Fig. S1) an induced fit mechanism in which the Phe370 and Asp371 side- shows that the Nb23 molecules are positioned far enough from chains flip over in opposite directions (Fig. 2F). Without this each other to allow 60 of them to attach and reach full 1:1 conformational adaptation, Asp371 would clash with Trp108Nb23, stoichiometric binding without bridging neighboring CPs. Con- which instead needs Phe370 to switch position to form stacking sistent with this finding and a size increase of the complex (16), interactions with Trp108, whereas Asp371 rotates to form a sta- we observe the same stoichiometry by native gel analysis upon bilizing salt bridge interaction with Arg55Nb23.Thisselection titration of GFLV with increasing amounts of Nb23 (SI Appen- mechanism may contribute to Nb23 specificity toward the GFLV dix, Fig. S3). antigen (16). Local resolution estimation shows that while Nb23 is moder- The structure shows that the interface between Nb23 and CP ately ordered at the periphery (3.5 to 5 Å resolution toward the is highly complementary, and also includes the C-terminal CP C terminus), it is very well defined in the region of interest, residue Val504 that is recognized by Lys54Nb23. One can there- enabling a precise analysis of the interactions within the interface fore hypothesize that disrupting the molecular interface of the between Nb23 and the capsid (Fig. 2). The part of Nb23 that is epitope region would lead to resistance loss. Interestingly, Nb- oriented toward the capsid exhibits clear side-chains densities (SI mediated resistance to GFLV in N. benthamiana lines that Appendix, Fig. S2) and includes the important antigen-binding constitutively express Nb23:EGFP displays various degrees of loops (Fig. 2 D and E). The viral capsid is well defined (including susceptibility to infection, indicating that GFLV could overcome the very N- and C-terminal ends) and comprises the A, B, and C Nb-mediated resistance (16). To further explore this partial re- domains of each CP (Fig. 2 A and B). The interaction region sistance breakdown, we forced our two resistant lines toward the between Nb23 and the capsid involves residues of the com- emergence of infection events by applying high inoculum pres- plementarity determining regions (CDRs) 2 and 3 of Nb23, as sure (3 μg vs. 300 ng of virus). Under such stringent conditions, well as the two neighboring CP domains A and B (2 of the 3 resistance was indeed overcome by 21 d postinoculation (dpi) in jelly roll β sandwiches of the CP). These structural elements 30% and 40% of plants from lines 23EG38-4 and 23EG16-9, from the same CP monomer polypeptide chain jointly form a respectively (Fig. 3 A and B). To address whether the infection composite binding site with two core interacting regions, events observed were due to the emergence of bona fide GFLV denoted1and2(Fig.2A, D,andE), adding up to a total in- RB variants and considering Nb23 is specific to GFLV (16), we teraction surface of ∼1,100 Å2. This large binding area is con- characterized the virus progeny by sequencing the CP from sistent with the formation of a stable Nb23–GFLV complex and Nb23-expressing plants that showed systemic infection. Re- with the high affinity of Nb23 for the virus, as determined by markably, the analysis reveals that all suspected RB events cor- microscale thermophoresis (Kd = 9.13 ± 2.91 nM; n = 3; SI respond to viruses with mutations in the CP coding sequence Appendix,Fig.S3). (Fig. 3C). The most frequent mutation (five independent events) The structure allows a precise mapping of the Nb23 epitope on is a nucleotide insertion near the 3′ end of the CP gene that leads the GFLV capsid and identifies the key residues of Nb23 CDR2 to a frameshift responsible for nonconservative mutations of CP and CDR3 and of GFLV involved in the specific molecular residues 503 and 504 and the addition of 12 extra amino acids at recognition events occurring on complex formation. Within the the C terminus of the CP (Fig. 3C). The second most represented two core interacting regions (Fig. 2 D and E), a high level of mutation (three occurrences) was the suppression of the stop specific interactions is provided through a series of noncovalent codon leading to a CP with three (CP+3) or five (CP+5) extra bonds (SI Appendix, Fig. S4). Region 1 (Fig. 2D) comprises loop C-terminal amino acids, the latter being the consequence of a region 212 to 216 (βC’’) of domain B of the CP in which Thr212 second mutation. The remaining two mutations are missense interacts with Asp100Nb23 and Tyr32Nb23, Lys214 forms hydrogen mutations mapping to residues 216 (Tyr216His) and 502 bonds with the carbonyl backbone of Ala101Nb23 and Ile102Nb23, (Phe502Leu) of the CP, both of which are directly involved in and Tyr216 interacts with the backbone and forms hydrophobic Nb23-virus recognition (Fig. 2 D and E). Remarkably, most RB contacts with the side chain of Leu104Nb23. Region 2 (Fig. 2E) variants (8 of 10) display a mutated CP with variable C-terminal

10850 | www.pnas.org/cgi/doi/10.1073/pnas.1913681117 Orlov et al. Downloaded by guest on October 2, 2021 BIOPHYSICS AND COMPUTATIONAL BIOLOGY

Fig. 2. Structural analysis of the Nb23 epitope on the GFLV capsid. (A and B) Ribbon diagram of a CP monomer and its associated Nb23, with side and top views of the CP and Nb23 (cyan), which sits onto a composite binding site comprising the 3 domains of the CP monomer shown in blue (A), red (B), and green (C); the C terminus of the CP is indicated. (C) Positions of CP and Nb23 (cyan) with respect to symmetry operators on the icosahedral reconstruction. (D and E) Close-up views of the boxed regions 1 and 2 in A, showing the detailed molecular interactions (hydrogen bonds are indicated by dotted lines; color code as in B). (F) Comparison of apo-capsid (in yellow) and Nb23-bound capsid (in blue) showing a conformational adaptation of the capsid residues Phe370 to Asn375, reminiscent of an induced fit mechanism.

extensions of 3 (CP+3), 5, or 12 residues (Fig. 3C); such an sequencing were unequivocally identified in our HTS datasets. extension would sterically clash with Nb23 and prevent recog- For example, the Tyr216His was found in 2 of the 12 grapevine nition of the free carboxylate group Val504, as evident from the samples tested. In contrast, STOP505Gln was detected in all structure (Fig. 2E). Accordingly, GFLV-CP+3 and GFLV- samples at an average rate close to 0.4%, together with other Tyr216His mutants are recognized only very poorly by Nb23 in missense mutations (e.g., STOP505Tyr, STOP505Ser, or STOP505- double antibody sandwich–enzyme-linked immunosorbent as- Glu), leading to a CP with extra C-terminal amino acid extensions. say (DAS-ELISA) (while they still are with conventional anti- Such assortment of missense mutations was observed at all ex- GFLV antibodies; Fig. 3D). To confirm that C-terminal ex- amined positions (Fig. 3F). Similarly, most RB mutations were tensions of the CP are RB mutations and to exclude the con- also detected in the viral RNA isolated from viruliferous vector tribution of non-CP residues in overcoming resistance, the nematodes (Fig. 3F), underscoring that most RB variants preexist CP+3 mutation was introduced into the GFLV-GHu infectious in natural viral populations at low frequencies as part of the viral clone (21). The resulting mutant fully infects both control and quasispecies, as defined in Domingo et al. (22). Nb23-expressing plants (100% infection), in contrast to wild- Finally, to address the question of whether the EVs would still type GFLV-GHu that infects only control plants (Fig. 3E), be active in vector transmission, we tested GFLV-Tyr216His, confirming that the CP+3 mutation is sufficient to overcome Phe502Leu, and CP+3 mutants for transmission by nematodes. Nb-mediated resistance. While both single-point mutants were still vectored, transmission We next investigated the possible preexistence and frequency of GFLV-CP+3 was abolished (Fig. 3G), despite its ability to of such mutations in natural viral populations from grapevines form viral particles (Fig. 3H). This