J.M. Kaper

Cucumber mosaic virus-associated RNA 5: I. Role of host plant and helper strain in determining amount of associated RNA 5 with virions

Abstract The low-molecular-weight fifth RNA component encapsidated in cucumber mosaic virus (CMV) has recently been identified as a satellite-like or defective RNA, dependent on a helper virus for its replication. Of six CMV strains studied, five were found to have this RNA 5 associated in major proportions after one or more serial transfers in tobacco. After passage(s) in squash, the viruses (with the exception of CMV strain R) contained only small amounts of RNA 5. In tobacco, increased RNA 5 proportions in the CMV preparations isolated were correlated with a decrease in symptoms of the plants, a decrease in specific infectivities, yields, and in the proportions of some of the genomic RNAs (particularly RNA 1) in the virus, indicating a general suppression of helper virus synthesis. The RNA 5 components of the five strains comigrated in polyacrylamide electrophoresis on 4% gels and thus seem to have very similar or identical molecular weights. In tobacco, addition of purified RNA 5 from different strains to inocula consisting of RNA 1 + 2 + 3 from CMV-S induced replication of RNA 5. Similarly, mixtures of RNA 5 and RNA 1 + 2 + 3 from CMV-S and CMV-R, and vice versa, were found to produce RNA 5 in the progeny. This made it possible to establish that, for these two strains, the amount of RNA 5 produced in squash was determined by the helper and not by the RNA 5 itself. Thus, with CMV the amount of RNA 5 associated with virions seems to be a characteristic determined by the interaction of the host plant with the helper virus and not with the RNA 5. This interaction could also be an important factor in the genesis of helper-dependent replicating RNAs. A code name CARNA 5 was introduced for CMV-associated RNA 5.

Some physical and chemical properties of cucumber mosaic virus (strain Y) and of its isolated ribonucleic acid

Abstract Strain Y of cucumber mosaic virus (CMV-Y) contains 1.8% phosphorus and 16.4% nitrogen. Its base ratio is: adenine 24.3%, guanine 23.4%, cytosine 23.2%, and uracil 29.0%. CMV-Y has a ribonucleic acid (RNA) content of 18.5%. The sedimentation constant is 92 Svedberg units (S). In 0.02 M phosphate buffer of pH 7, CMV-RNA preparations (obtained by phenol extraction of several different virus batches) sedimented as paucidisperse mixtures of three components (A, B, and C) with sedimentation rates of 13.3 S, 19.6 S, and 22.9 S in which the latter was usually the major component. The molecular weight of the 22.9 S component of CMV-RNA was estimated to be in the range of 0.9–1.2 million. On the assumption that this represented a full complement of CMV-RNA, the molecular weight of CMV-Y was calculated to fall in the range of 4.9–5.8 million. Highly infectious CMV-RNA with the same physical properties was also prepared by degrading CMV-Y in 1.5 M KCl. The two fastest-sedimenting species in CMV-RNA, B and C, had the same sedimentation characteristics under the influence of either high (0.1 M ) or low (6 × 10 −4 M ) phosphate buffer concentration and under the influence of traces of Mg ++ ions or spermine. These “mergers” could be reversed in all but the low phosphate concentration treatment to the original three-component system. After heating of CMV-RNA up to 10 minutes at 80°C or 20 minutes at 60°C or after reacting with formaldehyde, a three-component system was retained although the slowest sedimenting component A had become somewhat polydisperse and its amount had increased at the expense of the fastest sedimenting species C. No significant loss of infectivity occurred upon heating CMV-RNA for 20 minutes at 60°C. Hypochromicity studies on CMV-Y and on CMV-RNA suggested that the RNA inside the virus possessed a large amount of secondary structure, which was retained in the isolated RNA. This was confirmed in optical density-temperature profiles of CMV-RNA. The above findings are discussed in relation to two alternative models for CMV-RNA: (1) CMV-RNA is a mixture of three molecular species with different molecular weights. (2) CMV-RNA is also a mixture of three molecular species, but the fastest-sedimenting ones (B and C) are of equal molecular weight, component C being ring-structured.

Cucumber mosaic virus-associated RNA 5. III. Little nucleotide sequence homology between CARNA 5 and helper RNA.

