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PubMed1998

Relationship of the pelargonium flower break carmovirus (PFBV) coat protein gene with that of other carmoviruses.

The 3'-terminal 1500 nucleotides of the genome of pelargonium flower break carmovirus (PFBV) were sequenced from RT-PCR amplification products. One large ORF was found, encoding a 345 amino acid protein of Mr 37 kDa, which corresponds to the coat protein, as confirmed by immunoprecipitation of products of in vitro transcription and translation. The sequence also included the putative promoter of the coat protein gene subgenomic RNA, as well as its 5' and 3' untranslated regions. The PFBV coat protein was more similar to that of saguaro cactus virus and carnation mottle virus than to that of other carmoviruses. Despite the lower level of similarity of CP gene sequences compared to the RNA dependent RNA polymerase (RdRp) gene sequences of small icosahedral viruses used in taxonomic studies, PFBV CP sequence comparisons and alignments confirmed that PFBV is related to carmoviruses, tombusviruses and a dianthovirus, as previously concluded from the analysis of a PFBV RdRp gene fragment.

Base Sequence↗
PubMed1991

Point mutations in the turnip crinkle virus capsid protein affect the symptoms expressed by Nicotiana benthamiana.

In an effort to determine the biological function(s) of the capsid protein protruding domains unique to the plant carmo- and tombusviruses, we constructed turnip crinkle virus (TCV) mutants in which tandem, in-frame translation terminators replaced the first two codons of the five-amino acid hinge between the shell and the protruding domains of the TCV capsid protein. One of the mutants replicated in inoculated leaves and protoplasts without detectable accumulation of capsid protein. The mutant lacked the capacity to move systemically in Brassica campestris and Nicotiana benthamiana. After 8 weeks, revertant virions that had regained the capacity to move systemically were purified and found to have sense codons at the positions of the introduced translation terminators. One of the revertants, with amino acid substitutions in the hinge, elicited milder symptoms than those elicited by the wild-type virus, and another elicited more severe symptoms. Oligonucleotide-directed mutagenesis was used to show that the hinge mutations were sufficient to elicit the milder, but not the more severe, symptom syndrome. Single amino acid substitutions were also shown to be sufficient to elicit the milder, but not the more severe, symptoms.

Amino Acid Sequence↗
PubMed1984

Differences in the morphology of isometric particles of some plant viruses stained with uranyl acetate as an aid to their identification.

Purified preparations of viruses with small polyhedral particles representing ten distinct taxonomic groups were examined by electron microscopy after negative staining with uranyl acetate. Comparisons of the relative size, shape and surface appearance of the particles established that some of them could be distinguished at the group level. However, the particles of viruses belonging to the bromovirus group could not be distinguished from those of the cucumovirus group, the nepovirus group from those of the comovirus group, and the tombusvirus group from those of the dianthovirus group. Furthermore, the particles of viruses of the sobemovirus, lutoevirus and tobacco necrosis virus groups were indistinguishable. The usefulness of the observed differences in the particle morphologies to virus identification is discussed.

Microscopy, Electron↗
PubMed1985

Use of ELISA for measuring the extent of serological cross-reactivity between plant viruses.

The degree of antigenic relatedness between two plant viruses is commonly expressed by a serological differentiation index (SDI) which corresponds to the average number of two-fold dilution steps separating homologous from heterologous precipitin titers. Results obtained with several tobamo- and tombusviruses indicated that the indirect form of the enzyme-linked immunosorbent assay (ELISA) can also be used for calculating SDI values. This was achieved by comparing the antiserum dilutions that lead to the same absorbance measurements (for instance 1.0) when homologous and heterologous viruses are assayed by ELISA. SDI values calculated from ELISA were similar to those obtained from precipitin tests. Because of its greater sensitivity, ELISA is able to quantify weak cross-reactions that are not detectable by precipitin tests.

Antigens, Viral↗
PubMed2006

A simplified method for cloning of short interfering RNAs from Brassica juncea infected with Turnip mosaic potyvirus and Turnip crinkle carmovirus.

