Papers by Hélène Sanfaçon
Pathogens
Rapid apple decline disease (RAD) has been affecting orchards in the USA and Canada. Although the... more Rapid apple decline disease (RAD) has been affecting orchards in the USA and Canada. Although the primary cause for RAD remains unknown, viruses may contribute to the incidence or severity of the disease. We examined the diversity and prevalence of viruses in orchards affected by RAD in the Okanagan and Similkameen Valleys (British Columbia, Canada). Next-generation sequencing identified 20 previously described plant viruses and one viroid, as well as a new ilarvirus, which we named apple ilarvirus 2 (AIV2). AIV2 was related to subgroup 2 ilarviruses (42–71% nucleotide sequence identity). RT-PCR assays of 148 individual leaf samples revealed frequent mixed infections, with up to eight viruses or viroid detected in a single tree. AIV2 was the most prevalent, detected in 64% of the samples. Other prevalent viruses included three ubiquitous viruses from the family Betaflexiviridae and citrus concave gum-associated virus. Apple rubbery wood virus 1 and 2 and apple luteovirus 1 were also...
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<p><b>(</b>A) Amino acid alignment of the C-terminal region of the NTB domain o... more <p><b>(</b>A) Amino acid alignment of the C-terminal region of the NTB domain of relevant mutants of Rasp2 (as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162223#pone.0162223.g003" target="_blank">Fig 3A</a>). Mutated aa are underlined. In the case of the G<sup>578</sup>stop and A<sup>585</sup>stop mutants, stop codons were introduced at the indicated position resulting in the production of a truncated protein. The approximate deduced location of the SPase cleavage sites are shown with grey shadings, with darker shading corresponding to higher efficiency of SPase cleavage. The black arrow indicates the deduced position of the natural SPase cleavage site in the WT Rasp2 sequence while the grey arrow indicates the position of the additional SPase cleavage site detected in the M<sup>583</sup>A mutant. (B) <i>In vitro</i> membrane-associated modification assays of WT or mutant derivatives of Rasp2 S-cNV-H<sub>6</sub> were conducted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162223#pone.0162223.g002" target="_blank">Fig 2C</a>. (C and D) Experiments were conducted as in B, but reactions were allowed to proceed for four hours. (E and F) Experiments were conducted as in C, but using WT or mutant derivatives of Rasp2 HA-cNV-H<sub>6</sub>.</p
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Nature Microbiology, 2020
Virus taxonomy emerged as a discipline in the middle of the twentieth century. Traditionally, cla... more Virus taxonomy emerged as a discipline in the middle of the twentieth century. Traditionally, classification by virus taxonomists has been focussed on the grouping of relatively closely related viruses. However, during the past few years, the International Committee on Taxonomy of Viruses (ICTV) has recognized that the taxonomy it develops can be usefully extended to include the basal evolutionary relationships among distantly related viruses. Consequently, the ICTV has changed its Code to allow a 15-rank classification hierarchy that closely aligns with the Linnaean taxonomic system and may accommodate the entire spectrum of genetic divergence in the virosphere. The current taxonomies of three human pathogens, Ebola virus, severe acute respiratory syndrome coronavirus and herpes simplex virus 1 are used to illustrate the impact of the expanded rank structure. This new rank hierarchy of virus taxonomy will stimulate further research on virus origins and evolution, and vice versa, an...
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Archives of Virology
Plant-infecting viruses of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order ... more Plant-infecting viruses of the genus Nepovirus (subfamily Comovirinae, family Secoviridae, order Picornavirales) are bipartite positive-strand RNA viruses with each genomic RNA encoding a single large polyprotein. The RNA1-encoded 3C-like protease cleaves the RNA1 polyprotein at five sites and the RNA2 polyprotein at two or three sites, depending on the nepovirus. The specificity of nepovirus 3C-like proteases is notoriously diverse, making the prediction of cleavage sites difficult. In this study, the position of nepovirus cleavage sites was systematically re-evaluated using alignments of the RNA1 and RNA2 polyproteins, phylogenetic relationships of the proteases, and sequence logos to examine specific preferences for the P6 to P1’ positions of the cleavage sites. Based on these analyses, the positions of previously elusive cleavage sites, notably the 2a-MP cleavage sites of subgroup B nepoviruses, are now proposed. Distinct nepovirus protease clades were identified, each with diff...