Abstract Ribonuclease-resistant nucleic acid structures were isolated from plants infected with the S strain of cucumber mosaic virus (CMV). The behavior of these nucleic acids in relation to the corresponding single-stranded (ss) RNAs in rate zonal centrifugation experiments and/or in polyacrylamide gel electrophoresis suggested that they are the double-stranded (ds) RNAs of CMV and CMV-associated RNA 5 (CARNA 5). The yields of ds RNAs isolated from different host plants infected with CMV varied significantly. In host plant-inoculum combinations producing large amounts of CARNA 5, the dsRNAs consisted for 90% or more of dsCARNA 5, while yields of dsRNAs were two to five times higher than in the absence of dsCARNA 5. Separation of dsCARNA 5 from the viral dsRNAs was accomplished by rate zonal centrifugation on sucrose gradients, and resulted in a gel-electrophoretically pure product. This material hybridized with 3 H-labeled CARNA 5. In competition hybridization experiments only 9% of the radioactivity of [ 3 H]CARNA 5 would not have hybridized at infinitely high concentration of CMV RNA 1 + 2 + 3, and none with CMV RNA 4. Thus, CARNA 5 has no nucleotide sequence homology with CMV RNA 4, and, at most only 0.1 of its nucleotide sequence occurs in the genomic RNAs of CMV.

Physical and chemical differentiation of three strains of cucumber mosaic virus and peanut stunt virus.

Abstract Several physical and chemical properties of three strains of cucumber mosaic virus (CMV) and the more distantly related peanut stunt virus (PSV) were studied to see if these viruses, which can be distinguished easily by biological and serological criteria, also exhibited distinct physical-chemical characteristics. The four viruses showed clear differences in electrophoretic mobility, in reactivity with respect to the lysyl-specific reagent trinitrobenzenesulfonic acid, and in their stability with respect to high concentrations of LiCl. In most of these properties, the D strain of CMV was markedly different from the other viruses. Because the above characteristics reflect differences in the capsid structures of these strains, it was not surprising that there was an excellent correlation with the serological grouping of these viruses [ Devergne, J. C., and Cardin, L., Ann. Phytopathol. 5, 409–430 (1973) ; and Devergne, J. C., and Cardin, L., Ann. Phytopathol. , in press (1976) ]. A detailed comparison was also made of the RNA component composition of the four viruses, and the buoyant density distribution of their formaldehyde-stabilized nucleo-proteins after isopycnic centrifugation in Cs formate. Both the buoyant density profiles and RNA component composition were strain-characteristic, clearly reflecting qualitative and quantitative differences in the nucleoprotein composition of the strains. With none of the four viruses was it possible to propose a simple model of the RNA component distribution, as has been done for brome mosaic virus [ Lane, L. C., and Kaesberg, P., Nature New Biol. 232, 40–43 (1971) ]. This was due to the fact that with CMV strains D, R, and PSV, the molar ratios of RNA 3 to RNA 4 greatly exceeded unity while, in addition, strains D and R contained large quantities of the low molecular weight RNA 5. Also, it has been impossible to draw a correlation between the RNA component compositions of the four viruses and their buoyant density profiles.

Arrangement and identification of simple isometric viruses according to their dominating stabilizing interactions

Capsid-stabilized turnip yellow mosaic virus (TYMV) and protein-RNA linkage-stabilized cucumber mosaic virus (CMV) possess totally different stability characteristics which can serve as distinguishing characteristics to arrange and identify other simple isometric viruses according to their dominating stabilizing interactions.

Cucumber mosaic virus-associated RNA 5: II. In vitro translation in a wheat germ protein-synthesis system

Abstract Cucumber mosaic virus-associated RNA 5 (CARNA 5) has been shown to code for the synthesis of two low-molecular-weight polypeptides in a wheat germ in vitro protein-synthesis system. Polypeptide molecular weight estimates of 5200 and 3800 were obtained by SDS-polyacrylamide gel electrophoresis in the presence of 8 M urea. These two polypeptides appear to be initiated separately and do not share an obvious precursor-product relationship. The sensitivity of CARNA 5 translation to inhibition by 7-methyl-guanosine-5′-monophosphate strongly suggests that the 5′-terminus of CARNA 5 is “capped”. Possible relationships between these two products of CARNA 5 translation and a lethal necrotic disease of tomato [Kaper, J. M. and Waterworth, H. E. (1977) Science 196 , 429–431] are discussed.

Molecular organization and stabilizing forces of simple RNA viruses. IV. Selective interference with protein-RNA interactions by use of sodium dodecyl sulfate.

