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Anne E Simon - One of the best experts on this subject based on the ideXlab platform.
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RNA virus evasion of nonsense-mediated decay
PLOS Pathogens, 2018Co-Authors: Xuefeng Yuan, Erika Sawicki, Anne E SimonAbstract:Nonsense-mediated decay (NMD) is a host RNA control pathway that removes aberrant transcripts with long 3’ untranslated regions (UTRs) due to premature termination codons (PTCs) that arise through mutation or defective splicing. To maximize coding potential, RNA viruses often contain internally located stop codons that should also be prime targets for NMD. Using an agroinfiltration-based NMD assay in Nicotiana benthamiana, we identified two segments conferring NMD-resistance in the Carmovirus Turnip crinkle virus (TCV) genome. The ribosome readthrough structure just downstream of the TCV p28 termination codon stabilized an NMD-sensitive reporter as did a frameshifting element from umbravirus Pea enation mosaic virus. In addition, a 51-nt unstructured region (USR) at the beginning of the TCV 3’ UTR increased NMD-resistance 3-fold when inserted into an unrelated NMD-sensitive 3’ UTR. Several additional Carmovirus 3’ UTRs also conferred varying levels of NMD resistance depending on the construct despite no sequence similarity in the analogous region. Instead, these regions displayed a marked lack of RNA structure immediately following the NMD-targeted stop codon. NMD-resistance was only slightly reduced by conversion of 19 pyrimidines in the USR to purines, but resistance was abolished when a 2-nt mutation was introduced downstream of the USR that substantially increased the secondary structure in the USR through formation of a stable hairpin. The same 2-nt mutation also enhanced the NMD susceptibility of a subgenomic RNA expressed independently of the genomic RNA. The conserved lack of RNA structure among most Carmoviruses at the 5’ end of their 3’ UTR could serve to enhance subgenomic RNA stability, which would increase expression of the encoded capsid protein that also functions as the RNA silencing suppressor. These results demonstrate that the TCV genome has features that are inherently NMD-resistant and these strategies could be widespread among RNA viruses and NMD-resistant host mRNAs with long 3’ UTRs.
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Importance of sequence and structural elements within a viral replication repressor
Virology, 2005Co-Authors: Jiuchun Zhang, Anne E SimonAbstract:Abstract Efficient replication of plus-strand RNA viruses requires a 3′ proximal core promoter and an increasingly diverse inventory of supporting elements such as enhancers, repressors, and 5′ terminal sequences. While core promoters have been well characterized, much less is known about structure–functional relationships of these supporting elements. Members of the genus Carmovirus family Tombusviridae contain a hairpin (H5) proximal to the core promoter that functions as a repressor of minus-strand synthesis in vitro through an interaction between its large symmetrical internal loop (LSL) and 3′ terminal bases. Turnip crinkle virus satellite RNA satC with the H5 of Carmovirus Japanese iris necrosis virus or Cardamine chlorotic fleck virus (CCFV) did not accumulate to detectable levels even though 3′ end base-pairing would be maintained. Replacement of portions of the satC H5 with analogous portions from CCFV revealed that the cognate LSL and lower stem were of greater importance for satC accumulation than the upper stem. In vivo selex of the H5 upper stem and terminal GNRA tetraloop revealed considerable plasticity in the upper stem, including the presence of three- to six-base terminal loops, allowed for H5 function. In vivo selex of the lower stem revealed that both a stable stem and specific base pairs contributed to satC fitness. Surprisingly, mutations in H5 had a disproportionate effect on plus-strand accumulation that was unrelated to the stability of the mutant plus-strands. In addition, fitness to accumulate in plants did not always correlate with enhanced ability to accumulate in protoplasts, suggesting that H5 may be multifunctional.