uncovers an important and nematodes by high-throughput sequencing (HTS) data function of residues in the vicinity of the C-terminal Val504 in mining (Fig. 3F and Dataset S1), with a focus on the codons GFLV transmission by Xiphinema index. Remarkably, as can be encoding Tyr216, Phe502, and the amber stop codon identified seen in the structure, the Nb23 epitope (delineated in blue; to be at the origin of EVs (Fig. 3F). We also looked at the codons Fig. 4 A and B) and the ligand-binding pocket (LBP; delineated encoding Phe370 and Asp371 due to the importance of these in magenta; Fig. 4B) partially overlap on the capsid surface residues in the interaction with Nb23, as seen in the structure (Fig. 4 A and B). The LBP is a surface-exposed positively (Fig. 2; see list in SI Appendix, Fig. S4). As a control, we analyzed charged pocket near the 3-fold axis, whose walls are formed the codons encoding Met302 and Trp119, two outer-exposed essentially by the βG-βH, βB-βC, and βC’βC’’ loops within the residues of the CP excluded from the Nb23 epitope and likely B-domain of the CP (6). Previous mutagenesis and not involved in α/β structures nor in the virus transmission by loss-of-function studies have demonstrated the essential function nematodes (6, 17). All point mutations discovered via Sanger of residues from the βG-βH (6) and from the βB-βC (17) loops in

Orlov et al. PNAS | May 19, 2020 | vol. 117 | no. 20 | 10851 Downloaded by guest on October 2, 2021 Fig. 3. Molecular characterization of EV. (A) Symptom analysis of plants from lines EG11-3 (green), 23EG16-9 (red), and 23EG38-4 (orange) at 7, 14, and 21 dpi on inoculation with 3 μg of GFLV-GHu. (B) GFLV detection by DAS-ELISA at 21 dpi. Each dot corresponds to a single plant sample and represents the mean relative absorbance at 405 nm of experimental duplicates. Percentage of infections are indicated below each column. (C) Mapping of mutations found in EV. Mutations were found in the ORF encoding the CP of GFLV (red for substitutions and blue for insertions). In total, sequences from 10 independent EV were obtained and occurrence of mutations is provided for each type of mutant. CP sequence of GFLV-GHu from residues 215 to 217 and 501 to 517 is provided. CP coding sequences are boxed and corresponding protein sequences are provided here. (D) N. benthamiana infected with GFLV-GHu, GFLV-CP+3, or GFLV-

Tyr216His were tested by DAS-ELISA with either conventional antibodies or Nb23 for detection. Conventional antibodies recognize all GFLV isolates contrarily to Nb23 that fails to detect GFLV-CP+3 and GFLV-Tyr216His, indicating reduced binding of Nb23 to GFLV EV. (E) Susceptibility of N. benthamiana transgenic lines toward GFLV GHu or GFLV-CP+3. Plants were tested by DAS-ELISA for GFLV at 21 dpi. (F) HTS data mining on 13 samples infected with GFLV (12 from grapevines, upper histograms and 1 from nematodes, lower histograms). We focused on seven positions within the CP, with residues in red involved in Nb23- CP interactions, and found in EV, in orange, those involved in Nb23-CP interactions based on the structural analysis (Fig. 2), and in black, those exposedonthe outer surface of the capsid but not interacting with Nb23 nor involved in the viral transmission by nematodes. The underlined red residues were found mutated in EV. Each colored bar corresponds to the percentage of a missense mutation occurrence with ± SE (with the number of event per mutation/site being indicated above). (G) Transmission efficiency of GFLV-GHu and GFLV-CP+3 by nematodes. Dots correspond to DAS-ELISA results from individual bait plants and represent the mean relative absorbance at 405 nm of experimental replicates normalized against maximum assay values. The asterisk indicates statistically significant differences (two-tailed paired t test; P = 0.022). Number of plants tested (n) and percentage of infections (%) are provided below each column. NC, healthy plants. (H) Negative staining EM image of immunogold-labeled viral particles isolated from GFLV-CP+3 infected plants.

