Double-stranded cucumovirus associated RNA 5: experimental analysis of necrogenic and non-necrogenic variants by temperature-gradient gel electrophoresis

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Abstract
Cucumber mosaic virus (CMV) and peanut stunt virus (PSV) each contain a fifth major RNA in the size range of 334 to 393 nucleotides. This fifth RNA is a satellite capable of modulating the expression of viral disease symptoms. It is present in infected tissue in single-stranded and double-stranded form. Nucleotide sequence variants of the double-stranded CMV-asso-ciated RNA 5 (dsCARNA 5) and PSV-associated RNA 5 (dsPARNA 5) were analysed by temperature-gradient gel electrophoresis. Gels were 5% polyacrylamide, containing 8 M urea in 8.9 mM Tris-borate buffer, with temperature differences of 25–40°C establishing gradients either perpendicular or parallel to the direction of the electric field. For dsCARNA 5 two characteristic transitions were detected with increasing temperature: at temperatures between 40°C and 46°C a drastic retardation in electrophoretic mobility induced by partial dissociation of the duplex structure from the ends and at temperatures above 52°C an abrupt increase in mobility due to complete strand dissociation. dsPARNA 5 exhibited both transitions at up to 10°C higher temperatures and an additional retardation between the transitions mentioned. Seven different variants of dsCARNA 5, 4 necrogenic and 3 non-necrogenic, were analysed. Some showed only one single band, others gave rise to up to six well separated bands corresponding to six molecular species. From all experimental results a correlation between the temperature of the retardation transition and the necrogenicity of CARNA 5 was derived. The diagnostic application of the temperature-gradient gel analysis in agriculture, particularly for the use of non-necrogenic variants as biological control agents to impede CMV-infections, is discussed.