Differential interactions among strains of tomato aspermy virus and satellite RNAs of cucumber mosaic virus.

Tomato and tobacco plants were inoculated with either of two strains of tomato aspermy virus, 1-TAV or V-TAV, and each of six isolates of cucumber mosaic virus satellite RNA (CMV-satRNA), B1, B2, B3, Ix, or WL2. Ribonuclease protection assays, used to detect total satRNA and encapsidated satRNA, revealed that G-satRNA generated new satellite RNA not of the inoculated sequence. The other CMV-satRNAs were compared for their ability (1) to replicate, (2) to modulate symptoms, (3) to reduce TAV accumulation, and (4) to alter the extent of encapsidation of TAV genomic RNAs. The fraction of B2- and B3-satRNAs encapsidated was greater for 1-TAV than for V-TAV, although spread and accumulation of the satRNA were similar for both helper viruses. These results suggest that CMV-satRNA may spread in a nonencapsidated form. Accumulation of CMV-satRNA in systemically infected leaves was detected for all inoculum combinations except V-TAV and Ix-satRNA, for which the satellite RNA increased only in protoplasts and inoculated leaves of tobacco or tomato. In such inoculated leaves, Ix-satRNA was not detected in capsids. Thus the effectiveness of the TAV helpers of CMV-satRNAs may be controlled in at least some instances by the extent of satRNA spread or encapsidation rather than by the efficiency of satRNA replication. In contrast to infections initiated by inoculation of CMV and CMV-satRNA, inoculation of 1-TAV or V-TAV and CMV-satRNA did not alter the relative amounts of viral genomic RNAs encapsidated or result in accumulation of large amounts of double-stranded satRNA.     

Stability and structural transitions of tomato aspermy virus and cucumber mosaic virus

The properties of the S-strain of cucumber mosaic virus (S-CMV) and the B-strain of tomato aspermy virus (B-TAV) have been studied with respect to their (i) size and sedimentation behavior, (ii) requirement of divalent metal ions for stability, (iii) sensitivity towards chloride salts and the anionic detergent sodium dodecyl sulfate, (iv) solubility in ammonium sulfate-containing buffers, and (v) pH-dependent structural transitions. The results indicate that the coat protein of B-TAV is more hydrophobic than the other well-studied strains of TAV and CMV. Circular dichroism and uv absorption studies reveal pH-dependent structural transitions, although these do not result in particle swelling. These transitions appear to alter the strength of protein-nucleic acid interactions in these viruses.     

Comparative studies on tomato aspermy and cucumber mosaic viruses: V. Purification and properties of a cucumber mosaic virus inducing severe chlorosis

A cucumber mosaic virus (MCMV) inducing brilliant yellow mosaic symptoms in Nicotiana species could not be purified by methods previously used for other strains of cucumber mosaic virus (CMV). A method developed for purifying this virus is described. The physical and chemical properties of MCMV are typical of a cucumovirus, and an antigenic relationship with strains of CMV was demonstrated. Polyacrylamide-gel electrophoretic analysis of MCMV-RNA under nondenaturing conditions failed to reveal the presence of all four RNA components, but their resolution was achieved in the presence of formamide.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. II. Virus stability

Although tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) are structurally similar in many respects, the conditions required for their stability differ considerably. TAV is more resistant than CMV to degradation by pancreatic RNase and to precipitation by NaCl. Whereas TAV is stabilized by Mg²⁺, CMV precipitates in the presence of the cation. CMV is stabilized, but TAV is degraded by EDTA. Both viruses are dissociated by low concentrations of sodium dodecyl sulphate indicating that their capsid structures depend on RNA-protein interactions.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. VII. Serological relationships reinvestigated