10852 | www.pnas.org/cgi/doi/10.1073/pnas.1913681117 Orlov et al. Downloaded by guest on October 2, 2021 Fig. 4. Fingerprint of Nb23 on the GFLV capsid and mechanism of resistance loss. (A) Fingerprint of the Nb23 binding site on the capsid for one icosahedral asymmetric unit (black triangle, see also SI Appendix, Fig. S1) in which the polar angles θ and ϕ represent latitude and longitude, respectively. The coloring is according to the radial distance of the surface from the center of the particle; coloring of a CP monomer by domains as in Fig. 2B.(B) Fingerprint showing the projected surfaces on the GFLV capsid. The Nb23 footprints and projected surfaces are delineated in yellow and cyan, respectively, the LBP in magenta and mutated residues in GFLV EV shown in white, together with amino acid positions. The mosaic background shows the amino acids that form the viral surface. (C) Schematic representation of the molecular mechanism in which the C-terminal extension of GFLV EV would clash with Nb23, thus leading to resistance loss.

GFLV transmission by nematodes. However, the function of the extensions of the CP, also in grapevine upon natural transmission βC’βC’’ loops and neighboring residues in this mechanism was of the virus by nematodes. Their influence on the robustness and only predicted (6). Here we show that residues, which belong to durability of the resistance in grapevine under natural conditions this area in the LBP (in particular Val504), when mutated, abolish remains to be determined, especially when aiming at deploying GFLV transmission. The present structure–function analysis Nb-mediated resistance (16) as an antiviral strategy in vineyards. thus clarifies why RB mutations not only hinder Nb23 binding Finally, our structural data based on purified particles deco- but also, in the case of the C-terminal extensions, interfere with rated with 60 Nbs indicate that Nb23 has no virus-destabilizing BIOPHYSICS AND COMPUTATIONAL BIOLOGY GFLV transmission. activity in vitro. Hence the Nb-induced capsid destabilizing ac- Taken together, this study provides insights into the molecular tivity proposed for norovirus-specific Nbs (23) does not apply for mechanism of Nb23-mediated recognition of GFLV, including GFLV and likely does not contribute to the Nb-mediated re- the location of the Nb next to the fivefold symmetry axes on the sistance mechanism in planta. Whether Nb23 stabilizes viral capsid (Fig. 1) and the detailed description of the interface be- particles, interferes with cell to cell movement, and/or leads to tween the GFLV epitope and Nb23 (Fig. 2). The epitope consists the rapid degradation/clearance of the virus in vivo is unknown of a composite binding site involving domains A, B, and C of the and remains to be addressed in future studies. CP that interacts with the CDR2 and CDR3 of Nb23 (Fig. 2). Remarkably consistent with the epitope seen in the cryo-EM Methods structure, the RB mutations all map to neighboring residues Transgenic N. benthamiana Lines. Homozygous lines EG11-3 expressing EGFP exposed on the outer surface of the GFLV capsid (Fig. 3 and SI and 23EG16-9 and 23EG38-4 expressing Nb23 fused to EGFP were described Appendix, Fig. S4), which we show preexist at low frequency in in Hemmer et al. (16). virus quasispecies (Fig. 3). Specifically, the structure explains the mechanism of action of the C-terminal extension mutants in Inoculations. Plants were inoculated either mechanically by rub inoculation overcoming resistance: the extra three, five, or 12 residues at the using carborundum powder, or naturally via nematodes. Mechanical inoc- C terminus of the CP from GFLV EVs would interfere with ulations were performed with either purified virus at 300 ng or 3 μg per Nb23 binding due to steric clashes and loss of specific hydrogen inoculum or with crude sap from infected plants. bonds (in particular between the free carboxylate group of the CP last residue, Val504, and the Lys54 region; Fig. 2E). This GFLV DAS-ELISA Assessment of Nb23 In Vitro Binding to EV. Frozen leaf sam- Nb23 ples from infected plants were ground in extraction buffer (35 mM Na HPO , would prevent contacts with CP Val504 and occlude the highly 2 4 15 mM KH PO at pH 7.0) in a 1:5 wt/vol ratio. Virus detection was per- specific interface necessary for Nb23-GFLV recognition 2 4 C formed on twofold serial dilutions of clarified extracts by DAS-ELISA, using (Fig. 4 ). This manner of interfering with Nb23 binding and GFLV antibodies (Bioreba, catalog #120275) diluted 1,000-fold in coating

