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M J Roossinck

Publications and source records attributed to M J Roossinck.

At least 19 recordsLinked to original sources

Mutant clouds and occupation of sequence space in plant RNA viruses.

In nature, RNA viruses of plants often must adapt to ever-changing environments in the form of frequent host switches. This would favor a highly diverse population for transmission. However, most viruses that have been studied have been viruses of monocultural crops. In crop viruses, the mutation frequency of individual viral quasispecies varies greatly, both in experiment evolution studies and in populations of viruses within single field plants. There is some correlation between host range and mutation frequency in experimental evolution studies, but few viruses have been examined at the individual quasispecies level. Many questions about the nature of plant RNA virus populations and factors that affect the effective population sizes, such as genetic bottlenecks and postive and negative selection, have only begun to be studied. Many more analyses are required before generalized patterns can be determined.

Adaptation, Physiological↗

Genetic diversity in RNA virus quasispecies is controlled by host-virus interactions.

Many RNA viruses have genetically diverse populations known as quasispecies. Important biological characteristics may be related to the levels of diversity in the quasispecies (quasispecies cloud size), including adaptability and host range. Previous work using Tobacco mosaic virus and Cucumber mosaic virus indicated that evolutionarily related viruses have very different levels of diversity in a common host. The quasispecies cloud size for these viruses remained constant throughout serial passages. Inoculation of these viruses on a number of hosts demonstrated that quasispecies cloud size is not constant for these viruses but appears to be dependent on the host. The quasispecies cloud size remained constant as long as the viruses were maintained on a given host. Shifting the virus between hosts resulted in a change in cloud size to levels associated with the new host. Quasispecies cloud size for these viruses is related to host-virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases.

Genetic Variation↗

Cucumber mosaic virus D satellite RNA-induced programmed cell death in tomato.

D satellite RNA (satRNA) with its helper virus, namely, cucumber mosaic virus, causes systemic necrosis in tomato. The infected plant exhibits a distinct spatial and temporal cell death pattern. The distinct features of chromatin condensation and nuclear DNA fragmentation indicate that programmed cell death is involved. In addition, satRNA localization and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling show that cell death is initiated from the infected phloem or cambium cells and spreads to other nearby infected cells. Timing of the onset of necrosis after inoculation implicates the involvement of cell developmental processes in initiating tomato cell death. Analysis of the accumulation of minus- and plus-strand satRNAs in the infected plants indicates a correlation between high amounts of minus-strand satRNA and tomato cell death.

Apoptosis↗

Recognition of the core RNA promoter for minus-strand RNA synthesis by the replicases of Brome mosaic virus and Cucumber mosaic virus.

Replication of viral RNA genomes requires the specific interaction between the replicase and the RNA template. Members of the Bromovirus and Cucumovirus genera have a tRNA-like structure at the 3' end of their genomic RNAs that interacts with the replicase and is required for minus-strand synthesis. In Brome mosaic virus (BMV), a stem-loop structure named C (SLC) is present within the tRNA-like region and is required for replicase binding and initiation of RNA synthesis in vitro. We have prepared an enriched replicase fraction from tobacco plants infected with the Fny isolate of Cucumber mosaic virus (Fny-CMV) that will direct synthesis from exogenously added templates. Using this replicase, we demonstrate that the SLC-like structure in Fny-CMV plays a role similar to that of BMV SLC in interacting with the CMV replicase. While the majority of CMV isolates have SLC-like elements similar to that of Fny-CMV, a second group displays sequence or structural features that are distinct but nonetheless recognized by Fny-CMV replicase for RNA synthesis. Both motifs have a 5'CA3' dinucleotide that is invariant in the CMV isolates examined, and mutational analysis indicates that these are critical for interaction with the replicase. In the context of the entire tRNA-like element, both CMV SLC-like motifs are recognized by the BMV replicase. However, neither motif can direct synthesis by the BMV replicase in the absence of other tRNA-like elements, indicating that other features of the CMV tRNA can induce promoter recognition by a heterologous replicase.