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Analysis of a viral replication repressor: sequence requirements for a large symmetrical internal loop
Virology, 2004Co-Authors: Jiuchun Zhang, Robert M Stuntz, Anne E SimonAbstract:Nearly all members of the Carmovirus genus contain a structurally conserved 3′ proximal hairpin (H5) with a large internal symmetrical loop (LSL). H5 has been identified as a repressor of minus-strand synthesis in a satellite RNA (satC), which shares partial sequence similarity with its helper virus Turnip crinkle virus (TCV). Repression was due to sequestration of the 3′ end mediated by base pairing between 3′ end sequence and the 3′ side of the LSL (G. Zhang, J. Zhang and A. E. Simon, J. Virol., in press). Single site mutational analysis and in vivo genetic selection (SELEX) of the 14 base satC H5 LSL indicated specific sequences in the middle and upper regions on both sides of the LSL are necessary for robust satC accumulation in plants and protoplasts. Fitness of wild-type satC and satC LSL mutants to accumulate in plants, however, did not necessarily correlate with the ability of these RNAs to replicate in protoplasts. This suggests that the LSL might be involved in processes in addition to repression of minus-strand synthesis.
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analysis of cis acting sequences involved in plus strand synthesis of a turnip crinkle virus associated satellite rna identifies a new Carmovirus replication element
Virology, 2000Co-Authors: Hancheng Guan, Clifford D Carpenter, Anne E SimonAbstract:Abstract Satellite RNA C (satC) is a 356-base subviral RNA associated with turnip crinkle virus (TCV). A 3′-proximal element (3′-UCCCAAAGUAU) located 11 bases from the 3′ terminus of satC minus strands can function as an independent promoter in an in vitro RNA-dependent RNA polymerase (RdRp) transcription system. Furthermore, in the absence of a 5′-proximal element, the 3′-proximal element is required for complementary strand synthesis in vitro. Site-directed mutagenesis was conducted to investigate the functional significance of this element and the 3′ minus-strand terminal sequence “3′-OH-CCCUAU,” which contains the minus-strand 3′-end sequence “3′-OH-CC 1–2 (A/U)(A/U)(A/U)” found in all Carmovirus RNAs. Single mutations in the 3′-terminal sequence, which we have named the Carmovirus consensus sequence (CCS), suppressed satC plus-strand synthesis to undetectable levels in protoplasts while still permitting some minus-strand synthesis. However, single and multiple mutations introduced into the 3′-proximal element had little or no effect on satC accumulation in protoplasts. In vivo genetic selection (SELEX) of the minus-strand 3′-terminal 21 bases revealed that all satC species accumulating in plants contained the 3′ CCS. In addition, the 3′-proximal element preferentially contained a sequence similar to the CCS and/or polypurines, suggesting that this element may also contribute to accumulation of satC in vivo.
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Symptom attenuation by a satellite RNA in vivo is dependent on reduced levels of virus coat protein.
Virology, 1999Co-Authors: Jianlong Wang, Anne E SimonAbstract:Abstract Many plant RNA viruses provide replication and encapsidation functions for one or more satellite RNAs (sat-RNAs) that can modulate the symptoms of the associated helper virus. Sat-RNA C, a virulent sat-RNA associated with turnip crinkle virus (TCV), normally intensifies symptoms but can attenuate symptoms if the TCV coat protein (CP) is replaced with that of cardamine chlorotic fleck Carmovirus [Kong et al. (1995) Plant Cell 7, 1625–1634] or if TCV contains an alteration in the CP initiation codon (TCV-CPm) [Kong et al. (1997b) Plant Cell 9, 2051–2063]. To further elucidate the mechanism of symptom attenuation by sat-RNA C, the composition of the CP produced by TCV-CPm (CP CPm ) was determined. Our results reveal that CP CPm likely has two additional amino acids at its N-terminus compared with wild-type TCV CP. TCV-CPm produces reduced levels of CP, and this reduction, not the two additional residues at the CP N-terminus, is responsible for symptom attenuation by sat-RNA C.
Peter D. Nagy - One of the best experts on this subject based on the ideXlab platform.