prevent GFLV neutralization is likely more potent than single- buffer (15 mM Na2CO3, 35 mM NaHCO3 at pH 9.6) as capture antibody and residue missense mutations such as Tyr216His or Phe502Leu, either GFLV antibodies coupled with alkaline phosphatase (Bioreba, catalog which may explain why most escape mutations consist of #120275) diluted 1,000-fold or Strep II-tagged Nb23 at 1 μg/mL in conjugate C-terminal extensions of the CP. Remarkably, these specific RB buffer (10 mM phosphate-buffered saline [PBS], 0.1% wt/vol bovine serum mutations that lead to resistance loss simultaneously result in albumin [BSA], 0.05% vol/vol Tween 20 at pH 7.4) as detection antibody. In transmission deficiency due to the overlap between the epitope the latter case, an additional incubation with streptavidin-alkaline phos- and the LBP (Fig. 4). This demonstrates that the RB mechanism phatase (Jackson Immunoresearch, catalog #016-050-084) at 1:10,000 in observed here has its fitness cost: by maintaining its capacity to conjugate buffer was performed before detection with paranitrophenyl infect resistant plants, GFLV has evolved in a manner leading to phosphate (Interchim) at 1 mg/mL in substrate buffer (1 M diethanolamine loss of transmission by nematodes. Although the principles are at pH 9.8). Negative control consisted of noninoculated healthy plants. Ab- sorbance at 405 nm (A ) was recorded after 1 h of substrate hydrolysis. different, both Nb-mediated resistance and EVs that fail to be 405nm Results are presented as mean absorbance of experimental duplicates ±SE transmitted from plant to plant by nematodes contribute to normalized against maximum assay value. prevent the progression of the disease. Considering that the RB N. benthamiana mutants were isolated from a herbaceous host ( Characterization of RB Isolates. Leaf samples from Nb23-expressing N. ben- expressing Nb23:GFP) under laboratory conditions, it would be thamiana that tested positive by ELISA for GFLV were used for immuno- interesting to investigate the emergence of RB isolates, in par- capture RT-PCR and gene encoding the CP of GFLV sequenced by Sanger ticular the emergence of GFLV isolates with C-terminal sequencing, as described in Martin et al. (24).

Orlov et al. PNAS | May 19, 2020 | vol. 117 | no. 20 | 10853 Downloaded by guest on October 2, 2021 Synthetic GFLV-CP+3 Construct. GFLV-CP+3 point mutation was introduced quartz cuvettes. For every 20-μL sample, 10 successive measurements were into full-length GFLV-GHu RNA2 cDNA infectious clone (pG2) (21) as a BglII/ performed at 20 °C with constant amounts of GFLV (0.1 mg/mL). Data were SalI fragment following an overlap extension PCR amplification, using pG2 processed with manufacturer’s DTS software (version 6.01). GFLV-F13 as template. GFLV-GHu RNA1, GFLV-GHu RNA2, and GFLV-CP+3 RNA2 cap- remained soluble in 20 mM Tris·HCl at pH 8.3 to 8.5 upon addition of Nb23. ped transcripts were synthesized from corresponding SalI/XmaI or EcoRI/SalI The latter composition was chosen for the preparation of cryo-EM grids in linearized cDNAs clones by in vitro transcription with mMESSAGE which GFLV at 0.5 mg/mL led to exploitable images. mMACHINE T7 kit (Ambion) according to manufacturer’s instructions. For infections, combination of appropriate RNA1 and RNA2 transcripts was used Structure Determination. Nb23 was mixed with purified GFLV-F13 at a 70:1 or to mechanically inoculate N. benthamiana plants at the 4- to 6-leaf stage. 