De novo emergence of a novel satellite RNA of cucumber mosaic virus following serial passages of the virus derived from RNA transcripts.

Satellite RNA (satRNA) is often associated with cucumber mosaic virus (CMV); however, its origin remains unexplained and a subject for speculation. We passaged progeny of molecularly cloned CMV-Fny and CMV-LS in Nicotiana tabacum cv. Ky 14 under greenhouse conditions. A satRNA emerged after at least eight successive transfers of CMV-Fny, but no satRNA was recovered after eleven serial transfers of CMV-LS under the same conditions. The sequences of the newly emerged satRNA were determined, and an infectious cDNA clone was synthesized. Comparison of the sequences of the newly emerged satRNA with those of known CMV satRNAs showed that it is unique. This observation raises interesting questions regarding the enigmatic nature of the origin of CMV satRNAs.

Aspergillus mycoviruses are targets and suppressors of RNA silencing.

RNA silencing can function as a virus defense mechanism in a diverse range of eukaryotes, and many viruses are capable of suppressing the silencing machinery targeting them. However, the extent to which this occurs between fungal RNA silencing and mycoviruses is unclear. Here, three Aspergillus dsRNA mycoviruses were partially characterized, and their relationship to RNA silencing was investigated. Aspergillus virus 1816 is related to Agaricus bisporus white button mushroom virus 1 and suppresses RNA silencing through a mechanism that alters the level of small interfering RNA. Aspergillus virus 178 is related to RNA virus L1 of Gremmeniella abietina and does not appear to affect RNA silencing. The third virus investigated, Aspergillus virus 341, is distantly related to Sphaeropsis sapinea RNA virus 2. Detection of mycovirus-derived siRNA from this mycovirus demonstrates that it is targeted for degradation by the Aspergillus RNA silencing machinery. Thus, our results indicate that Aspergillus mycoviruses are both targets and suppressors of RNA silencing. In addition, they suggest that the morphological and physiological changes associated with some mycoviruses could be a result of their antagonistic relationship with RNA silencing.

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.

Expression of hepatitis B viral core region in mammalian cells.

We studied the expression of the core region of the hepatitis B virus genome in mammalian cells with recombinant plasmid vectors. Stably transformed rat fibroblast cell lines were established by transfection with vectors containing subgenomic and genome-length hepatitis B virus DNA, followed by G418 selection. The RNA transcripts directed by the core region were characterized by Northern blot hybridization and S1 nuclease mapping. Using the chloramphenicol acetyltransferase gene expression system, the promoter activity located upstream of the core open reading frame was confirmed. The synthesis of core and e polypeptides was studied with a commercial radioimmunoassay. These studies show that partial deletion of the precore sequences abolished secretion of the e antigen, but there was pronounced synthesis of the core antigen in transfected cells.

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.

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.

Genetic Diversity of Panicum mosaic virus Satellite RNAs in St. Augustinegrass.

ABSTRACT St. Augustine decline is a viral disease caused by Panicum mosaic virus (PMV) alone or in combination with a satellite virus (SPMV) and/or satellite RNAs (satRNAs). A ribonuclease protection assay (RPA) was used to evaluate the genetic diversity of PMV satRNAs isolated from 100 naturally infected St. Augustinegrass plants (Stenotaphrum secundatum). Distinctive satRNA RPA profiles were observed for 40 of 52 samples from College Station (CS) and 37 of 48 samples from Corpus Christi (CC), Texas. A dendrogram constructed from the RPA data revealed that satRNAs were grouped in two distinct clusters based on their place of origin. From 100 samples, only 4 satRNAs from CS were placed in the CC group, and only 2 satRNAs from CC were placed in the CS group. The data show that there is genetic variability in PMV satRNAs in naturally occurring infections, and distinct geographically separate populations can be identified from CC and CS.

Field Survey of Cucumber mosaic virus Subgroups I and II in Crop Plants in Costa Rica.

Leaf samples were collected from cucurbit and solanaceous crop plants and Musa spp. in 28 locations in five provinces of Costa Rica during the period from January to October 1996. Sampling sites were selected in dry, humid, and moist tropical regions ranging in altitude from 50 to 2,100 m above sea level. RNA-enriched total nucleic acid solutions were spotted onto nylon membranes and hybridized to RNA probes specific for Cucumber mosaic virus (CMV) subgroups I or II. The presence of CMV was confirmed in 13 crops in 23 of the 28 sampling sites. CMV subgroup I was found to predominate in Costa Rica. CMV subgroup II was detected in the Atlantic region only, and in only 1 out of 113 CMV-positive samples.

Cucumber mosaic virus, a model for RNA virus evolution.