Distant antigenic relationships between Australian strains of tomato aspermy virus (TAV) and cucumber mosaic virus (CMV) were investigated by three serological methods: radial double immunodiffusion in agar, enzyme-linked immunosorbent assays (ELISA), and immune-electron microscopy (IEM) using the antibody decoration technique. Based on the work with TAV and CMV presented in this paper, it appears that the ELISA method is especially suitable for studying antigenic relationships among viruses. For distinguishing closely related viruses, heterologous antibodies were used for both coating the ELISA plates and for conjugating with enzyme. On the other hand, for establishing distant antigenic relationships, heterologous and homologous antibodies were used for coating and coupling to enzyme, respectively. Immunodiffusion tests were less efficient than ELISA in distinguishing minor antigenic differences between strains of CMV and also, and distant relationship between TAV and CMV was not as clearly detected. Decoration of the virus particles with antibodies in IEM was the least satisfactory method for investigating antigenic relationships among the viruses.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. I. Physical and chemical properties

Some physical and chemical properties of the V strain of tomato aspermy virus (TAV) and the Q strain of cucumber mosaic virus (CMV) have been compared. The size, morphology, sedimentation rate, RNA base ratio, and buoyant density of the two viruses are indistinguishable. Preparations of RNA from both viruses were each resolved into four distinct species by polyacrylamide-gel electrophoresis. TAV-RNA preparations contained species with molecular weights of 1.26 × 10⁶,1.10 × 10⁶, 0.90 × 10⁶, and 0.43 × 10⁶ daltons, and CMV-RNA, species of 1.26 × 10⁶, 1.10 × 10⁶, 0.77 × 10⁶, and 0.34 × 10⁶ daltons. Analysis of sodium dodecyl sulphate (SDS)-treated viral proteins by polyacrylamide-gel electrophoresis showed that both viruses have protein subunits of molecular weight 24,500 daltons. The amino acid compositions of proteins from the two viruses, although similar, were distinguishable, and the calculated molecular weights of protein subunits were 26,100 and 26,300 daltons for TAV and CMV, respectively. The two viruses were serologically distinct. On the data presented it is suggested that in preparations of both TAV and CMV three distinct particles are present in each with identical protein shells, but different RNA cores.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. IV. Immunogenic and serological properties.

Immunogenicity of TAV was not significantly different from that of CMV when compared by intraperitoneal injections of purified virus preparations into mice in doses ranging from 5 to 320 μg of antigen per injection per mouse. It is concluded that both viruses are poor immunogens. However, their immunogenicities could be enhanced by fixation with formaldehyde. Anti-TAV sera always contained antibodies specific to viral capsids and sometimes also to viral coat protein subunits, whereas anti-CMV sera invariably contained antibodies only to intact capsids. Both capsid-specific and protein coat-subunit-specific antibodies were shown to be immunoglobulins of the IgG class.Preparations of protein coat subunits from CMV failed to elicit antibody production in mice. However, those from TAV were weakly immunogenic producing low-titered antisera containing antibodies specific to TAV coat protein subunits but not to intact capsids.TAV capsid-specific and TAV protein coat subunit-specific antibodies were shown to be antigenically distinct. No antigenic relationship could be demonstrated between TAV and CMV.     

Comparative studies on two satellite RNAs of cucumber mosaic virus

Cucumber mosaic virus (CMV) satellite RNA (Sat-RNA, D. W. Mossop and R. I. B. Francki, 1978, Virology86, 562-566) is similar in many of its physical and biological properties to CMV associated RNA 5 (CARNA 5) described by Kaper and Tousignant (1977, Virology85, 323-327). However, CARNA 5, unlike Sat-RNA, causes a serious necrotic disease of tomato. Sat-RNA when inoculated together with various CMV or tomato aspermy virus strains not only failed to increase the severity of symptoms in infected tomato plants, but ameliorated them in some instances. Comparisons of the two RNAs by hybridization analysis using 32P-labelled complementary DNA probes, indicate that they have partial nucleotide sequence homology. It is suggested that the difference in their primary structure is reflected in their biological properties.     