Base Sequence↗

Efficient and specific initiation of subgenomic RNA synthesis by cucumber mosaic virus replicase in vitro requires an upstream RNA stem-loop.

We defined the minimal core promoter sequences responsible for efficient and accurate initiation of cucumber mosaic virus (CMV) subgenomic RNA4. The necessary sequence maps to positions -28 to +15 relative to the initiation cytidylate used to initiate RNA synthesis in vivo. Positions -28 to -5 contain a 9-bp stem and a 6-nucleotide purine-rich loop. Considerable changes in the stem and the loop are tolerated for RNA synthesis, including replacement with a different stem-loop. In a template competition assay, the stem-loop and the initiation cytidylate are sufficient to interact with the CMV replicase. Thus, the mechanism of core promoter recognition by the CMV replicase appears to be less specific in comparison to the minimal subgenomic core promoter of the closely related brome mosaic virus.

Base Sequence↗

Evolutionarily related Sindbis-like plant viruses maintain different levels of population diversity in a common host.

The levels of population diversity of three related Sindbis-like plant viruses, Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus (CCMV), in infections of a common host, Nicotiana benthamiana, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in N. benthamiana. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.

Base Sequence↗

Rearrangements in the 5' nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups.

Cucumber mosaic virus (CMV) has been divided into two subgroups based on serological data, peptide mapping of the coat protein, nucleic acid hybridization, and nucleotide sequence similarity. Analyses of a number of recently isolated strains suggest a further division of the subgroup I strains. Alignment of the 5' nontranslated regions of RNA 3 for 26 strains of CMV suggests the division of CMV into subgroups IA, IB, and II and suggests that rearrangements, deletions, and insertions in this region may have been the precursors of the subsequent radiation of each subgroup. Phylogeny analyses of CMV using the coat protein open reading frame of 53 strains strongly support the further division of subgroup I into IA and IB. In addition, strains within each subgroup radiate from a single point of origin, indicating that they have evolved from a single common ancestor for each subgroup.

5' Untranslated Regions↗

Structural analysis of a necrogenic strain of cucumber mosaic cucumovirus satellite RNA in planta.

Structural studies of plant viral RNA molecules have been based on in vitro chemical and enzymatic modification. That approach, along with mutational analysis, has proven valuable in predicting structural models for some plant viruses such as tobacco mosaic tobamovirus and brome mosaic bromovirus. However, in planta conditions may be dramatically different from those found in vitro. In this study we analyzed the structure of cucumber mosaic cucumovirus satellite RNA (sat RNA) strain D4 in vivo and compared it to the structures found in vitro and in purified virions. Following a methodology developed to determine the structure of 18S rRNA within intact plant tissues, different patterns of adenosine and cytosine modification were found for D4-sat RNA molecules in vivo, in vitro, and in virions. This chemical probing procedure identifies adenosine and cytosine residues located in unpaired regions of the RNA molecules. Methylation data, a genetic algorithm in the STAR RNA folding program, and sequence alignment comparisons of 78 satellite CMV RNA sequences were used to identify several helical regions located at the 5' and 3' ends of the RNA molecule. Data from previous mutational and sequence comparison studies between satellite RNA strains inducing necrosis in tomato plants and those strains not inducing necrosis allowed us to identify one helix and two tetraloop regions correlating with the necrogenicity syndrome.

Base Sequence↗

Support of a cucumber mosaic virus satellite RNA maps to a single amino acid proximal to the helicase domain of the helper virus.

