Studies on the stabilizing forces of simple RNA viruses: I. Selective interference with protein—RNA interactions in turnip yellow mosaic virus

Abstract

To allow an experimental study of the disengagement of the RNA from its linkage with the protein in turnip yellow mosaic virus, several methods were developed which affected the capsid structure to such an extent that upon breakage of the protein-RNA linkage, the RNA was allowed enough mobility to undergo either internal rearrangements or to be released in the reaction mixture. These “capsid-expanding” methods encompassed alkali treatment, moderate heating, and the treatment with 12 m-formamide, all in the presence of high ionic strength (1.0) m-KCl). Moderate alkali treatment of turnip yellow mosaic virus gave rise to an in situ fragmentation of the phosphodiester chain of the RNA followed by a complexing of the RNA fragments inside the intact capsid. In alkali treatment with pH above 11, a large proportion of the virus particles released their fragmented RNA into the reaction mixture. The release of RNA was an all-or-nothing process, and followed single-hit kinetics for the majority of particles in a population. The rate of RNA release increased as the pH was raised, but appeared to be independent of the degree of fragmentation and/or complexing that preceded it. Hence, the release of RNA is primarily controlled by the degree of capsid expansion. Moderate heating or treatment with 12 m-formamide provided a means of capsid perturbation which allowed a direct analysis of the breakage of protein-RNA linkages as a function of pH. With both methods it was found that at pH values under neutrality turnip yellow mosaic virus retained its stability, but that a pH of 7 or higher induced dissociation into capsids and free RNA. The same pH dependence of dissociation of turnip yellow mosaic virus was also found after high doses of ultraviolet light irradiation. Whereas the viral capsid degraded into slowly sedimenting fragments upon irradiation under slightly acid as well as under neutral conditions, the majority of the virus particles remained stable at pH 6, but not at pH 7. It was concluded that in turnip yellow mosaic virus the integrity of the protein-RNA linkages is dependent upon the pH, and that these linkages are broken between pH 6 and 7. The type of interaction compatible with such behavior would be an ionic linkage between protonated histidinyl residues and negatively charged nucleic acid phosphates, or could consist of juxtaposed protein carboxylate and nucleotide amino groups, sharing a hydrogen-bonded proton. The latter model would provide a structural role for the extraordinarily large number of cytidilic acid residues in turnip yellow mosaic virus RNA.