Biophysical and biochemical properties of tomato aspermy virus

A virus isolated from tomato in British Columbia was identified as tomato aspermy virus (TAV). The virus was purified and compared with an isolate of TAV from chrysanthemum (CV-L) and with an isolate of cucumber mosaic virus (CMV). TAV and CV-L were serologically identical but CMV was only distantly related. The amino acid composition of TAV was similar to that of CV-L, showing only four amino acid exchanges, whereas there were 18 amino acid exchanges between TAV and CMV. TAV had a sedimentation coefficient of 98 S, a diffusion coefficient of 1.49 × 10^?7 cm²/sec and a molecular weight of 5.3 million. The molecular weight of the viral protein subunit, determined on polyacrylamide gel, was 26,300. The base percentage composition of the viral RNA was G = 23.1, A = 26.2, C = 21.6, and U = 29.0. Phosphorus analysis of the RNA indicated that the nucleic acid content of the virus was 17.7%.     

The RNAs of cucumoviruses: 3'-terminal sequence analysis of two strains of tomato aspermy virus

The sequences of 189 residues from the 3' terminus of three RNAs of one strain and two RNAs of another strain of tomato aspermy virus have been determined; there was almost complete sequence homology between the RNAs. A base-paired transfer RNA-like structure is proposed for tomato aspermy virus RNAs which is similar in many aspects to the structure proposed for the 3'-terminal 172 residues of RNA 4 of the Q-strain of cucumber mosaic virus (R. H. Symons, Nucleic Acids Res.7, 825-837, 1979). These 172 residues of cucumber mosaic virus RNA 4 can be aligned to show 73% sequence homology with the 3'-terminal 189 residues of the tomato aspermy virus RNAs.     

Cross-protection and interference between electrophoretically distinct strains of cucumber mosaic virus in tomato

Cucumber mosaic virus (CMV) was partially purified by polyethylene glycol precipitation from extracts of tomato leaflets. The presence and quantity of two strains with different electrophoretic mobilities was analyzed by polyacrylamide gel (2.5%) electrophoresis of preparations from as little as 50 mg of tissue. This method was used to analyze interference and cross-protection between the strains. Coinoculation resulted in local and systemic mixed infections and reductions in the synthesis of both strains. Systemic symptoms and accumulation of the strain with the more severe symptoms were not detected in up to 13 newly formed leaves in five of six plants preinfected with the mild strain and challenged with the severe strain. Systemic accumulation of the mild strain was not detected in each of six plants preinfected with the severe strain and challenged with the mild strain. Both strains could accumulate in challenge-inoculated leaves. The level or absence of interference in these leaves did not affect systemic cross-protection. Cross-protection between these strains of CMV involves considerable inhibition of virus accumulation in addition to absence of symptoms.     

Satellite RNAs of cucumber mosaic virus: characterization of two new satellites

Two new satellite RNAs of cucumber mosaic virus (CMV) which did not induce necrosis on tomato in the presence of CMV, B-sat RNA, and WL-sat RNA, were shown to be related by sequence to two well-characterized satellite RNAs of CMV: G-sat RNA (non-necrotic on tomato) and n-CARNA 5 (necrotic on tomato). Using the techniques of molecular hybridization analysis, RNA fingerprinting and partial RNA sequencing, B-sat RNA and WL-sat RNA were shown to be more closely related to each other (probably differing by only a small number of nucleotides) than to the other two satellite RNAs. Furthermore, B-sat RNA and WL-sat RNA showed greater sequence homology with G-sat RNA than with n-CARNA 5. WL-sat RNA, which induces a "white-leaf" disease on tomato in the presence of CMV [Gonsalves et al. (1982)., exhibited heterogeneity of sequence in at least one nucleotide position.     

Hybrid brome mosaic virus RNAs express and are packaged in tobacco mosaic virus coat protein in vivo

Brome mosaic virus (BMV) is an icosahedral virus with a tripartite RNA genome which infects monocotyledonous plants, while the cowpea or legume strain of tobacco mosaic virus (CcTMV) is a rod-shaped virus with a single component RNA genome which infects dicotyledonous plants. To examine the potential for exchanging entire genes between RNA viruses, biologically active cDNA clones were used to replace the natural coat gene of BMV RNA3 with the coat gene and encapsidation origin of CcTMV. In protoplasts coinoculated with BMV RNAs 1 and 2, the resulting hybrid RNA3 was replicated by BMV trans-acting factors but was packaged in TMV coat protein to give rod-shaped particles rather than the usual BMV icosahedra. When the CcTMV encapsidation origin was suitably inserted in derivatives of BMV RNAs 1 and 2, these RNAs were also packaged in a ribonuclease-resistant form in protoplasts coinoculated with the hybrid RNA3 expressing TMV rather than BMV coat protein. Thus, despite the markedly divergent nature of BMV and TMV, replicating hybrids bearing characters derived from both parent viruses were produced. Such hybrid viruses could be of considerable value for studying specific steps in infection and for assigning functions to particular virus genes.     