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Journal of Virology, 2006
Replication of nepoviruses (family Comoviridae ) occurs in association with endoplasmic reticulum... more Replication of nepoviruses (family Comoviridae ) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleoside triphosphate-binding protein (NTB) of Tomato ringspot nepovirus is an integral membrane protein with two ER-targeting sequences and have suggested that it anchors the viral replication complex (VRC) to the membranes. A second highly hydrophobic protein domain (X2) is located immediately upstream of the NTB domain in the RNA1-encoded polyprotein. X2 shares conserved sequence motifs with the comovirus 32-kDa protein, an ER-targeted protein implicated in VRC assembly. In this study, we examined the ability of X2 to associate with intracellular membranes. The X2 protein was fused to the green fluorescent protein and expressed in Nicotiana benthamiana by agroinfiltration. Confocal microscopy and membrane flotation experiments suggested that X2 is targeted to ER membranes. Mutagenesis studies revealed that X2 contai...
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<p><b>(</b>A) Amino acid (aa) alignment of the C-terminal region of the NTB dom... more <p><b>(</b>A) Amino acid (aa) alignment of the C-terminal region of the NTB domain for relevant mutants of Rasp2. The Rasp1 sequence is also shown for reference. Mutated aa are underlined. The alignment starts with the four C-terminal aa of the TM2 domain and ends with the C-terminal aa of the NTB domain. (B and D) <i>In vitro</i> membrane-associated modification assays of WT or mutant derivatives of Rasp2 S-cNV-H<sub>6</sub> were conducted as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162223#pone.0162223.g002" target="_blank">Fig 2C</a>. (C) <i>In vitro</i> membrane-associated modification assays of the WT or ΔTV mutant derivative of Rasp2 HA-cNV-H<sub>6</sub>. Reactions were conducted for 4h.</p
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Archives of virology, Jan 11, 2017
We mark the 50th anniversary of the International Committee on Taxonomy of Viruses (ICTV) by pres... more We mark the 50th anniversary of the International Committee on Taxonomy of Viruses (ICTV) by presenting a brief history of the organization since its foundation, showing how it has adapted to advancements in our knowledge of virus diversity and the methods used to characterize it. We also outline recent developments, supported by a grant from the Wellcome Trust (UK), that are facilitating substantial changes in the operations of the ICTV and promoting dialogue with the virology community. These developments will generate improved online resources, including a freely available and regularly updated ICTV Virus Taxonomy Report. They also include a series of meetings between the ICTV and the broader community focused on some of the major challenges facing virus taxonomy, with the outcomes helping to inform the future policy and practice of the ICTV.
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PloS one, 2016
The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein as... more The NTB-VPg polyprotein from tomato ringspot virus is an integral membrane replication protein associated with endoplasmic reticulum membranes. A signal peptidase (SPase) cleavage was previously detected in the C-terminal region of NTB-VPg downstream of a 14 amino acid (aa)-long hydrophobic region (termed TM2). However, the exact location of the cleavage site was not determined. Using in vitro translation assays, we show that the SPase cleavage site is conserved in the NTB-VPg protein from various ToRSV isolates, although the rate of cleavage varies from one isolate to another. Systematic site-directed mutagenesis of the NTB-VPg SPase cleavage sites of two ToRSV isolates allowed the identification of sequences that affect cleavage efficiency. We also present evidence that SPase cleavage in the ToRSV-Rasp2 isolate occurs within a GAAGG sequence likely after the AAG (GAAG/G). Mutation of a downstream MAAV sequence to AAAV resulted in SPase cleavage at both the natural GAAG/G and the m...