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use of double stranded rna templates by the tombusvirus replicase in vitro implications for the mechanism of plus strand initiation
Virology, 2006Co-Authors: Tadas Panavas, Jozsef Stork, Peter D. NagyAbstract:Abstract Plus-stranded RNA viruses replicate efficiently in infected hosts producing numerous copies of the viral RNA. One of the long-standing mysteries in RNA virus replication is the occurrence and possible role of the double-stranded (ds)RNA formed between minus- and plus-strands. Using the partially purified Cucumber necrosis virus (CNV) replicase from plants and the recombinant RNA-dependent RNA polymerase (RdRp) of Turnip crinkle virus (TCV), in this paper, we demonstrate that both CNV replicase and the related TCV RdRp can utilize dsRNA templates to produce viral plus-stranded RNA in vitro. Sequence and structure of the dsRNA around the plus-strand initiation site had a significant effect on initiation, suggesting that initiation on dsRNA templates is a rate-limiting step. In contrast, the CNV replicase could efficiently synthesize plus-strand RNA on partial dsRNAs that had the plus-strand initiation promoter “exposed”, suggesting that the polymerase activity of CNV replicase is strong enough to unwind extended dsRNA regions in the template during RNA synthesis. Based on the in vitro data, we propose that dsRNA forms might have functional roles during tombus- and Carmovirus replication and the AU-rich nature of the terminus could be important for opening the dsRNA structure around the plus-strand initiation promoter for tombus- and Carmoviruses and possibly many other positive-strand RNA viruses.
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heterologous rna replication enhancer stimulates in vitro rna synthesis and template switching by the Carmovirus but not by the tombusvirus rna dependent rna polymerase implication for modular evolution of rna viruses
Virology, 2005Co-Authors: Chiping Cheng, Tadas Panavas, Peter D. NagyAbstract:The viral RNA plays multiple roles during replication of RNA viruses, serving as a template for complementary RNA synthesis and facilitating the assembly of the viral replicase complex. These roles are coordinated by cis-acting regulatory elements, such as promoters and replication enhancers (REN). To test if these RNA elements can be used by related viral RNA-dependent RNA polymerases (RdRp), we compared the potential stimulatory effects of homologous and heterologous REN elements on complementary RNA synthesis and template-switching by the tombus- (Cucumber necrosis virus, CNV), Carmovirus (Turnip crinkle virus, TCV) and hepatitis C virus (HCV) RdRps in vitro. The CNV RdRp selectively utilized its cognate REN, while discriminating against the heterologous TCV REN. On the contrary, RNA synthesis by the TCV RdRp was stimulated by the TCV REN and the heterologous tombusvirus REN with comparable efficiency. The heterologous REN elements also promoted in vitro template-switching by the TCV and HCV RdRps. Based on these observations, we propose that REN elements could facilitate intervirus recombination and postrecombinational amplification of new recombinant viruses.
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partial purification and characterization of cucumber necrosis virus and tomato bushy stunt virus rna dependent rna polymerases similarities and differences in template usage between tombusvirus and Carmovirus rna dependent rna polymerases
Virology, 2000Co-Authors: Peter D. Nagy, Judit PoganyAbstract:Tombusviruses are small, plus-sense, single-stranded RNA viruses of plants. RNA-dependent RNA polymerases (RdRp) of two tombusviruses, Tomato bushy stunt virus (TBSV) and Cucumber necrosis virus (CNV), have been partially purified from infected Nicotiana benthamiana plants. The obtained RdRp complexes are capable of de novo initiation of complementary RNA synthesis using either plus- or minus-strand templates derived from tombusvirus defective interfering (DI) RNAs. In addition to template-sized products, shorter than full-length products were also generated efficiently apparently because of internal initiation of RNA synthesis by the tombusvirus RdRp. This property could be important for the formation of DI RNAs that are observed in tombusvirus infections. The tombusvirus RdRp is also able to use heterologous RNAs derived from satellite RNAs associated with Turnip crinkle virus (TCV) as templates. Generation of full-length, complementary RNA by the tombusvirus RdRp suggests that it can correctly and efficiently recognize the heterologous TCV-specific promoters. Reduced generation of a 3′-terminal extension product in the preceding assay suggests that the previously characterized replication enhancer present in sat-RNA C (Nagy et al., 1999, EMBO J. 18, 5653–5665) does not stimulate tombusvirus RdRp activity. Taken together, these results suggest that template usage by the tombusvirus and Carmovirus RdRps are similar, but not identical.