RNA silencing is a plant defense mechanism in which virus infected plants produce short interfering RNAs (siRNAs) derived from viral RNA, that attack the virus at the post-transcriptional level. In a previous study on Cymbidium ringspot tombusvirus (CymRSV) infection in Nicotiana benthamiana, siRNAs (determined by cloning and sequencing) predominantly originated from the sense (+) strand of the viral RNA, suggesting that the majority of siRNAs are produced through the direct cleavage of the virus single strand (ss) RNA by the plant Dicer-like enzyme. To test whether this asymmetry in strand polarity is a generic rule for all plant viruses, siRNAs from Brassica juncea, either singly infected by Turnip mosaic potyvirus (TuMV, the family Potyviridae), or doubly infected with TuMV and Turnip crinkle carmovirus (TCV, the family Tombusviridae) were investigated. A simplified siRNA cloning method was developed, using a single ligation reaction to attach both 5' and 3' adapters to the target short RNAs followed by one-step RT-PCR amplification. In the TCV infection, as for the CymRSV infection, siRNAs were produced predominantly (97.6%) from the +ss RNA. However, for TuMV infections, siRNAs were derived from both strands (+/-, 58.1-41.9%), indicating the presence of alternative siRNA production mechanisms.

Carmovirus↗
PubMed2005

RNA silencing suppressor p21 of Beet yellows virus forms an RNA binding octameric ring structure.

Many plant viruses encode proteins that suppress the antiviral RNA silencing response mounted by the host. The suppressors p19 from tombusvirus and p21 from Beet yellows virus appear to block silencing by directly binding siRNA, a critical mediator in the process. Here, we report the crystal structure of p21, which reveals an octameric ring architecture with a large central cavity of approximately 90 A diameter. The all alpha-helical p21 monomer consists of N- and C-terminal domains that associate with their neighboring counterparts through symmetric head-to-head and tail-to-tail interactions. A putative RNA binding surface is identified in the conserved, positive-charged inner surface of the ring. In contrast to the specific p19-siRNA duplex interaction, p21 is a general nucleic acid binding protein, interacting with 21 nt or longer single- and double-stranded RNAs in vitro. This study reveals an RNA binding structure adopted by the p21 silencing suppressor.

Amino Acid Sequence↗
PubMed2004

Structure of Cowpea mottle virus: a consensus in the genus Carmovirus.

Cowpea mottle virus (CPMoV) is a T = 3 virus that belongs to Carmovirus genus of the Tombusviridae family. Here, we report the crystal structure of CPMoV determined to a resolution of 7.0 angstroms. The structures and sequences of three Carmoviruses, CPMoV, Turnip crinkle virus (TCV), and Carnation mottle virus (CarMV) have been compared to TBSV from the Tombusvirus genus. CPMoV, TCV, and CarMV all have a deletion in betaC strand in the S domain relative to TBSV that may be distinctive to the genus. Although CPMoV has an elongated C-terminus like TBSV, it does not interact with the icosahedrally related P domain as observed in TBSV. In CPMoV, the termini of A and B interact with the icosahedrally related shell domains of A and C, respectively, to form a chain of interactions around the 5-fold axes. The C subunit terminus does not, however, interact with the B subunit because of quasi-equivalent differences in the P domain orientations.

Amino Acid Sequence↗
PubMed2006

Sequence analysis of coat protein gene of Wuhan nodavirus isolated from insect.

Wuhan nodavirus (WhNV) particles are isometric, non-enveloped, and about 29 nm in diameter. In the previous study, we determine its physiochemical characterization and the nucleotide sequence of the larger genomic segment, RNA1 and identify it a nodavirus. WhNV RNA1 is 3,149 nt in length, encoding protein A, catalytic subunit of RNA-dependent RNA polymerase (RdRp). In this report, we complete the sequence determination of the smaller genomic segment, RNA2 of WhNV. WhNV RNA2 is determined to be 1,562 nt long, containing a 430-amino-acid open reading frame (ORF) encoding the coat protein of WhNV with a calculated molecular mass of 47,856 Da. The homology of the coat protein of WhNV and the homologous proteins of other nodaviruses either alphanodaviruses or betanodaviruses is very low. WhNV coat protein shares the highest identity (24%) with that of Lates calcarifer encephalitis virus (LCEV), a betanodavirus, and shares less than 16% identical amino acids with each of the alphanodaviruses. Furthermore, the prediction of WhNV capsid structure by 3D-PSSM shows that the capsid structure of WhNV resembles that of tomato bushy stunt virus (TBSV), a tombusvirus, which contains two domains, rather than the expected single-domain capsid protein of insect nodaviruses. The phylogenetic analysis indicates that WhNV is the most distantly related of both the alphanodaviruses and betanodaviruses, which provides significant new data for understanding the evolution of the nodavirus family.

Amino Acid Sequence↗
PubMed1983

The fine structure of Cymbidium ringspot virus infections in host tissues. III. Role of peroxisomes in the genesis of multivesicular bodies.