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Virus Genes, 1989
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Phytopathology™, 2020
Given the importance of and rapid research progress in plant virology in recent years, this Focus... more Given the importance of and rapid research progress in plant virology in recent years, this Focus Issue broadly emphasizes advances in fundamental aspects of virus infection cycles and epidemiology. This Focus Issue comprises three review articles and 18 research articles. The research articles cover broad research areas on the identification of novel viruses, the development of detection methods, reverse genetics systems and functional genomics for plant viruses, vector and seed transmission studies, viral population studies, virus–virus interactions and their effect on vector transmission, and management strategies of viral diseases. The three review articles discuss recent developments in application of prokaryotic clustered regularly interspaced short palindromic repeats/CRISPR-associated genes (CRISPR/Cas) technology for plant virus resistance, mixed viral infections and their role in disease synergism and cross-protection, and viral transmission by whiteflies. The following br...
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Members of the family Secoviridae are non-enveloped viruses with monoor bipartite (RNA-1 and RNA-... more Members of the family Secoviridae are non-enveloped viruses with monoor bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes of 9 to 13.7 kilobases in total (Table 1.Secoviridae). Secoviruses are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus).
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PLoS ONE, 2021
Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bip... more Tomato ringspot virus (ToRSV, genus Nepovirus, family Secoviridae, order Picornavirales) is a bipartite positive-strand RNA virus, with each RNA encoding one large polyprotein. ToRSV RNAs are linked to a 5’-viral genome-linked protein (VPg) and have a 3’ polyA tail, suggesting a non-canonical cap-independent translation initiation mechanism. The 3’ untranslated regions (UTRs) of RNA1 and RNA2 are unusually long (~1.5 kb) and share several large stretches of sequence identities. Several putative in-frame start codons are present in the 5’ regions of the viral RNAs, which are also highly conserved between the two RNAs. Using reporter transcripts containing the 5’ region and 3’ UTR of the RNA2 of ToRSV Rasp1 isolate (ToRSV-Rasp1) and in vitro wheat germ extract translation assays, we provide evidence that translation initiates exclusively at the first AUG, in spite of a poor codon context. We also show that both the 5’ region and 3’ UTR of RNA2 are required for efficient cap-independen...
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Plum pox virus (PPV) is a serious viral disease of Prunus species. Few sources of natural resista... more Plum pox virus (PPV) is a serious viral disease of Prunus species. Few sources of natural resistance to PPV are available. In this light, biotechnology is a strong option to develop PPV resistance in plants. We have previously shown that a transgene consisting of a region of the PPV HC-Pro sequence in an intron-spliced hairpin conformation could provide high level of resistance to PPV in Nicotiana benthamiana, a herbaceous model host. We have now generated transgenic plum lines expressing the same transgene. Plants were inoculated with PPV virus using a chip bud grafting method and were subjected to three cycles of dormant/growth periods. Plants were analyzed for PPV resistance after each cold treatment (dormant period) using a real-time polymerase chain reaction (PCR) assay. After the first cold treatment, transgenic plum plants remained PPV-free whereas all the control plants were PPV positive. After three cycles of cold treatment, many transgenic plants still exhibited strong res...
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Journal of Virology
Strawberry mottle virus (SMoV) belongs to the family Secoviridae (order Picornavirales) and has a... more Strawberry mottle virus (SMoV) belongs to the family Secoviridae (order Picornavirales) and has a bipartite genome with each RNA encoding one polyprotein. All characterized secovirids encode a single protease related to the picornavirus 3C-protease. The SMoV 3C-like protease was previously shown to cut the RNA2 polyprotein (P2) at a single site between the predicted movement protein and coat protein (CP) domains. However, the SMoV P2 polyprotein includes an extended C-terminal region with a coding capacity of up to 70 kDa downstream of the presumed CP domain, an unusual characteristic for this family. In this study, we identified a novel cleavage event at a P↓AFP sequence immediately downstream of the CP domain. Following deletion of the PAFP sequence, the polyprotein was processed at or near a related PKFP sequence 40 kDa further downstream, defining two protein domains in the C-terminal region of the P2 polyprotein. Both processing events were dependent on a novel protease domain ...