100:1 molecular ratio, so as to saturate the theoretical maximum number of 60 possible binding Nbs per capsid. Samples of the freshly prepared GFLV– Transmission Assays. Transmission assays by nematodes were performed using Nb23 complex (2.5 μL of a 0.5 mg/mL solution) were applied to 300 mesh a two-step transmission procedure according to Marmonier et al. (25). holey carbon Quantifoil R1.2/1.3 grids (Quantifoil Micro Tools, Jena, Ger- During a 6-wk acquisition access period, ca. 300 aviruliferous X. index per many), blotted with filter paper from both sides in the temperature- and plant were left in contact with roots of systemic infected N. benthamiana humidity-controlled Vitrobot apparatus (FEI, Eindhoven, Netherlands), T = grown in a 3:1:1 vol/vol ratio of sand, loess, and clay pebbles soil. Source 20 °C, humidity 99%, blot force 4, blot time 0.5 s, and vitrified in liquid plants were then substituted with healthy N. benthamiana for an 8- to ethane precooled by liquid nitrogen. Images were collected on the in-house 10-wk inoculation access period, at the end of which virus transmission was spherical aberration-corrected Titan Krios S-FEG instrument (FEI) operating tested on roots of bait plants by DAS-ELISA. GFLV-GHu was used as positive at 300 kV acceleration voltage and at an actual underfocus of −0.6 to −6.0 and healthy source plants as negative controls. Values exceeding those of μm, using the second-generation back-thinned direct electron detector negative controls by a factor of 2.4 were considered positive. CMOS (Falcon II) 4,096 × 4,096 camera and automated data collection with His6-tagged and Strep-Tagged II Nb23 were produced as described in EPU software (FEI), using seven frames over a 1-s exposure (dose rate of − − − Hemmer et al. (16). ∼40 e Å 2·s 1). The calibrated magnification (based on the fit of the GFLV crystal structure into the cryo-EM map) is 127,272×, resulting in 0.527 Å pixel Native Agarose Gel Electrophoresis. Native gel electrophoresis of purified size at the specimen level (which is close to the nominal pixel size of 0.533 Å, GFLV was performed in 1% wt/vol agarose gels in 1.0× TA buffer (20 mM 1.1% difference). Before semiautomatic particle picking using IMAGIC (19), TrisBase, 0.06% vol/vol acetic acid at pH 9). For nucleic acids detection, 5 μg stack alignment was performed using the whole image motion correction of virus particles were diluted in loading buffer (10% vol/vol glycerol, 25 mM method (28), and correction of the contrast transfer function was done by Hepes at pH 9) supplemented with ethidium bromide (EtBr) at 0.1 μg/mL phase flipping using IMAGIC. After centering by alignment against the total After electrophoretic separation, the EtBr-prestained gel was first processed sum reference image, 2D classification in IMAGIC was used to remove bad or for nucleic acid content, using the Gel Doc system (Bio-Rad) equipped with a empty particles, leaving 6,139 of 9,502 particles (selected from 1,055 im- 302-nm excitation source and a 520- to 640-nm band-pass emission filter ages). For the first steps of image processing, the images were coarsened by before Instant Blue protein staining. a factor of four (binned 4×), and further refinement was achieved with two times coarsened data. The structure was determined via common lines angle Microscale Thermophoresis. Microscale thermophoresis (MST) measurements assignment and refined with the anchor set angular reconstitution pro- were performed using a Monolith NT115 instrument (NanoTemper Tech- cedure, as implemented in IMAGIC, using final search angles of 0.05°. nologies GmbH), using Nb23 and purified GFLV-derived virus-like particles in Thus, the structure determination, and also refinement, were done en- which enhanced GFP is encaged instead of mRFP, as described in Belval et al. tirely using common line methods for Euler angle assignments and re- (26). The concentration of the fluorescent virus-like particles was kept con- finement, which are less biased and require no starting reference. The stant at a concentration of 100 nM, while the concentration of Nb23 varied resolution was estimated by Fourier shell correlation (FSC) calculations, in- from 0.07 to 1.0 μM. Samples were diluted in MST optimized buffer (20 mM dicating an average resolution of 2.8 Å according to the 0.143 and half-bit Tris·HCl buffer at pH 9.5). Each experiment consisted of 15-point titration resolution criteria (29, 30), using IMAGIC (19); map-model correlation cal-