Differential roles of the 5' untranslated regions of cucumber mosaic virus RNAs 1, 2, 3 and 4 in translational competition

RNA species of plant tripartite RNA viruses show distinct translational activities in vitro when the viral RNA concentration is high. However, it is not known what causes the differential translation of virion RNAs. Using an in vitro wheat germ translation system, we investigated the translation efficiencies and competitive activities of chimeric cucumber mosaic virus (CMV) RNAs that contained viral untranslated regions (UTRs) and a luciferase-coding sequence. The chimeric RNAs exhibited distinct translation efficiencies and competitive activities. For example, the translation of chimeric CMV RNA 4 was about 40-fold higher than that of chimeric CMV RNA 3 in a competitive environment. The distinct translation resulted mainly from differences in competitive activities rather than translation efficiencies of the chimeric RNAs. The differential competitive activities were specified by viral 5 UTRs, but not by 3 UTRs or viral proteins. The competitive translational activities of the 5 UTRs were as follows: RNA 4 (coat protein)>RNAs 2 and 1 (2a and 1a protein, or replicase )> RNA 3 (3a protein).     

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

Summary 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 °C is shown to be impaired. CARNA 5 accumulation in tomato at 32 °C is reduced about 100-fold compared to accumulation in plants held at 24 °C, while viral RNA accumulation is reduced about 5-fold. CMV-infected tomato held for 3 days at 24 °C prior to shift to 32 °C do not develop lethal necrosis. Longer incubations at 24 °C prior to shift to 32 °C allow necrosis to develop. CMV-infected plants held for up to 4 weeks at 32 °C required an additional 8–10 days at 24 °C to develop necrosis. Necrogenic CMV-infected plants held at 24 °C and analyzed 3 days p.i. contained detectable amounts of ss- and ds-CARNA 5; upon shift to 32 °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.     

RNA sequence heterogeneity in natural populations of three satellite RNAs of cucumber mosaic virus

Sequence heterogeneity within populations of three satellite RNAs of cucumber mosaic virus (CMV) was assessed using two different approaches. Comparison of the nucleotide sequences of several cDNA clones of each satellite RNA revealed microsequence heterogeneity, which is often seen in populations of RNA genomes. RNase protection assays using minus-sense satellite RNA probes were used to detect major sites of heterogeneity within natural populations of each satellite RNA. In RNase protection assays of WL1-sat RNA populations, no major sites of heterogeneity were detected within seven different populations, including preparations from four different host plant species. In contrast, RNase protection assays of nine populations of B1-sat RNA showed three different patterns, which were most likely due to the existence of the B1-sat RNA populations as mixtures in which different sequence variants predominated in different preparations. Assays of five independent populations of D-sat RNA revealed a single major site of heterogeneity which was common to each population and was localized at approximately nucleotide 225 of the 335-nucleotide satellite sequence. This common site of heterogeneity is a feature of the D-sat RNA population structure which may represent an equilibrium between alternative nucleotides at a selectively neutral position, or may be actively selected for and maintained.     

Comparative studies on tomato aspermy and cucumber mosaic viruses. VI. Partial compatibility of genome segments from the two viruses

Isolated RNA genomic segments (RNAs 1, 2, and 3) of tomato aspermy virus and of three strains of cucumber mosaic virus (CMV) were inoculated to leaves of Chenopodium amaranticolor Costs and Reyn in all combinations. The data presented demonstrate that whereas RNA 3 is readily interchangeable between the two viruses to produce infections resulting in lesion development, RNAs 1 and 2 are not, although they are interchangeable between the three CMV strains. The possible significance of these observations to virus taxonomy is discussed.