Ultrastructural studies of Nicotiana clevelandii plants systemically infected with Cymbidium ringspot virus, a member of the tombusvirus group, have shown that a clear-cut relationship exists between perioxisomes and multivesicular bodies (MVB). In infected cells, peroxisomes undergo a progressive vesiculation of the bounding membrane through the possible addition of membranous material by the endoplasmic reticulum and become very plastic. Portions of the ground cytoplasm are engulfed either through the invagination of the limiting membrane or the production of membranous appendages that fold back on the main body. Cytochemical tests have shown MVB to possess catalase and glycolate oxidase activity in the matrix. The vesicles contain RNA, a substantial amount of which is double stranded, as indicated by differential RNase digestion tests in high- and low-salt media. The double-stranded RNA may consist of replicative forms or replicative intermediates of the viral nucleic acid. If so, MVB (i.e., modified peroxisomes) may be directly involved in the replication of Cymbidium ringspot virus.

Catalase↗
PubMed2003

Size selective recognition of siRNA by an RNA silencing suppressor.

RNA silencing in plants likely exists as a defense mechanism against molecular parasites such as RNA viruses, retrotransposons, and transgenes. As a result, many plant viruses have adapted mechanisms to evade and suppress gene silencing. Tombusviruses express a 19 kDa protein (p19), which has been shown to suppress RNA silencing in vivo and bind silencing-generated and synthetic small interfering RNAs (siRNAs) in vitro. Here we report the 2.5 A crystal structure of p19 from the Carnation Italian ringspot virus (CIRV) bound to a 21 nt siRNA and demonstrate in biochemical and in vivo assays that CIRV p19 protein acts as a molecular caliper to specifically select siRNAs based on the length of the duplex region of the RNA.

Amino Acid Sequence↗
PubMed2000

A century of plant virus management in the Salinas valley of California, 'East of Eden'.

The mild climate of the Salinas Valley, CA lends itself well to a diverse agricultural industry. However, the diversity of weeds, crops and insect and fungal vectors also provide favorable conditions for plant virus disease development. This paper considers the incidence and management of several plant viruses that have caused serious epidemics and been significant in the agricultural development of the Salinas Valley during the 20th century. Beet curly top virus (BCTV) almost destroyed the newly established sugarbeet industry soon after its establishment in the 1870s. A combination of resistant varieties, cultural management of beet crops to provide early plant emergence and development, and a highly coordinated beet leafhopper vector scouting and spray programme have achieved adequate control of BCTV. These programmes were first developed by the USDA and still operate. Lettuce mosaic virus was first recognized as causing a serious disease of lettuce crops in the 1930s. The virus is still a threat but it is controlled by a lettuce-free period in December and a seed certification programme that allows only seed lots with less than one infected seed in 30000 to be grown. 'Virus Yellows' is a term used to describe a complex of yellows inducing viruses which affect mainly sugarbeet and lettuce. These viruses include Beet yellows virus and Beet western yellows virus. During the 1950s, the complex caused significant yield losses to susceptible crops in the Salinas Valley. A beet-free period was introduced and is still used for control. The fungus-borne rhizomania disease of sugarbeet caused by Beet necrotic yellow vein virus was first detected in Salinas Valley in 1983. Assumed to have been introduced from Europe, this virus has now become widespread in California wherever beets are grown and crop losses can be as high as 100%. Movement of infested soil and beets accounts for its spread throughout the beet-growing regions of the United States. Control of rhizomania involves several cultural practices, but the use of resistant varieties is the most effective and is necessary where soils are infested. Rhizomania-resistant varieties are now available that perform almost as well as the non-resistant varieties under non-rhizomania conditions. Another soil-borne disease termed lettuce dieback, caused by a tomato bushy stunt-like tombusvirus, has become economically limiting to romaine and leaf lettuce varieties. The virus has no known vector and it seems to be moved through infested soil and water. Heavy rains in the past 4 years have caused flooding of the Salinas River and lettuce fields along the river have been affected severely by dieback. Studies are now in progress to characterize this new virus and identify sources of resistance. Agriculture in the Salinas Valley continues to grow and diversify, driven by demands for 'clean', high quality food by the American public and for export. The major aspects of plant virus control, including crop-free periods, breeding for resistance, elimination of inoculum sources, and vector control will continue to be vital to this expansion. Undoubtedly, the advances in crop production through genetic manipulation and advances in pest management through biological control will eventually become an important part of agricultural improvement.

Animals↗
PubMed2002

A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs.