series in which the Nb concentrations were generated as a 1:1 dilution of culations [using Phenix (20)] suggest an average resolution of ∼3.5 Å (dmodel μ virus-like particles. For the measurements, 10 L of the different dilutions of in Phenix mtriage, d99 is 3.2 Å resolution for the unmasked reconstruction), samples were transferred into hydrophilic-treated capillaries (premium probably because there is averaging in the resolution estimation between coated capillaries, NanoTemper Technologies) and measured after a 10-min the well-defined CP and CP–Nb23 interface and the less ordered solvent- equilibration at room temperature. The measurements were performed (n = exposed parts of Nb23 (3.5 to 5 Å resolution toward the C terminus but 3) at 40% light-emitting diode (LED), and 20%, 40%, and 80% MST power. still well visible in medium-resolution maps [SI Appendix, Fig. S1 A and B]; we Laser-on time was 30 s, and laser-off time was 5 s. MST data were analyzed kept the atomic model of these Nb23 regions in the overall structure as the using the program PALMIST (biophysics.swmed.edu/MBR/software.html), fold is known from other structures and places well into the nonfiltered and evaluated using the appropriate model (1:1), according to Scheuermann map). Because the exterior part of the Nb23 structure is less well ordered, et al. (27). and therefore less resolved (Fig. 1B), this might also have an influence on the shape of the FSC curve. However, there is no doubt that the features re- Immunosorbent Electron Microscopy. GFLV-CP+3-infected leaves were ground solved in the map allow visualizing side-chains in the capsid and in the in-

in phosphate buffer (35 mM Na2HPO4,15mMKH2PO4) and centrifuged at terface region with Nb23, which is required for the description of the 3,000 × g for 5 min, and clarified samples were incubated for 2 h on carbon- residues at the CP–Nb23 interface, consistent also with the fact that the coated Formvar nickel grids (300 mesh; Electron Microscopy Science, PA) atomic model refined well, as illustrated from the good fit in the well- coated with anti-GFLV polyclonal antibodies (Bioreba AG, catalog #120475) resolved regions (SI Appendix, Fig. S2) and the overall good geometry (SI at 1:200 dilution. After blocking with 2% wt/vol BSA, 10% vol/vol normal Appendix, Fig. S1E). Local resolution values were calculated with ResMap goat serum, 0.05% Triton-X100 in 22.5 mM Hepes at pH 8 for 30 min, grids (31). Map interpretation was done using Chimera (32) and COOT (33), were incubated with monoclonal anti-GFLV (Bioreba, catalog #120475) at starting from the available atomic model of the GFLV crystal structure (PDB 1:150 dilution for 30 min and immunogold labeling performed using anti- IDs: 4V5T and 2Y7T) (6) and of a homology model of Nb23 that we derived mouse antibodies conjugated to 10 nm colloidal gold particles at 1:50 di- from the cAb-Lys3 VHH antibody domain, using the Swiss-Model Server lution for 30 min (British Biocell International, catalog #EM.GAMA10). The (https://swissmodel.expasy.org/, starting model: PDB ID: 1XFP; ref. 34, 68% experiment was performed at room temperature, and washes were done sequence identity with Nb23). Initial atomic model building was done by a with phosphate buffer between all steps. Observations were realized after rigid body fitting, followed by extensive manual model building in COOT negative staining with 1% m/v ammonium molybdate, using a Philips EM208 and real space refinement of the atomic model against the experimental transmission electron microscope. map, using Phenix, including restrained temperature factor refinement (20). The final atomic model comprises 295,800 atoms (60 × 4,930 atoms exclud- Sample Preparation for Cryo-EM. GFLV was purified from infected N. ben- ing hydrogens across the 504 and 137 amino acids for each monomer of the thamiana plants according to Schellenberger et al. (6). GFLV-specific single- CP and Nb23, respectively). Protein residues of the final atomic model show domain antibody Nbs (Nb23) were generated as described in Hemmer et al. well-refined geometrical parameters (most favored regions, 96.7%; addi- (16). Dynamic light-scattering analyses were used both to find the buffer tionally allowed regions, 3.3%; and 0.0% of outliers in Ramachandran plots, compositions and pH values at which GFLV and GFLV bound to Nb23 are r.m.s. bond deviations of 0.009 Å and angle deviations of 0.8°; SI Appendix, soluble and to determine their diameters and monodispersity. Measure- Fig. S1 for data collection and structure refinement statistics). Solvent- ments were performed using a Malvern Zetasizer NanoZS instrument and accessible surface was calculated with the program GetArea (probe radius