Cucumber mosaic virus (CMV) is a tripartite RNA virus that can support the replication of satellite RNAs, small molecular parasites of the virus. Satellite RNAs can have a dramatic effect on the helper virus and the host plant in a manner specific to the helper, satellite, and host. Previously, we showed that the Sny-CMV strain is not able to support the replication of the WL1 satellite RNA in zucchini squash and that this phenotype maps to RNA 1. In the present study, we use recombinant cDNA clones of Fny- and Sny-CMV RNA 1 and a site-directed mutant of Fny-CMV RNA 1 to demonstrate that the inability to support WL1 satellite RNA maps to a single amino acid at residue 978 in the 1a protein, proximal to the helicase domain VI. Support of satellite RNA in whole plants and in protoplasts of zucchini squash is analyzed.

Amino Acid Sequence↗

Mechanisms of plant virus evolution.

Plant viruses utilize several mechanisms to generate the large amount of genetic diversity found both within and between species. Plant RNA viruses and pararetroviruses probably have highly error prone replication mechanisms, that result in numerous mutations and a quasispecies nature. The plant DNA viruses also exhibit diversity, but the source of this is less clear. Plant viruses frequently use recombination and reassortment as driving forces in evolution, and, occasionally, other mechanisms such as gene duplication and overprinting. The amount of variation found in different species of plant viruses is remarkably different, even though there is no evidence that the mutation rate varies. The origin of plant viruses is uncertain, but several possible theories are proposed. The relationships between some plant and animal viruses suggests a common origin, possibly an insect virus. The propensity for rapid adaptation makes tracing the evolutionary history of viruses difficult, and long term control of virus disease nearly impossible, but it provides an excellent model system for studying general mechanisms of molecular evolution.

Journal Article↗

Spontaneous change of a benign satellite RNA of cucumber mosaic virus to a pathogenic variant.

Plant satellite RNAs generally reduce the level of helper virus accumulation and attenuate the disease symptoms induced by the helper virus that they depend upon for replication and packaging. As such, satellite RNAs could be used as biocontrol agents to reduce the level of disease in field crops, either by the application of a viral vaccine to healthy plants, or by the transgenic expression of satellite RNA in transformed plants. One such virus/satellite RNA system already under use in field tests is cucumber mosaic virus (CMV) and its satellite RNAs. However, in this system, some satellite RNAs also intensify viral disease in particular host plants. We passaged a satellite RNA of CMV with its helper virus to determine whether a satellite RNA that attenuates CMV-induced disease on tobacco plants could mutate to a pathogenic form, which might then be selected. In several experiments involving strains of CMV from each of the two subgroups, the satellite rapidly mutated to a pathogenic form, which was selected. This demonstrates an inherent risk associated with the use of attenuating satellite RNAs as a form of biocontrol of CMV.

Base Sequence↗

Interspecific reassortment of genomic segments in the evolution of cucumoviruses.

Segmented genomes of RNA viruses are thought to evolve and be maintained in analogy to sexual recombination and reassortment in eukaryotic systems. If reassortment among genomes is an important event in cucumoviral evolution, then such events should be detectable among extant viruses. In this study, phylogenetic analyses of cucumoviruses were performed using aligned amino acid sequences. The results reveal different relationships among species when the three genomic segments are compared, suggesting that reassortment events have given rise to extant forms. In addition, we describe a cucumovirus isolate that is composed of genomic segments from two distinct viral species. These results indicate that reassortment events may provide a mechanism for speciation in cucumoviruses.

Amino Acid Sequence↗

Variation in the hypervariable region of cucumber mosaic virus satellite RNAs is affected by the helper virus and the initial sequence context.

The D satellite RNA (sat RNA) of cucumber mosaic virus (CMV) was previously shown to contain a region of hypervariability around nucleotide 230, in wild-type populations and in cDNA clones and progeny of one such clone (pDsat4) after passage with the subgroup I strain Fny-CMV. This hypervariable region (HVR) consists of a series of consecutive A and/or U residues. We found that variability is also generated in the HVR of transcript derived from pDsat4 after passage with the subgroup II strain LS-CMV and with tomato aspermy virus (TAV). However, the progeny differ with respect to the sequence of the HVR after passage with both LS-CMV and TAV. Another D-sat RNA cDNA clone that contains a C residue in the HVR, pDsat1, was previously shown not to develop variability in the HVR upon passage with Fny-CMV. However, when the C (position 231) was changed to an A residue, variability developed by the third passage with Fny-CMV. An additional cDNA clone derived from the B1-sat RNA, pBsat5, also contains a C residue in the region analogous to the D-sat RNA HVR and did not develop variability upon passage with either Fny- or LS-CMV. Changing this C to a U residue did not result in the development of hypervariability in the progeny of transcript from this mutant. Models to explain the generation of hypervariability are discussed.