Posttranscriptional gene silencing (PTGS) processes double-stranded (ds) RNAs into 21-25 nucleotide (nt) RNA fragments that direct ribonucleases to target cognate mRNAs. In higher plants, PTGS also generates mobile signals conferring sequence-specific silencing in distant organs. Since PTGS acts as an antiviral system in plants, successful virus infection requires evasion or suppression of gene silencing. Here we report that the 19 kDa protein (p19) of tombusviruses is a potent silencing suppressor that prevents the spread of mobile silencing signal. In vitro, p19 binds PTGS-generated, 21-25 nt dsRNAs and 21-nt synthetic dsRNAs with 2-nt 3' overhanging end(s), while it barely interacts with single-stranded (ss) RNAs, long dsRNAs or blunt-ended 21-nt dsRNAs. We propose that p19 mediates silencing suppression by sequestering the PTGS-generated 21-25 nt dsRNAs, thus depleting the specificity determinants of PTGS effector complexes. Moreover, the observation that p19-expressing transgenic plants show altered leaf morphology might indicate that the p19-targeted PTGS pathway is also important in the regulation of plant development.

Gene Silencing↗
PubMed2026

A Novel Approach to Engineering Tomato Spotted Wilt Virus Infectious Clones by Disarming Key Nodes in Antiviral Defenses.

Tomato spotted wilt virus (TSWV) is an economically devastating pathogen that rapidly overcomes genetic resistance in major crops. Reverse genetic systems are crucial for investigating plant-virus interactions and resistance-breaking mechanisms, and developing these tools for segmented ambisense RNA viruses remains a crucial challenge. Current TSWV clones rely on extensively modified Asian isolates requiring co-delivery of multiple replication helpers and viral silencing suppressors. Streamlining these systems for regionally significant strains with minimal genetic alterations is essential. Here, we developed the first infectious clone of a U.S. TSWV isolate (PA01). Three binary plasmids contain cDNAs for the antigenomic L and S segments, as well as the genomic M segment, with enhanced GFP replacing NSs on the S segment. Co-delivery of the cucumovirus 2b alone or in combination with tombusvirus P19 or begomovirus AL2 achieved a high proportion of systemically infected Nicotiana benthamiana and Capsicum annuum plants. In N. tabacum, co-delivering the Caenorhabditis elegans cell death suppressor CED-9 or using NahG transgenic plants produced 30 to 33% systemically infected plants. Co-delivery of 2b boosted infection levels in NahG plants to 62%. These data indicate that in addition to the antiviral RNA-silencing machinery, additional host defense pathways influence TSWV rescue and systemic infection from cDNA. [Formula: see text] Copyright © 2026 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Tospovirus↗
PubMed1991

De novo generation of cymbidium ringspot virus defective interfering RNA.

Nicotiana clevelandii plants were inoculated with cymbidium ringspot tombusvirus RNA synthesized in vitro, after which further passages were made by sap inoculation. During the third passage, low Mr RNA species appeared which had the characteristics of deletion mutants of genomic RNA. Sequence analysis of several of these defective interfering RNAs suggested a possible evolution of smaller from larger molecules. Computer-generated secondary structures of sequences surrounding recombination sites were extensive and stable and these sites occurred in interior or hairpin loops, thus providing a possible explanation for discontinuous RNA transcription and the formation of deletions in genomic RNA.

Base Sequence↗
PubMed1991

Phylogeny of capsid proteins of small icosahedral RNA plant viruses.

Statistically significant alignment was generated between the amino acid sequences of the (putative) shell (S) domains of the capsid proteins of small RNA plant viruses with icosahedral capsids in the tombusvirus, carmovirus, dianthovirus, sobemovirus and luteovirus groups. Inspection of the alignment showed good correspondence between the experimentally defined beta-strands and alpha-helices of the capsid proteins of tomato bushy stunt, southern bean mosaic and turnip crinkle viruses, allowing prediction of the secondary structure elements in proteins with unresolved tertiary structure. It is concluded that this set of viral capsid proteins forms a tight evolutionary cluster. Comparison of the alignment of the proteins of this family with the sequences of other capsid proteins of icosahedral RNA viruses revealed more distant similarities to the satellites of tobacco necrosis, panicum mosaic, tobacco mosaic and maize white line mosaic viruses, as well as to nepo- and comoviruses. The tentative phylogenetic tree derived from the capsid protein alignment separated into three main lineages: (I) carmo-, tombus- and dianthoviruses, (II) southern bean mosaic, tobacco necrosis and maize chlorotic mottle viruses, and (III) luteoviruses. Comparison of this tree topology with the tentative evolutionary schemes for the respective virus RNA-dependent RNA polymerases suggested that gene shuffling is the universal trend in the evolution of small RNA plant virus genomes.