10854 | www.pnas.org/cgi/doi/10.1073/pnas.1913681117 Orlov et al. Downloaded by guest on October 2, 2021 1.4 Å, ref. 35). Figures were prepared using the software Chimera (32) and ACKNOWLEDGMENTS. We thank J. Michalon and R. Fritz for IT support, and Pymol (36). J.-F. Ménétret for technical support, J. Misbach, L. Ley, and the Institut Na- tional de la Recherche Agronomique and Centre National de la Recherche HTS Data Mining. To determine whether RB mutations preexisted within Scientifique greenhouse teams for technical support and plants production natural viral populations or were acquired in Nb23-expressing plants, we and Jean-Yves Sgro (Institute for Molecular Virology, University of Wiscon- sin) for helpful advice in stereographic roadmap projections; B.P.K. thanks analyzed HTS datasets (2 × 150 bp RNAseq performed on an Illumina Hiseq the late Michael G. Rossmann for many exciting discussions. This work was 3000, Illumina, San Diego, CA) from a previous study by Hily et al. (37). We supported by the Centre National de la Recherche Scientifique, the Agence specifically focused on 12 repetitions of grafted and nongrafted grapevines Nationale de la Recherche (ANR); grant ANR-COMBiNiNG ANR-14-CE19- naturally infected by a vineyard population of GFLV. In addition, total RNA 0022), Institut National de la Recherche Agronomique, Université de Stras- was extracted from a single sample containing 300 viruliferous nematodes bourg, Institut National de la Santé et de la Recherche Médicale, the Interreg previously fed on a GFLV-F13-infected plant according to Demangeat et al. Rhin Supérieur V project “Vitifutur” (http://www.interreg-rhin-sup.eu/projet/ (38), and a cDNA library was made as described in Hily et al. (37). Analyses of vitifutur-reseau-transnational-de-recherche-et-de-formation-en-viticulture/), datasets were performed using CLC Genomics Workbench 8.5.1 software and the Conseils Interprofessionels des vins d’Alsace, de Champagne, de Bour- (Qiagen), with mapping parameters set at length fraction of 0.5 and simi- gogne et de Bordeaux. C.H. was supported by a fellowship from the Région larity of 0.7. Postmapping, SNPs at each position were recovered (only SNPs Alsace and the “Plant Health and the Environment” Institut National de la above the Illumina error rate of 0.1% per base were recorded), and missense Recherche Agronomique division. L.A. was supported by a CIFRE grant from mutations (consisting of one SNP per codon) were reported with their per- the Institut Français de la Vigne et du Vin, subsidized by the Association Natio- centage rate. Data are available in Dataset S1. nale Recherche Technologie (ANRT) (Conventions Industrielles de Formation par la Recherche [CIFRE] number 2012/0929). B.P.K. acknowledges ANR and University of Strasbourg Institute for Advanced Study (USIAS). This work was Statistical Analyses. For the GFLV-CP+3 transmission assay, statistical analysis supported by the ANR-10-LABX-0030-INRT grant under the frame program was performed by two-tailed paired t test (P = 0.022). Investissements d’Avenir ANR-10-IDEX-0002-02. The electron microscope facil- ity at the Centre for Integrative Biology (CBI) was supported by the Région Data Availability. Materials are available upon reasonable request. Raw data Alsace, the Fondation pour la Recherche Médicale, the Infrastructures en Biol- of the HTS data mining are available in the Dataset S1 file. Cryo-EM map and ogie Santé et Agronomie (IBiSA) platform program, Inserm, Centre National coordinates of the atomic model can be found in the Protein Data Bank and de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, the Electron Microscopy Data Bank under the following accession numbers: the French Infrastructure for Integrated Structural Biology ANR-10-INSB-05-01, PDB, 5FOJ; EMD, 3246. and Instruct-ERIC.

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