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RNA recombination in turnip crinkle virus: its role in formation of chimeric RNAs, multimers, and in 3′-end repair
Seminars in Virology, 1996Co-Authors: Anne E Simon, Peter D. NagyAbstract:Abstract RNA recombination is well documented for an increasing number of viruses and it is thought to have affected viral evolution and adaptation. However, molecular mechanisms mediating crossover events have been studied in only a few viral systems due to difficulties such as the low frequency of the event, and the scattered distribution of junction sites. Therefore, the uniquely high recombination frequency and nonrandom crossover site distribution for recombination among turnip crinkle Carmovirus (TCV) RNAs make TCV an excellent model recombination system. Characterization of large numbers of junction sites between two species of TCV satellite RNAs and between the TCV genomic RNA and one of the satellite RNAs revealed for the first time the role of specific sequences and structures in recombination. Also, recombination was found to play a role in formation of novel chimeric RNAs, multimeric forms of satellite RNAs as well in 3′ end repair of mutated satellite RNAs. A replicase-driven template-switching model is presented to explain many common features occurring during recombination in TCV infections.
Jae Sun Moon - One of the best experts on this subject based on the ideXlab platform.
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First Report of Soybean yellow mottle mosaic virus in Soybean in North America
Plant Disease, 2009Co-Authors: Shuxian Li, Jae Sun Moon, Leslie L. DomierAbstract:Soybean yellow mottle mosaic virus (SYMMV) is a soybean-infecting virus recently discovered in Korea that initially induces bright yellow mosaic on leaves followed by stunting and reduced growth of older leaves (1). Nucleotide sequence analysis of genomic RNA of the Korean SYMMV isolate suggested that the virus is a new member of the genus Carmovirus in the family Tombusviridae. To determine whether SYMMV is present in the United States, single leaflets were collected without regard for symptoms from 7 to 10 plants in each of 136 plots in August 2008 from a research field in Stoneville, MS that contained 16 plant introductions (including five from Korea) and ‘Williams 82’. Samples were grouped into 10 pools of 100 leaves from which total RNA was extracted with the Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScript III Platinum SYBR Green One-Step Quantitative Real-time Reverse Transcriptase-PCR Kit (Invitrogen, Carlsbad, CA) and two pairs of oligonucleotide ...
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Nucleotide sequence and genomic organization of a newly identified member of the genus Carmovirus, soybean yellow mottle mosaic virus, from soybean
Archives of Virology, 2009Co-Authors: Leslie L. Domier, Jung Kyung Moon, Hong-soo Choi, Jae Sun MoonAbstract:The viral genome of soybean yellow mottle mosaic virus (SYMMV) from infected soybean ( Glycine max ) in Korea was cloned and sequenced. The complete monopartite single-stranded RNA genome of SYMMV consists of 4009 base pairs with six putative open reading frames and includes 5′- and 3′-untranslated regions of 39 and 229 nucleotides, respectively. The nucleotide and coat protein sequences of SYMMV share the highest sequence identity with those of cowpea mottle virus. Based on its genomic organization, its predicted amino acid sequence, and its phylogenetic relatedness to known Carmoviruses, we report that SYMMV is a new member of the genus Carmovirus in the family Tombusviridae .
Vicente Pallás - One of the best experts on this subject based on the ideXlab platform.
