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Helene Sanfacon - One of the best experts on this subject based on the ideXlab platform.
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Mapping of sequences in the 5' region and 3' UTR of Tomato Ringspot Virus RNA2 that facilitate cap-independent translation of reporter transcripts in vitro.
PloS one, 2021Co-Authors: Dinesh Babu Paudel, Helene SanfaconAbstract: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-independent translation of these transcripts. We identify translation-enhancing elements in the 5' proximal coding region of the RNA2 polyprotein and in the RNA2 3' UTR. Cap-dependent translation of control reporter transcripts was inhibited when RNAs consisting of the RNA2 3' UTR were supplied in trans. Taken together, our results suggest the presence of a CITE in the ToRSV-Rasp1 RNA2 3' UTR that recruits one or several translation factors and facilitates efficient cap-independent translation together with the 5' region of the RNA. Non-overlapping deletion mutagenesis delineated the putative CITE to a 200 nts segment (nts 773-972) of the 1547 nt long 3' UTR. We conclude that the general mechanism of ToRSV RNA2 translation initiation is similar to that previously reported for the RNAs of blackcurrant reversion Virus, another nepoVirus. However, the position, sequence and predicted structures of the translation-enhancing elements differed between the two Viruses.
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Expression and antiviral function of ARGONAUTE 2 in Nicotiana benthamiana plants infected with two isolates of Tomato Ringspot Virus with varying degrees of virulence
Virology, 2018Co-Authors: Dinesh Babu Paudel, Basudev Ghoshal, Sushma Jossey, Márta Ludman, Károly Fátyol, Helene SanfaconAbstract:Abstract 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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Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus
2016Co-Authors: Ting Wei, Joan Chisholm, Helene SanfaconAbstract: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 mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3–7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s) oriented parallel to the membrane inner surface.
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Complete genome sequence of three Tomato Ringspot Virus isolates: evidence for reassortment and recombination
Archives of virology, 2014Co-Authors: Melanie Walker, Joan Chisholm, Ting Wei, Basudev Ghoshal, Hanna Saeed, Michael Rott, Helene SanfaconAbstract:The genome sequence of Tomato Ringspot Virus (ToRSV, a subgroup C nepoVirus) is currently available for one raspberry isolate. In this study, we describe the complete genome sequence of three additional isolates from raspberry (Rasp1-2014), grapevine (GYV-2014) and prunus (13C280). The degree of nucleotide sequence identity shared between RNA1 and RNA2 in the 5′-terminal 900 nucleotides and 3′ untranslated region varied from 98-99 % (13C280, GYV-2014) to 80 % (Rasp1-2014). Phylogenetic studies revealed distinct origins for Rasp1-2014 RNA1 and RNA2, suggesting reassortment. Two recombination events were also identified in the 3′ UTR and 5′-terminal region of RNA1.
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Tomato Ringspot Virus coat protein binds to argonaute 1 and suppresses the translation repression of a reporter gene
Molecular Plant-microbe Interactions, 2014Co-Authors: Rajita A Karran, Helene SanfaconAbstract:RNA silencing regulates plant gene expression and antiviral defenses and functions by cleaving target RNAs or repressing translation. As a counter defense, many plant Viruses encode suppressor proteins that sequester small RNAs or inactivate Argonaute (AGO) proteins. All known plant Virus silencing suppressor activities eventually inhibit the degradation of target mRNAs. Using a transiently expressed green fluorescent protein (GFP) reporter gene, we show that Tomato Ringspot Virus (ToRSV) coat protein (CP) is a suppressor of RNA silencing that enhances GFP expression but does not prevent the degradation of the GFP mRNA or the accumulation of GFP small interfering RNAs (siRNAs). Coexpression of the CP with GFP resulted in increased association of residual GFP mRNAs with polysome fractions and reduced association of GFP siRNAs with monosome fractions. AGO1 was co-immunoprecipitated with the CP and CP expression destabilized AGO1. A WG motif within the CP was critical for the enhanced GFP expression, AGO1 interaction, and AGO1 destabilization, suggesting that the ToRSV CP acts as an AGO-hook protein and competes for AGO binding with a plant cellular GW/WG protein involved in translation repression.
Joan Chisholm - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a Non-Canonical Signal Peptidase Cleavage Site in a Replication Protein from Tomato Ringspot Virus
2016Co-Authors: Ting Wei, Joan Chisholm, Helene SanfaconAbstract: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 mutated AAA/V sequences. Given that there is a distance of seven aa between the two cleavage sites, this indicates that there is flexibility in the positioning of the cleavage sites relative to the inner surface of the membrane and the SPase active site. SPase cleavage sites are typically located 3–7 aa downstream of the hydrophobic region. However, the NTB-VPg GAAG/G cleavage site is located 17 aa downstream of the TM2 hydrophobic region, highlighting unusual features of the NTB-VPg SPase cleavage site. A putative 11 aa-long amphipathic helix was identified immediately downstream of the TM2 region and five aa upstream of the GAAG/G cleavage site. Based on these results, we present an updated topology model in which the hydrophobic and amphipathic domains form a long tilted helix or a bent helix in the membrane lipid bilayer, with the downstream cleavage site(s) oriented parallel to the membrane inner surface.