Base Sequence↗

Genetic analysis of helper virus-specific selective amplification of cucumber mosaic virus satellite RNAs.

Satellite RNAs (sat-RNAs) are small molecular parasites associated with a number of plant RNA viruses. The cucumber mosaic virus (CMV) sat-RNAs are ca. 335 nucleotides and have evolved to produce a large number of closely related sat-RNAs. Different cucumoviruses can act as helper viruses in the amplification of CMV sat-RNAs. We have found that different helper viruses show a preference for a particular sat-RNA in a mixed infection. In this study the specificity of WL47 sat-RNA amplification by LS-CMV and of D4 sat-RNA amplification by tomato aspermy virus in mixed infections was examined. Recombinant cDNA clones of D4 sat-RNA and WL47 sat-RNA were used to map the sat-RNA sequences responsible for the helper virus selection of a specific sat-RNA for amplification.

Base Sequence↗

Characterization of defective RNAs derived from RNA 3 of the Fny strain of cucumber mosaic cucumovirus.

Two defective RNAs (designated D RNA 3 alpha and D RNA 3 beta) were found to be associated with the Fny strain of cucumber mosaic cucumovirus but not with the Sny strain after serial passages in a tobacco host. The D RNAs were derived from RNA 3 by single, in-frame deletions within the 3a open reading frame. A full-length cDNA clone from which biologically active transcripts can be produced in vitro has been constructed for D RNA 3 beta. This transcript can be replicated in tobacco plants infected with subgroup I and II cucumber mosaic cucumovirus strains and with peanut stunt cucumovirus. Translation of D RNA 3 beta in vitro produced a 20-kDa peptide, which was consistent with the predicted coding capacity of the deleted 3a open reading frame. D RNA 3 beta was also associated with polyribosomes isolated from infected tobacco plants. The presence of the D RNAs had no apparent effect upon helper virus yield or symptom production.

Amino Acid Sequence↗

The kinetics of infection of zucchini squash by cucumber mosaic virus indicate a function for RNA 1 in virus movement.

The differential rate of systemic symptom induction in zucchini squash by the Fny- and Sny-strains of cucumber mosaic virus (CMV) previously was mapped to RNA 1, which encodes a protein (1a) involved in virus replication. Examination of the kinetics of accumulation of the RNAs and the four encoded proteins in the inoculated cotyledons showed that the Fny-CMV-associated products generally appeared earlier than the Sny-CMV-associated products. In the systematically infected leaves, this difference was magnified, with a 2-day delay in the appearance of the Sny-CMV RNAs and encoded proteins. However, both Fny-CMV and Sny-CMV RNAs showed similar kinetics of RNA, 2a, 3a, and coat protein accumulation in protoplasts prepared from zucchini squash cotyledons. These data indicate that the differential rate of systemic symptom development was due to a difference in the rate of movement rather than the rate of replication. This was confirmed by a leaf-detachment assay, which showed a difference in the rate of systemic movement by Fny-CMV vs Sny-CMV, and by leaf-press blot hybridization of the inoculated cotyledons at different days postinoculation, which showed a difference in the rate of cell-to-cell movement by the two strains of CMV. Taken together, these data show that the rates of cell-to-cell and long-distance movement can be regulated by sequences in CMV RNA 1, previously thought to be involved only in virus replication.

Cucumovirus↗