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Biomedical subjects

J M Kaper

Publications and source records attributed to J M Kaper.

At least 19 recordsLinked to original sources
PubMed1995

The replication of a necrogenic cucumber mosaic virus satellite is temperature-sensitive in tomato.

Lethal necrosis development in tomato plants infected with cucumber mosaic virus (CMV) strain D containing the necrogenic satellite D-CARNA 5 and held at 32 degrees C is shown to be impaired. CARNA 5 accumulation in tomato at 32 degrees C is reduced about 100-fold compared to accumulation in plants held at 24 degrees C, while viral RNA accumulation is reduced about 5-fold. CMV-infected tomato held for 3 days at 24 degrees C prior to shift to 32 degrees C do not develop lethal necrosis. Longer incubations at 24 degrees C prior to shift to 32 degrees C allow necrosis to develop. CMV-infected plants held for up to 4 weeks at 32 degrees C required an additional 8-10 days at 24 degrees C to develop necrosis. Necrogenic CMV-infected plants held at 24 degrees C and analyzed 3 days p.i. contained detectable amounts of ss- and ds-CARNA 5; upon shift to 32 degrees C, such CARNA 5 declined to undetectable levels and lethal necrosis did not occur. There appear to be temperature-sensitive factors that are required for efficient satellite replication which are not required for efficient viral RNA replication. Whether these factor(s) are of host or satellite origin is uncertain.

Cucumovirus↗
PubMed1995

Effect of temperature on cucumber mosaic virus satellite-induced lethal tomato necrosis is helper virus strain dependent.

The effect of temperature on the response of tomato (Lycopersicon esculentum Mill. cv. Rutgers) to infections with the necrogenic cucumber mosaic virus (CMV) satellite D-CARNA 5 was investigated with each of four CMV strains D, 1, Y and S functioning as helper virus. At 24 degrees C lethal necrosis was observed in all infections. However, at 32 degrees C the response varied from total absence or reduction of necrosis with some strains to accelerated lethal necrosis with others. The total lack of necrotic response with CMV-S and the aggravated necrosis with CMV-Y at the higher temperature both turned out to be independent of the coinfecting satellite, and rather to correlate with the changing rate of viral RNA accumulation in tomato, which probably was responsible for the changes in pathogenic response. However, when CMV-D was helper virus, satellite accumulation decreased, while with CMV-1 it increased, respectively, while viral RNA accumulations were not seriously affected. Although these profound effects of temperature seem to link the necrotic response of tomato to the competitive replication dynamics of the infecting virus/satellite combination in the case of CMV-D/D-CARNA 5, temperature effects at other levels of disease induction probably play an important role as well.

Cucumovirus↗
PubMed1995

Small satellite of arabis mosaic virus: autolytic processing of in vitro transcripts of (+) and (-) polarity and infectivity of (+) strand transcripts.

In vitro RNA transcripts of both (+) and (-) polarities were obtained from a circularly permuted dimer clone of the small satellite of arabis mosaic virus (sArMV). The transcripts show efficient self-cleavage at the two specific sites in the sequence where the monomers were joined. Autolytic processing of the full-length transcript in both orientations releases promoter-proximate fragments (+) or (-) P, promoter-distal fragments (+) or (-) D, and the monomer fragments (+) or (-) M. The presence of an OH group at their 5' ends and a 2',3' cyclophosphate at their 3' ends suggests that (+) and (-) M originated via two self-cleavage reactions within the full-length transcript of corresponding polarity. Infectivity assays showed that the (+) M fragment but not the (-) M fragment initiates replication as efficiently as the natural linear sArMV in Chenopodium quinoa. Two (-) fragments were identified which are the result of religation activity: a P-D fragment formed by religation of P and D, and c-M, which is the result of efficient self-ligation of (-) M. In contrast, linear (+) M self-ligates in vitro to a very limited extent but could be circularized enzymically in a wheat germ extract.

Base Sequence↗
PubMed1995

The complete sequence of a cucumber mosaic virus from Ixora that is deficient in the replication of satellite RNAs.