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Complete genome sequence of three Tomato Ringspot Virus isolates: evidence for reassortment and recombination
Archives of virology, 2014Co-Authors: Melanie Walker, Joan Chisholm, Ting Wei, Basudev Ghoshal, Hanna Saeed, Michael Rott, Helene SanfaconAbstract:The genome sequence of Tomato Ringspot Virus (ToRSV, a subgroup C nepoVirus) is currently available for one raspberry isolate. In this study, we describe the complete genome sequence of three additional isolates from raspberry (Rasp1-2014), grapevine (GYV-2014) and prunus (13C280). The degree of nucleotide sequence identity shared between RNA1 and RNA2 in the 5′-terminal 900 nucleotides and 3′ untranslated region varied from 98-99 % (13C280, GYV-2014) to 80 % (Rasp1-2014). Phylogenetic studies revealed distinct origins for Rasp1-2014 RNA1 and RNA2, suggesting reassortment. Two recombination events were also identified in the 3′ UTR and 5′-terminal region of RNA1.
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Characterization of proteinase cleavage sites in the N-terminal region of the RNA1-encoded polyprotein from Arabis mosaic Virus (subgroup A nepoVirus)
Virology, 2008Co-Authors: Thierry Wetzel, Joan Chisholm, Alexandra Bassler, Helene SanfaconAbstract:Arabis mosaic Virus is a subgroup A nepoVirus. The RNA1-encoded polyprotein (P1) contains the domains for the NTP-binding protein (NTB), VPg, proteinase (Pro) and polymerase at its C-terminus. Putative cleavage sites delineating these domains have been proposed. However, the number and location of cleavage sites upstream of the NTB domain are not known. Using in vitro processing assays, we have confirmed proteolytic cleavage at the NTB-VPg and VPg-Pro sites. In addition, we have identified two cleavage sites in the N-terminal region of P1. Site-directed mutagenesis and immunoprecipitation experiments using inserted peptide tags confirmed that the position of these cleavage sites corresponds to that of cleavage sites delineating the X1 and X2 domains in Tomato Ringspot Virus (subgroup C nepoVirus). Amino acid alignments implied the presence of similar cleavage sites in the P1 polyprotein of other nepoViruses. Our results suggest that the presence of two protein domains upstream of NTB is a common feature of nepoViruses.
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Peripheral association of a polyprotein precursor form of the RNA-dependent RNA polymerase of Tomato Ringspot Virus with the membrane-bound viral replication complex.
Virology, 2007Co-Authors: Joan Chisholm, Guangzhi Zhang, Aiming Wang, Helene SanfaconAbstract:Replication of Tomato Ringspot Virus (ToRSV) occurs in association with endoplasmic reticulum (ER)-derived membranes. We have previously shown that the putative nucleotide triphosphate-binding protein (NTB) of ToRSV is an ER-targeted protein and that an intermediate polyprotein containing the domains for NTB and for the genome-linked viral protein (VPg) is associated with the replication complex. We now report the detection of a 95-kDa polyprotein that contains the domains for the RNA-dependent RNA polymerase (Pol), the proteinase (Pro) and the VPg. This polyprotein appears to be a truncated version of the full-length 111-kDa VPg-Pro-Pol polyprotein and was termed VPg-Pro-Pol'. A subpopulation of VPg-Pro-Pol' was peripherally associated with ER-derived membranes active in viral replication. However, the VPg, Pro and Pol domains did not target to membranes in the absence of viral infection. We propose a model in which VPg-Pro-Pol' is brought to the site of replication through interaction with a viral membrane protein.
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Interaction in vitro between the proteinase of Tomato Ringspot Virus (genus NepoVirus) and the eukaryotic translation initiation factor iso4E from Arabidopsis thaliana.
The Journal of general virology, 2002Co-Authors: Simon Léonard, Joan Chisholm, Jean-françois Laliberté, Helene SanfaconAbstract:Eukaryotic initiation factor eIF(iso)4E binds to the cap structure of mRNAs leading to assembly of the translation complex. This factor also interacts with the potyVirus VPg and this interaction has been correlated with Virus infectivity. In this study, we show an interaction between eIF(iso)4E and the proteinase (Pro) of a nepoVirus (Tomato Ringspot Virus; ToRSV) in vitro. The ToRSV VPg did not interact with eIF(iso)4E although its presence on the VPg-Pro precursor increased the binding affinity of Pro for the initiation factor. A major determinant of the interaction was mapped to the first 93 residues of Pro. Formation of the complex was inhibited by addition of m(7)GTP (a cap analogue), suggesting that Pro-containing molecules compete with cellular mRNAs for eIF(iso)4E binding. The possible implications of this interaction for translation and/or replication of the Virus genome are discussed.