Summary Taxonomic relationships: Cucumber mosaic virus (CMV) is the type member of the Cucumovirus genus, in the family Bromoviridae. Additional members of the genus are Peanut stunt virus (PSV) and Tomato aspermy virus (TAV). The RNAs 3 of all members of the genus can be exchanged and still yield a viable virus, while the RNAs 1 and 2 can only be exchanged within a species. Physical properties: The virus particles are about 29 nm in diameter, and are composed of 180 subunits (T = 3 icosahedral symmetry). The particles sediment with an s value of approximately 98. The virions contain 18% RNA, and are highly labile, relying on RNA-protein interactions for their integrity. The three genomic RNAs, designated RNA 1 (3.3 kb in length), RNA 2 (3.0 kb) and RNA 3 (2.2 kb) are packaged in individual particles; a subgenomic RNA, RNA 4 (1.0 kb), is packaged with the genomic RNA 3, making all the particles roughly equivalent in composition. In some strains an additional subgenomic RNA, RNA 4A is also encapsidated at low levels. The genomic RNAs are single stranded, plus sense RNAs with 5' cap structures, and 3' conserved regions that can be folded into tRNA-like structures. Satellite RNAs: CMV can harbour molecular parasites known as satellite RNAs (satRNAs) that can dramatically alter the symptom phenotype induced by the virus. The CMV satRNAs do not encode any proteins but rely on the RNA for their biological activity. Hosts: CMV infects over 1000 species of hosts, including members of 85 plant families, making it the broadest host range virus known. The virus is transmitted from host to host by aphid vectors, in a nonpersistent manner. Useful web sites: http://mmtsb.scripps.edu/viper/1f15.html (structure); http://www.ncbi.nlm.nih.gov/ICTVdb/ICTVdB/10040001.htm (general information).

Temperature-sensitive replication of cucumber mosaic virus in muskmelon (Cucumis melo cv. Iroquois), maps to RNA 1 of a slow strain.

Several strains of cucumber mosaic virus have been categorized as either 'fast' or 'slow', based on the time of appearance of symptoms after inoculation onto zucchini squash (Curbita pepo cv. Black Beauty). These strains were examined for their ability to replicate in muskmelon (Cucumis melo cv. Iroquois) at elevated temperatures. All of the fast strains were able to replicate at 37 degrees C in muskmelon, whereas all of the slow strains were unable to replicate to detectable levels at 37 degrees C, but replicated efficiently at 27 degrees C. Using previously constructed pseudorecombinants between a fast and a slow strain, Fny- and Sny-CMV, temperature sensitivity was mapped to RNA 1 of the Sny-CMV strain.

In vivo phosphorylation and protein analysis of hepatitis B virus core antigen.

The C open reading frame of the hepatitis B virus contains two in-frame ATG codons that are separated by the precore region and encodes two major polypeptides that are antigenically distinct and that are probably synthesized from individual mRNAs. The precore region directs the secretion of the e antigen, whereas the core antigen can be expressed in the absence of these sequences. In this report a transient expression system was used to study the hepatitis B virus core antigen. By using a chimeric complex of adenovirus major late promoter-simian virus 40 enhancer sequences, we were able to achieve high levels of core antigen expression in transfected cells, permitting characterization of this protein and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The core polypeptide is a 20.9-kilodalton protein, and we show in this study that it is phosphorylated in vivo. Cell fractionation studies, the results of which are supported by indirect immunofluorescence, localized the phosphocore in the cytosol and the nucleus and indicated that it is associated with the membrane of transfected cells. Results of Triton X-114 solubilization studies indicated that the phosphocore is peripherally associated with cytoplasmic membranes. Expression of the membrane-associated phosphocore occurred in the absence of the precore sequences. The phosphocore also assembled into particles in the absence of other viral gene products or intact DNA.

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.

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.

Differential replication in zucchini squash of a cucumber mosaic virus satellite RNA maps to RNA 1 of the helper virus.

Cucumber mosaic virus (CMV) supports the replication and encapsidation of its satellite RNA, both in solanaceous and cucurbit host plants; however, different strains of CMV support the replication of satellite RNAs with different efficiency. In addition, replication of satellite RNA is very efficient in solanaceous host plants and generally poor in cucurbit host plants. The WL1-satellite (WL1-sat) RNA is an exception, and replicates to high levels in both solanaceous and curcubit host plants with most CMV strains as the helper virus. Two strains of CMV were used in this study: Fny-CMV, which replicates the WL1-sat RNA efficiently in all hosts tested; and Sny-CMV, which does not replicate the WL1-sat RNA to detectable levels in zucchini squash (Cucurbita pepo), but does replicate WL1-sat RNA efficiently in other hosts. Using pseudorecombinants constructed between Fny-CMV and Sny-CMV we have mapped to RNA 1 the ability to support the efficient replication of WL1-sat RNA in zucchini squash.

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.

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.

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.

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.

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.