A cucumber mosaic virus (CMV-Ix) from Ixora is unusual in that it does not support the accumulation of some well-characterized CMV satellite RNAs in plants. CMV-Ix can support a particular satellite RNA variant which causes lethal tomato necrosis when inoculated with other CMV strains but not when inoculated with CMV-Ix. This difference in ability to support accumulation of specific satellite variants is apparent even when their sequences differ by only 10 nucleotides. Electroporation of tomato protoplasts with combinations of CMV-Ix or CMV-1 RNA plus the same satellite variants showed similar differences in accumulation, indicating a defect in satellite RNA replication and not movement or encapsidation. Pseudorecombinant virus infections between CMV-1 and CMV-Ix indicated that the genomic determinants responsible for this phenotype reside on RNA 1 since only combinations with CMV-Ix RNA 1 failed to replicate satellite RNA. The complete genome of CMV-Ix was cloned, sequenced and compared with the genomes of other cucumoviruses. CMV-Ix is most similar in RNA and protein sequence to subgroup 1 CMV-Fny and CMV-Y but slightly less similar than they are to each other. CMV-Ix and all cucumovirus strains sequenced thus far share a domain in the 3' untranslated portion of their genomic RNAs in which 39 of 40 bases are completely conserved.

Amino Acid Sequence↗
PubMed1994

Requirement of 3'-terminal guanosine in (-)-stranded RNA for in vitro replication of cucumber mosaic virus satellite RNA by viral RNA-dependent RNA polymerase.

The 3' terminus of the (-) RNA strand in the replicative forms of several (+)-stranded RNA viruses possesses an unpaired guanosine with unknown function. This unpaired guanosine is also found at the 3' terminus of the (-) strand in the double-stranded form of two cucumoviral satellite RNAs. Using a cucumber mosaic virus (CMV) RNA-dependent RNA polymerase capable of replicating the satellite RNA in vitro, the 3'-terminal guanosine of the satellite (-) strand was shown to be an absolute requirement for satellite (+) strand synthesis. If genomic RNA synthesis of CMV and other members of the alphavirus-like superfamily that produce (-) strands terminating in an unpaired 3' guanosine follows a similar strategy, the work reported here would represent the first experimental support for the notion of 3'-terminal guanosine functioning as an essential recognition signal for viral replicases, enabling (+) strand RNA synthesis to be initiated internally from a (-) strand template.

Base Sequence↗
PubMed1993

Replication of satellite RNA in vitro by homologous and heterologous cucumoviral RNA-dependent RNA polymerases.

The RNA-dependent RNA polymerases (RdRp) of cucumber mosaic virus (CMV) and peanut stunt virus (PSV), members of the cucumovirus group, have been purified from virus infected plants and were used to study RNA synthesis in vitro using different viral RNAs, two cucumoviral satellites, and chimeric satellite cDNA clone transcripts as templates. The results show that solubilized RdRp preparations of CMV and PSV have a high degree of template dependency and catalyze (-) strand synthesis of the homologous cucumoviral RNAs with greater efficiency than the RNAs of heterologous cucumoviruses, although the PSV RdRp exhibits a lesser specificity than the CMV RdRp. On the other hand, both (-) and (+) strands of the satellite RNAs of CMV and PSV are synthesized by their homologous but not by the heterologous viral RdRps, indicating that recognition of satellites by the viral RdRp determines their replicative dependence upon specific helper viruses. Cucumoviral RdRp reactions using chimeric satellite transcripts suggest that the promoter structure for the satellite (-) strand synthesis resides in regions harboring the 3' termini of the two satellites.

Base Sequence↗
PubMed1993

Tomato necrosis and the 369 nucleotide Y satellite of cucumber mosaic virus: factors affecting satellite biological expression.

To determine which factors can affect biological expression of the Y satellite RNA of cucumber mosaic virus (CMV) in tomato, three laboratories collaboratively exchanged their natural satellite variants, the corresponding recombinant DNA clones and helper virus strains, as well as tomato varieties, on which different observations previously reported were based. The effects of these materials and the influence of temperature on symptom expression were systematically studied. The results show that in a standardized tomato bioassay at 24 degrees C, the Y satellite, when supported by either CMV-1 or CMV-Y, did not induce tomato necrosis in the Rutgers variety but elicited a slower necrotic response in the Best of All variety that was variably lethal, as compared to the faster inevitably lethal response induced by a prototype necrogenic D satellite variant in both tomato varieties. At higher temperatures (26.5 to 32 degrees C) an extremely fast-killing necrosis caused by CMV-Y itself was observed. The study demonstrates that in experiments on virus symptom modulation induced by CMV satellites, the nature of the helper virus, host plant varieties, as well as the environmental conditions should be precisely defined, and the effects of each parameter change determined separately.