R P Schreiner - One of the best experts on this subject based on the ideXlab platform.
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epidemiology of xiphinema americanum and Tomato Ringspot Virus on red raspberry rubus idaeus
Plant Disease, 2008Co-Authors: John N. Pinkerton, Robert R. Martin, J Kraus, R P SchreinerAbstract:ABSTRACT Population dynamics of Xiphinema americanum and transmission of Tomato Ringspot Virus (ToRSV) were studied in a red raspberry field in Washington State. Population densities of X. americanum were highest in the winter, lowest in the summer, and were correlated with precipitation (R2 = 0.42). All nematode stages were present throughout the year. Gravid females were observed only in the spring, indicating one generation per year. The sequence of the coat protein of the ToRSV from this field was similar to those of other raspberry isolates from the Pacific Northwest. Cucumber seedlings were planted in soil collected monthly from the field and were evaluated for nematode transmission of ToRSV by enzyme-linked immunosorbent assay (ELISA). The proportion of assay plants infected with ToRSV was negatively correlated with nematode densities (R2 = 0.31). In another study, ToRSV was detected by ELISA in fine roots of raspberry plants 5 months after planting in field soil infested with viruliferous nematode...
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epidemiology of xiphinema americanum and Tomato Ringspot Virus on red raspberry rubus idaeus
Plant Disease, 2008Co-Authors: John N. Pinkerton, Robert R. Martin, J Kraus, R P SchreinerAbstract:Population dynamics of Xiphinema americanum and transmission of Tomato Ringspot Virus (ToRSV) were studied in a red raspberry field in Washington State. Population densities of X. americanum were highest in the winter, lowest in the summer, and were correlated with precipitation (R2 = 0.42). All nematode stages were present throughout the year. Gravid females were observed only in the spring, indicating one generation per year. The sequence of the coat protein of the ToRSV from this field was similar to those of other raspberry isolates from the Pacific Northwest. Cucumber seedlings were planted in soil collected monthly from the field and were evaluated for nematode transmission of ToRSV by enzyme-linked immunosorbent assay (ELISA). The proportion of assay plants infected with ToRSV was negatively correlated with nematode densities (R2 = 0.31). In another study, ToRSV was detected by ELISA in fine roots of raspberry plants 5 months after planting in field soil infested with viruliferous nematodes, in all subterranean portions of plants after 12 months, and in all aerial portions the second year. The rate of spread of ToRSV in a raspberry field was 70 cm per year. These results suggest that the rate of ToRSV spread is limited by systemic spread of Virus in plants when nematode-infested soil is not transported in the field.
Sandra Wickizer - One of the best experts on this subject based on the ideXlab platform.
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localization of transmissible and nontransmissible Viruses in the vector nematode xiphinema americanum
Phytopathology, 2002Co-Authors: Shouhua Wang, R C Gergerich, Sandra WickizerAbstract:ABSTRACT The inner lining of the food canal of nematodes that transmit plantinfecting Viruses is regarded as the retention region of Viruses. To characterize the location of transmissible and nontransmissible Viruses in the vector nematode Xiphinema americanum, three nepoViruses, Tobacco Ringspot Virus (TRSV), Tomato Ringspot Virus(TomRSV), and Cherry leaf roll Virus(CLRV), and one non-nematode-transmissible Virus, Squash mosaic Virus (SqMV), were evaluated for transmission efficiency and localization sites in the nematode. Transmission trials showed highest transmission efficiency for TomRSV (38% with 1 and 100% with 10 nematodes, respectively), intermediate efficiency for TRSV (27% with 1 and 65% with 10 nematodes, respectively), and no transmission for CLRV and SqMV. Electron microscopy and immunofluorescent labeling revealed that TRSV was primarily localized to the lining of the lumen of the stylet extension and the anterior esophagus, but only rarely in the triradiate lumen. Within a nematode populat...
Robert R. Martin - One of the best experts on this subject based on the ideXlab platform.
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Occurrence of Tomato Ringspot Virus and Tobacco Ringspot Virus in highbush blueberry in New York State.