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Virology, 2018
ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown... more ARGONAUTEs (notably AGO1 and AGO2) are effectors of plant antiviral RNA silencing. AGO1 was shown to be required for the temperature-dependent symptom recovery of Nicotiana benthamiana plants infected with tomato ringspot virus (isolate ToRSV-Rasp1) at 27 °C. In this study, we show that symptom recovery from isolate ToRSV-GYV shares similar hallmarks of antiviral RNA silencing but occurs at a wider range of temperatures (21-27 °C). At 21 °C, an early spike in AGO2 mRNAs accumulation was observed in plants infected with either ToRSV-Rasp1 or ToRSV-GYV but the AGO2 protein was only consistently detected in ToRSV-GYV infected plants. Symptom recovery from ToRSV-GYV at 21 °C was not prevented in an ago2 mutant or by silencing of AGO1 or AGO2. We conclude that other factors (possibly other AGOs) contribute to symptom recovery under these conditions. The results also highlight distinct expression patterns of AGO2 in response to ToRSV isolates and environmental conditions.
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The Journal of general virology, 2017
Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RN... more Members of the family Secoviridae are non-enveloped viruses with mono- or bipartite (RNA-1 and RNA-2) linear positive-sense ssRNA genomes with the size of the RNAs combined ranging from 9 to 13.7 kb. They are related to picornaviruses and are classified in the order Picornavirales. The majority of known members infect dicotyledonous plants and many are important plant pathogens (e.g. grapevine fanleaf virus and rice tungro spherical virus). This is a summary of the current International Committee on Taxonomy of Viruses (ICTV) report on the taxonomy of the family Secoviridae available at www.ictv.global/report/secoviridae.
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Viruses, 2015
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METHODS IN MOLECULAR BIOLOGY-CLIFTON …, 2008
Replication of the genome of positive-strand RNA plant viruses takes place in membrane-bound comp... more Replication of the genome of positive-strand RNA plant viruses takes place in membrane-bound complexes that contain viral replicase proteins, viral RNA, and host proteins. Many viral replicase proteins play a crucial role in the assembly of replication complexes at intracellular membranes. They are integral membrane proteins that interact directly with the membranes and bring other proteins and the viral RNA to the complex via protein-protein or protein-RNA interactions. In this chapter, we describe subcellular fractionation methods that determine whether viral proteins are integral membrane proteins in planta. Differential centrifugation techniques are used to produce membrane-enriched fractions, which can then be analyzed for the presence of viral replicase proteins by immunoblotting. Confirmation of the membrane-association is obtained by membrane flotation assays and treatment of membrane-enriched fractions with high salt or high pH followed by detection of the viral proteins. Because many plant viruses replicate in association with the endoplasmic reticulum (ER), we also discuss two techniques to specifically analyze the interaction of viral proteins with these membranes. These techniques are continuous sucrose-gradient fractionation in the presence or absence of 3 mM Mg2+ and glycosylation assays.
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... Capote, Plant Protection and Biotechnology Centre, Instituto Valenciano de Investigaciones Ag... more ... Capote, Plant Protection and Biotechnology Centre, Instituto Valenciano de Investigaciones Agrarias (IVIA), Carretera Moncada a Náquera km 5, 46113 Moncada, Valencia, Spain AO Charkowski, Department of Plant Pathology, University of Wisconsin-Madison, 1630 Linden ...
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Papers by Hélène Sanfaçon