Mosaic Viruses↗
PubMed1992

Replication footprint analysis of cucumber mosaic virus electroporated into tomato protoplasts.

Total RNA extracted from cucumber mosaic virus (CMV) strains WT, with its associated satellite CARNA 5 (CMV-associated RNA 5), was successfully electroporated into isolated tomato protoplasts. At various time intervals samples were extracted for total nucleic acids and analyzed by semidenaturing polyacrylamide gel electrophoresis (PAGE). Sequence-specific hybridization probes were used for the detection of viral and satellite RNAs following Northern transfer. The resulting PAGE patterns and/or autoradiographs depict the proportional presence of viral and satellite RNAs in the extracts over time and have been referred to as "replication footprint profiles" (RFPs) of specific CMV/CARNA 5 combinations. The effective isolation and infection of tomato protoplasts, combined with the ability to follow virus/satellite titers during the infection by RFP analysis, yield results similar to those of infected plants and reduces experiments of 21 or more days in whole plants to less than 72 h in protoplasts.

Autoradiography↗
PubMed1992

Widely separated sequence elements within cucumber mosaic virus satellites contribute to their ability to induce lethal tomato necrosis.

To determine the structural requirements for cucumber mosaic virus (CMV) satellites to elicit lethal tomato necrosis, three satellite variants D, S and Y were used in the construction and cloning of chimeric cDNAs. D and S are necrogenic and non-necrogenic 'prototype' variants, respectively, and Y possesses the 3' conserved necrosis-determining region but does not cause lethal tomato necrosis. Its 5' half harbours an insertion/deletion region that results in a molecule about 30 nucleotides longer than other variants. Tomato bio-assays were conducted with RNA transcripts of all six chimeric combinations of the 5' and 3' halves of the three satellite variants divided by a common restriction site, as well as with a mutated chimera. None of the chimeras containing the 5' half of Y induced lethal necrosis in tomato even when their 3' halves were that of the D variant with the conserved necrogenic element. Chimeras with the 3' half of Y elicited only partial or restricted necrosis which was much less severe than that induced by prototype variant D, and often was not lethal. Site-directed mutation of a single nucleotide in proximity to the necrogenic element of such a chimera containing the 3' half of Y restored much lethal necrogenicity. The results revealed the presence of structural elements in CMV satellite variant Y that modulate or even suppress the expression of the 3' conserved necrosis-determining element. They indicate that in CMV satellites widely separated sequence elements constituting a three-dimensional requirement are responsible for eliciting lethal necrosis in tomato.

Base Sequence↗
PubMed1991

Replication of cucumber mosaic virus satellite RNA in vitro by an RNA-dependent RNA polymerase from virus-infected tobacco.

An RNA-dependent RNA polymerase purified from tobacco infected with cucumber mosaic virus catalyzes the synthesis of (-) and (+) strands of the viral satellite RNA, CARNA 5, but fails to replicate the satellite RNA of peanut stunt virus (PSV). The enzyme replicates the genomic RNAs of the three principal cucumoviruses CMV, PSV and tomato aspermy virus (TAV) with varying efficiencies. The specificity with which CMV RdRp replicates different sequence-unrelated RNA templates suggests that the site of their recognition requires secondary or higher level structural organization.

Autoradiography↗
PubMed1990

Transformed tomato plants express a satellite RNA of cucumber mosaic virus and produce lethal necrosis upon infection with viral RNA.

Tomato plants transformed with a single copy of a tomato necrosis causing satellite RNA of cucumber mosaic virus (CMV) express the satellite sequence, but the plants show no disease symptoms and have a normal appearance. Upon challenge infection of the F1 progeny with a CMV strain free of any detectable encapsidated satellite the plants accumulated single and double-stranded forms of satellite RNA and developed lethal necrosis.

Blotting, Northern↗
PubMed1990

In-vitro translation of cucumoviral satellites. III. Translational efficiencies of cucumber mosaic virus-associated RNA 5 sequence variants can be related to the predicted secondary structures of their first 55 nucleotides.