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Short Communication Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants
2020Co-Authors: Silvia Garcı́a-castillo, M. Amelia Sánchez-pina, Vicente PallásAbstract:Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus.
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An Update on the Intracellular and Intercellular Trafficking of Carmoviruses.
Frontiers in Plant Science, 2017Co-Authors: José A. Navarro, Vicente PallásAbstract:: Despite harboring the smallest genomes among plant RNA viruses, Carmoviruses have emerged as an ideal model system for studying essential steps of the viral cycle including intracellular and intercellular trafficking. Two small movement proteins, formerly known as double gene block proteins (DGBp1 and DGBp2), have been involved in the movement throughout the plant of some members of Carmovirus genera. DGBp1 RNA-binding capability was indispensable for cell-to-cell movement indicating that viral genomes must interact with DGBp1 to be transported. Further investigation on Melon necrotic spot virus (MNSV) DGBp1 subcellular localization and dynamics also supported this idea as this protein showed an actin-dependent movement along microfilaments and accumulated at the cellular periphery. Regarding DGBp2, subcellular localization studies showed that MNSV and Pelargonium flower break virus DGBp2s were inserted into the endoplasmic reticulum (ER) membrane but only MNSV DGBp2 trafficked to plasmodesmata (PD) via the Golgi apparatus through a COPII-dependent pathway. DGBp2 function is still unknown but its localization at PD was a requisite for an efficient cell-to-cell movement. It is also known that MNSV infection can induce a dramatic reorganization of mitochondria resulting in anomalous organelles containing viral RNAs. These putative viral factories were frequently found associated with the ER near the PD leading to the possibility that MNSV movement and replication could be spatially linked. Here, we update the current knowledge of the plant endomembrane system involvement in Carmovirus intra- and intercellular movement and the tentative model proposed for MNSV transport within plant cells.
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RNA-binding properties and membrane insertion of Melon necrotic spot virus (MNSV) double gene block movement proteins.
Virology, 2006Co-Authors: José-antonio Navarro, Ainhoa Genovés, J. Climent, Ana Saurí, Luis Martínez-gil, Ismael Mingarro, Vicente PallásAbstract:Abstract Advances in structural and biochemical properties of Carmovirus movement proteins (MPs) have only been obtained in p7 and p9 from Carnation mottle virus (CarMV) . Alignment of Carmovirus MPs revealed a low conservation of amino acid identity but interestingly, similarity was elevated in regions associated with the functional secondary structure elements reported for CarMV which were conserved in all studied proteins. Nevertheless, some differential features in relation with CarMV MPs were identified in those from Melon necrotic virus (MNSV) (p7A and p7B). p7A was a soluble non-sequence specific RNA-binding protein, but unlike CarMV p7, its central region alone could not account for the RNA-binding properties of the entire protein. In fact, a 22-amino acid synthetic peptide whose sequence corresponds to this central region rendered an apparent dissociation constant ( K d ) significantly higher than that of the corresponding entire protein (9 mM vs. 0.83–25.7 μM). This p7A-derived peptide could be induced to fold into an alpha-helical structure as demonstrated for other Carmovirus p7-like proteins. Additionally, in vitro fractionation of p7B transcription/translation mixtures in the presence of ER-derived microsomal membranes strongly suggested that p7B is an integral membrane protein. Both characteristics of these two small MPs forming the double gene block (DGB) of MNSV are discussed in the context of the intra- and intercellular movement of Carmovirus.
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Spatio-temporal analysis of the RNAs, coat and movement (p7) proteins of Carnation mottle virus in Chenopodium quinoa plants.