Abstract The effect of the anionic detergent sodium dodecyl sulfate (SDS) on a number of simple isometric RNA viruses and empty capsids was tested. Some viruses showed extreme sensitivity, e.g., cucumber mosaic (CMV), brome mosaic (BMV) and alfalfa mosaic (AMV) virus, some extreme resistance, e.g., turnip yellow mosaic (TYMV) and tomato bushy stunt (TBSV) virus, and some intermediate resistance, e.g., southern bean mosaic virus (SBMV) and bacteriophage f2, to dissociation into components by this detergent. In the viruses most sensitive to SDS, virion dissociation is apparently caused by disruption of the electrostatic protein-RNA interactions which are responsible for stabilizing the virions. It is proposed that dodecyl sulfate (DS−) ions bind by means of the hydrocarbon chain to specific binding sites on the virion so that the sulfate groups are near lysine- or arginine-phosphate interaction points; these interactions are neutralized and the phosphates are repulsed, resulting in virion dissociation. Based on this hypothesis several predictions were made, tested experimentally and found valid: (1) Virions become more resistant to SDS as the contribution of the protein-protein interactions to virus stability increases. This was confirmed by testing the sensitivities of a number of viruses, including those mentioned above, to SDS. (2) Capsids devoid of nucleic acid are less sensitive to SDS than the respective intact virions. The behavior of TYMV, BMV and bacteriophage f2 virions and capsids with SDS confirmed this prediction. (3) Positive and neutral detergents do not cause dissociation of the SDS-sensitive viruses. (4) Reassembly of viruses stabilized by protein-RNA interactions is inhibited by SDS but not by positive detergents. Predictions (3) and (4) were confirmed by appropriate experiments with CMV or BMV and SDS, dodecyltrimethylammonium chloride (DTAC) and Triton-X-100. Measurements of amounts of detergent bound at low SDS concentrations showed that CMV and BMV have much greater affinitives than TYMV for DS− ions. It is suggested that SDS is useful as a probe for protein-RNA interactions and that relative sensitivity to SDS could be used in categorizing viruses according to stabilizing interactions.

Further studies on the RNA component distribution among the nucleoproteins of cucumber mosaic virus.

Abstract Several attempts to obtain a more reliable definition of the RNA component distribution among the nucleoproteins of cucumber mosaic virus (CMV) are described. Preparative isopycnic centrifugation of formaldehyde-stabilized CMV could not be applied because CMV component cross-linking precluded its further dissociation by SDS or Pronase for RNA component analysis. However, in salt stability tests, preparations of CMV-D were found to be incompletely degraded in 2 M RbCl, and the undegraded virus had the same RNA component ratio as untreated CMV-D. The undegraded CMV-D was fractionated following isopycnic centrifugation in RbCl on a preparative scale. RNA 1 (1.07 million daltons) was found predominantly in the heavy nucleoprotein fractions, and RNA 2 (0.95 million daltons) in the light ones. RNA 3 (0.69 million daltons) and RNA 4 (0.33 million daltons) were found in slightly larger proportions in the middle density fractions. RNA 5 (about 0.1 million daltons) was almost evenly distributed among all nucleoprotein fractions. It was concluded that in CMV-D, with its RNA components basically distributed like brome mosaic virus, there are probably other types of nucleoproteins which have RNA 2 and RNA 3 combined with RNA 5, or RNA 5 molecules without the other components.

Highly infectious nucleic acid from crude and purified preparations of cucumber mosaic virus (Y strain)

Abstract When tobacco leaves infected with the Y strain of cucumber mosaic virus (CMV) were extracted with pyrophosphate buffer in the presence of phenol, the resulting preparations were nearly as infectious as or more infectious than preparations made with phosphate buffer. The infectious entities responsible for the high infectivity of the phenol-treated preparations were not destroyed in phosphate buffer extracts and were not ribonuclease sensitive before phenol treatment. Citrate-chloroform extracts of CMV-infected leaves were more infectious than phosphate buffer extracts, but phenol treatment of citrate-chloroform extracts led to reduced infectivity. The infectious entities in phosphate buffer extracts sedimented more readily than those in citrate-chloroform extracts. These results indicate that most or all of the infectivity of phenol-treated tissue extracts originates from complete virus particles. Control experiments with healthy leaves to which purified CMV was added before extraction confirmed this conclusion. Nucleic acid prepared from purified CMV retained approximately half of the infectivity of an equal weight of nucleic acid in CMV particles if the virus was suspended in a medium of relatively high ionic strength during treatment with phenol. Nucleic acid preparations had ultraviolet spectra typical of nucleic acid and were ribonu-clease sensitive, but analytical ultracentrifugation disclosed two major and two minor components. The faster moving major component moved at about a 10% faster rate than the slower moving major component. Only the faster moving major component appeared to be infectious. The minor components were not infectious.

Comparison of molecular weights of single-stranded viral RNAs by two empirical methods

Abstract The molecular weights of four well-characterized single-stranded viral RNAs (turnip yellow mosaic virus-RNA, bacteriophage MS2-RNA, and cucumber mosaic virus-RNA components B and A) were obtained by sedimentation velocity ultracentrifugation after reaction with formaldehyde. The logarithms of their molecular weights were linearly related to the electrophoretic migration distances of the non-formylated RNAs in 2.4% polyacrylamide gels. This relationship was used to estimate the molecular weights of the RNAs from tobacco ringspot virus, southern bean mosaic virus, carnation mottle virus, tobacco necrosis virus, and brome mosaic virus by electrophoresis, and to compare them with the values obtained by means of formylation and sedimentation velocity analysis.