Journal of Plant Pathology, 2010Co-Authors: Marc Fuchs, G.s. Abawi, P. Marsella-herrick, R. Cox, Kerik D. Cox, Juliet Carroll, Robert R. MartinAbstract:SUMMARY A survey for Viruses by DAS-ELISA of highbush blueberry (Vaccinium corymbosum L.) showing Viruslike symptoms and decline in vigor, showed the occurrence of Tomato Ringspot Virus (ToRSV) and Tobacco Ringspot Virus (TRSV) in New York State (USA). The presence of the two Viruses was confirmed in leaf samples by RT-PCR and IC-RT-PCR with appropriate primer pairs that amplify a 320 bp and a 585 bp fragment of the RNA-dependent RNA polymerase gene of TRSV and ToRSV, respectively. Comparative sequence analysis of viral amplicons of New York isolates indicated moderate (80.7-99.7%) and high (90.8-99.7%) nucleotide sequence identities with other ToRSV and TRSV strains, respectively. Analysis of soil samples from the root zone of blueberry bushes for the occurrence of dagger nematodes revealed the presence of Xiphinema americanum-group nematodes at low population densities. Cucumber bait plants potted in soil samples containing X. americanum became infected with ToRSV or TRSV in a greenhouse. Together, these findings show the occurrence of ToRSV and, to a lesser extent, TRSV in highbush blueberry plantings in New York, as well as of the vector X. americanum sensu lato.
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epidemiology of xiphinema americanum and Tomato Ringspot Virus on red raspberry rubus idaeus
Plant Disease, 2008Co-Authors: John N. Pinkerton, Robert R. Martin, J Kraus, R P SchreinerAbstract:Population dynamics of Xiphinema americanum and transmission of Tomato Ringspot Virus (ToRSV) were studied in a red raspberry field in Washington State. Population densities of X. americanum were highest in the winter, lowest in the summer, and were correlated with precipitation (R2 = 0.42). All nematode stages were present throughout the year. Gravid females were observed only in the spring, indicating one generation per year. The sequence of the coat protein of the ToRSV from this field was similar to those of other raspberry isolates from the Pacific Northwest. Cucumber seedlings were planted in soil collected monthly from the field and were evaluated for nematode transmission of ToRSV by enzyme-linked immunosorbent assay (ELISA). The proportion of assay plants infected with ToRSV was negatively correlated with nematode densities (R2 = 0.31). In another study, ToRSV was detected by ELISA in fine roots of raspberry plants 5 months after planting in field soil infested with viruliferous nematodes, in all subterranean portions of plants after 12 months, and in all aerial portions the second year. The rate of spread of ToRSV in a raspberry field was 70 cm per year. These results suggest that the rate of ToRSV spread is limited by systemic spread of Virus in plants when nematode-infested soil is not transported in the field.
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epidemiology of xiphinema americanum and Tomato Ringspot Virus on red raspberry rubus idaeus
Plant Disease, 2008Co-Authors: John N. Pinkerton, Robert R. Martin, J Kraus, R P SchreinerAbstract:ABSTRACT Population dynamics of Xiphinema americanum and transmission of Tomato Ringspot Virus (ToRSV) were studied in a red raspberry field in Washington State. Population densities of X. americanum were highest in the winter, lowest in the summer, and were correlated with precipitation (R2 = 0.42). All nematode stages were present throughout the year. Gravid females were observed only in the spring, indicating one generation per year. The sequence of the coat protein of the ToRSV from this field was similar to those of other raspberry isolates from the Pacific Northwest. Cucumber seedlings were planted in soil collected monthly from the field and were evaluated for nematode transmission of ToRSV by enzyme-linked immunosorbent assay (ELISA). The proportion of assay plants infected with ToRSV was negatively correlated with nematode densities (R2 = 0.31). In another study, ToRSV was detected by ELISA in fine roots of raspberry plants 5 months after planting in field soil infested with viruliferous nematode...
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Management of Tomato Ringspot Virus in Red Raspberry with Crop Rotation
International Journal of Fruit Science, 2005Co-Authors: John N. Pinkerton, Robert R. MartinAbstract:Abstract Tomato Ringspot Virus (ToRSV) is an important disease of red raspberry which is vectored by the dagger nematode, Xiphinema americanum. A field study was conducted to evaluate crop rotation as an alternative to soil fumigation for the management of ToRSV. The effects of treatments on nematode population densities and ToRSV were evaluated for 18 months during growth of rotation crops, and for 36 months after replanting raspberries. Treatments included the rotational crops rapeseed (Brassica napus) and tall fescue (Festuca arundinacea) (nonhosts for ToRSV), clean fallow, fumigation with methyl bromide, and controls in which raspberries followed raspberries. At planting, X. americanum densities were highest in fescue and control plots and lowest in fumigated plots, but densities decreased and remained low in all plots after planting ‘Meeker’ raspberries. Raspberry leaves were collected in each plot and assayed for ToRSV with ELISA for 3 years. ToRSV was detected only in the control plots. Rotations w...