Journal of General Virology, 2003Co-Authors: Silvia Garcı́a-castillo, M. Amelia Sánchez-pina, Vicente PallásAbstract:Time-course and in situ hybridization analyses were used to study the spatio-temporal distribution of Carnation mottle virus (CarMV) in Chenopodium quinoa plants. Genomic and subgenomic RNAs of plus polarity accumulated linearly with time, whereas the corresponding minus strands reached a peak during infection in inoculated leaves. Analyses of serial tissue sections showed that plus polarity strands were localized throughout the infection area, whereas minus strands were localized at the borders of the chlorotic lesions. The accumulation kinetics of the coat protein (CP) and the p7 movement protein (MP) as well as their subcellular localization were also studied. Unlike most MPs, CarMV p7 showed a non-transient expression and a mainly cytosolic location. However, as infection progressed the presence of p7 in the cell wall fraction increased significantly. These results are discussed on the basis of a recent model proposed for the mechanism of cell-to-cell movement operating in the genus Carmovirus.
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in vitro evidence for rna binding properties of the coat protein of prunus necrotic ringspot ilarvirus and their comparison to related and unrelated viruses
Archives of Virology, 1999Co-Authors: Vicente Pallás, J A Sancheznavarro, Juana DiezAbstract:The RNA binding properties of the prunus necrotic ringspot virus (PNRSV) coat protein (CP) were demonstrated by northwestern and dot-blot analyses. The capability to bind PNRSV RNA 4 was compared with viruses representing three different interactions prevailing in the assembly and architecture of virions. The results showed that cucumber mosaic virus (CMV) and PNRSV CPs, which stabilise their virions mainly through RNA-protein interactions bound PNRSV RNA 4 even at very high salt concentrations. The CP of cherry leaf roll nepovirus, whose virions are predominantly stabilised by protein-protein interactions did not bind even at the lowest salt concentration tested. Finally the CP of carnation mottle Carmovirus, that has an intermediate position in which both RNA-protein and protein-protein interactions are equally important showed a salt-dependent RNA binding.
Leslie L. Domier - One of the best experts on this subject based on the ideXlab platform.
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First Report of Soybean yellow mottle mosaic virus in Soybean in North America
Plant Disease, 2009Co-Authors: Shuxian Li, Jae Sun Moon, Leslie L. DomierAbstract:Soybean yellow mottle mosaic virus (SYMMV) is a soybean-infecting virus recently discovered in Korea that initially induces bright yellow mosaic on leaves followed by stunting and reduced growth of older leaves (1). Nucleotide sequence analysis of genomic RNA of the Korean SYMMV isolate suggested that the virus is a new member of the genus Carmovirus in the family Tombusviridae. To determine whether SYMMV is present in the United States, single leaflets were collected without regard for symptoms from 7 to 10 plants in each of 136 plots in August 2008 from a research field in Stoneville, MS that contained 16 plant introductions (including five from Korea) and ‘Williams 82’. Samples were grouped into 10 pools of 100 leaves from which total RNA was extracted with the Qiagen RNeasy Plant Mini Kit (Germantown, MD), reverse transcribed, and amplified with SuperScript III Platinum SYBR Green One-Step Quantitative Real-time Reverse Transcriptase-PCR Kit (Invitrogen, Carlsbad, CA) and two pairs of oligonucleotide ...
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Nucleotide sequence and genomic organization of a newly identified member of the genus Carmovirus, soybean yellow mottle mosaic virus, from soybean
Archives of Virology, 2009Co-Authors: Leslie L. Domier, Jung Kyung Moon, Hong-soo Choi, Jae Sun MoonAbstract:The viral genome of soybean yellow mottle mosaic virus (SYMMV) from infected soybean ( Glycine max ) in Korea was cloned and sequenced. The complete monopartite single-stranded RNA genome of SYMMV consists of 4009 base pairs with six putative open reading frames and includes 5′- and 3′-untranslated regions of 39 and 229 nucleotides, respectively. The nucleotide and coat protein sequences of SYMMV share the highest sequence identity with those of cowpea mottle virus. Based on its genomic organization, its predicted amino acid sequence, and its phylogenetic relatedness to known Carmoviruses, we report that SYMMV is a new member of the genus Carmovirus in the family Tombusviridae .