The cucumber mosaic virus (CMV) satellites D- and S-CARNA 5 (CARNA 5 = Cucumber mosaic virus-Associated RNA 5), their full-length cDNA clone transcripts, and DNA clone transcripts of their open reading frames (ORFs) were used as mRNAs in the wheat-germ in-vitro translation system. Natural D-CARNA 5 yielded an anomalously large polypeptide, while transcripts made from cDNA clones of D-CARNA 5 or its first ORF had no mRNA activity. Transcripts made from the second major ORF in D-CARNA 5 yielded a smaller product, consistent with its size. Natural S-CARNA 5 and its cDNA clone transcripts both yielded the two polypeptides previously reported, while transcripts of its only major ORF yielded exclusively the smaller of the two products. The potential for an alternate initiation codon, 36 nucleotides upstream, being the source of the larger of the two polypeptides was tested. The differences in the translational properties of D- and S-CARNA 5 were related to the predicted secondary structures of the first 55 nucleotides in these CARNA 5 sequence variants. The calculated free energies of the predicted hairpins correlated inversely with their in-vitro translational activities.

Base Sequence↗
PubMed1990

Nucleotide sequence and structural analysis of two satellite RNAs associated with chicory yellow mottle virus.

The two satellite RNAs associated with CYMV infections were sequenced. The larger (sCYMV-L1) has only linear molecules 1145 nucleotides long, a poly(A) tail, a long open reading frame (ORF) coding for a protein of Mr 39,636 resembling in composition those of other large nepovirus satellite RNAs, a 5' leader sequence of 16 nucleotides and a 3' non-coding region of 40 nucleotides. In vitro translation of sCYMV-L1 yielded a protein product with a size that corresponded to that predicted from the sequence. The smaller satellite (sCYMV-S1) is 457 nucleotides long, has no ORF of significant length and no in vitro messenger activity. Both linear and circular forms of this satellite RNA were detected in infected tissues. Comparison of the sCYMV-S1 primary structure with the sequences of other small nepoviral satellites reveals large regions of homology. Analysis of the secondary structures derived from the sequences of the plus and minus strands suggests possible consensus sequences for their self-cleavage.

Amino Acid Sequence↗
PubMed1989

A simple method for detection of viral satellite RNAs in small plant tissue samples.

A procedure is described that allows extraction and can estimate the total amount of single-stranded and double-stranded viral satellite RNAs and viral RNA present in a minimal amount of infected plant tissue, and is capable of distinguishing different cucumber mosaic viral and nepoviral satellites by polyacrylamide gel electrophoresis under semi-denaturing or fully-denaturing conditions.

Blotting, Northern↗
PubMed1988

Nucleotide sequence predicts circularity and self-cleavage of 300-ribonucleotide satellite of arabis mosaic virus.

The nucleotide sequence of the satellite of arabis mosaic virus was determined using the satellite RNA encapsidated in virions. The 300-nucleotide long sequence showed extensive homology (50%) with that of the 359-nucleotide satellite RNA of tobacco ringspot virus, which occurs both in a linear and a circular form. This homology also revealed the presence of conceived sequences believed to mediate self-cleavage of the latter as well as other viral satellite RNAs. A circular form of the arabis mosaic virus satellite can be isolated from infected tissues and partially converts to the linear form upon elution from denaturing gels.

Base Sequence↗
PubMed1988

Cucumber mosaic virus-associated RNA 5. XI. Comparison of 14 CARNA 5 variants relates ability to induce tomato necrosis to a conserved nucleotide sequence.

Seven CARNA 5 (Cucumber mosaic virus-Associated RNA 5) variants were obtained from an Indonesian cucumber mosaic virus isolate (designated CMV-In) by serial passage of this virus in squash, Chenopodium quinoa, tobacco, and tomato. Bioassays of the isolated CARNA 5 variants for their capability to induce lethal necrosis in tomato allowed categorization of the CARNA 5 variants as either necrogenic or nonnecrogenic. Comparison of the CARNA 5 sequences with sequences previously published shows that in general CMV satellites display a high degree of nucleotide sequence conservation. Except for two sharply defined domains of variability, the sequences of the necrogenic CARNA 5s are essentially conserved, whereas those of the nonnecrogenic CARNA 5s possess at least nine additional hypervariable domains. In eight positions of the CARNA 5 sequences all necrogenic variants differ uniquely from those that are nonnecrogenic. All CMV satellites compared in this work possess open reading frames (ORFs), with equivalent initiation codons, capable of encoding polypeptides of significant lengths. Two equivalent or identical ORFs were found only in the necrogenic CARNA 5s, whereas a variable-length ORF was found in the sequences of all the CARNA 5 variants compared. These findings constitute a basis for experiments aimed at determining the biological significance, if any, of these ORFs.

Base Sequence↗