Amino Acid Sequence↗
PubMed1991

The complete nucleotide sequence of tobacco necrosis virus strain D.

The complete sequence of the RNA genome of tobacco necrosis virus strain D (TNV-D) consisting of 3759 nucleotides has been determined. The positive strand contains five open reading frames (ORFs). The 5'-proximal ORF encodes a 22K protein terminating with an amber codon which may be read through to produce a 82K protein (p82). Two small centrally located ORFs each encode two out-of-frame 7K proteins (p7a and p7b). The 3'-proximal ORF encodes the 29K coat protein (CP), the N terminus of which has been sequenced directly. The genomic organization of TNV-D is very similar to that of TNV-A but differs in the placement of the p7a ORF, which does not overlap the p82 ORF in TNV-D, and in the absence of an ORF downstream of the CP gene in TNV-D. The p82 ORF shows extensive sequence similarity with the putative polymerases of the carmovirus group. This ORF is also as closely related to the corresponding ORF of TNV-A as it is to the corresponding ORF of the tombusvirus cucumber necrosis virus. The amino acid sequence of the TNV-D CP gene is similar to both the TNV-A and southern bean mosaic virus CP genes. Of the two p7 ORFs, p7a exhibits amino acid sequence similarity with corresponding proteins from TNV-A, melon necrotic spot virus, carnation mottle virus, turnip crinkle virus and maize chlorotic mottle virus, whereas the p7b ORF appears to be unique to TNV-A and TNV-D. Only the 3'-terminal three nucleotides of TNV-D genomic RNA are identical to the 3'-terminal nucleotides of the TNV satellite virus.

Amino Acid Sequence↗
PubMed1993

The chimeric nature of the genome of pea enation mosaic virus: the independent replication of RNA 2.

The genome of pea enation mosaic virus (PEMV) consists of two plus-sense RNAs, both of which are required for mechanical transmission. RNA 1 (5706 nucleotides) has strong sequence similarity with members of the luteovirus group, a similarity that is also manifested in the symptomatology, cytopathology and vector transmission of this virus. RNA 2 (4253 nucleotides) is hypothesized to facilitate systemic invasion and mechanical transmission, attributes that distinguish PEMV from the phloem-limited luteoviruses. Sequence analysis of RNA 2 has demonstrated that PEMV is unique among multicomponent viruses in that it lacks 3'- and 5'-terminal homology between its genomic RNAs. Sequence analysis of RNA 2 has identified an open reading frame encoding a putative product of 65K that contains a series of polymerase-like motifs typical of viral RNA-dependent RNA polymerases. This protein sequence lacks homology with the polymerase encoded on RNA 1 of PEMV, instead being more closely affiliated with the polymerases of viruses related to the carmo- and tombusvirus groups. Inoculation of pea protoplasts with RNA transcripts derived from a full-length cDNA clone of RNA 2 has demonstrated that RNA 2 replicates autonomously in the absence of RNA 1, although comparable inoculation of whole plants failed to establish a systemic infection. There is no evidence that RNA 2 encodes structural proteins, suggesting that encapsidation functions are supplied in trans by RNA 1, comparable to the helper-dependent complexes occurring within the luteovirus group. These data suggest that the PEMV genome can be characterized as a symbiotic association of two taxonomically distinct viral RNAs cooperatively interacting in the establishment of a systemic virus infection.

Amino Acid Sequence↗
PubMed1993

Sequence and structure of defective interfering RNAs associated with cucumber necrosis virus infections.

The presence of symptom-attenuating defective interfering (DI) RNAs in a laboratory culture of cucumber necrosis tombusvirus (CNV) was confirmed. Sequencing of cDNA clones of these DI RNAs revealed that CNV DI RNAs retained sequences from the CNV 5'-untranslated and 3'-terminal regions as well as a portion of the coding region for the 92K protein. Similar sequence arrangements were also observed in symptom-attenuating DI RNAs generated de novo from synthetic wild-type CNV transcripts. A comparison of the sequences and biological activities of these CNV DI RNAs is presented. In co-infections of synthetic wild-type CNV and CNV DI RNAs, prominent RNA species of a higher M(r) than the DI RNA used in the co-infection were found. The possible nature of these RNA species is discussed.

Base Sequence↗