Interactions in biological polyelectrolytes: Studies on the dissociation and assembly of two simple RNA viruses

Simple spherical viruses consist of two types of biological polyelectrolytes, proteins and nucleic acids. In their integrated state, these components form a geometrically highly organized superstructure in which the protein subunits are arranged in quasi-equivalent positions on an icosahedral surface lattice to form the viral capsid, and the RNA is interlaced with the capsid structure in a highly regular fashion. The resulting superstructure is in a state of minimum free energy, and is called the virion. The forces which stabilize the virion probably cover the whole range of noncovalent interactions which are recognized to exist in biological polymers. However, in view of the polyelectrolyte nature of the viral components, ionic interactions might be expected to make significant contributions to the minimum free energy state. Two simple ribonucleic acid (RNA) viruses with similar geometry, the turnip yellow mosaic virus (TYMV) and the cucumber mosaic virus (CMV), were selected for analysis of their principal stabilizing forces. In TYMV the in vivo occurrence of RNA-devoid capsids as well as the ease with which they may be obtained in vitro already indicated that interprotein subunit linkages made a major contribution to the stability of the virion. TYMV capsids were found to be very stable at high ionic strength, despite very adverse conditions of pH or temperature or in the presence of a denaturant such as 12 M formamide. However, stoichiometric reaction of the protein sulfhydryl groups with organic mercurials possessing apolar substituents degraded the TYMV capsids. The salt stabilization and the mercurial sensitivity suggested that the capsid stabilization of TYMV was brought about by hydrophobic bonding. A comparative analysis of the stability and dissociation properties of CMV with those of TYMV and a third virus, the cowpea chlorotic mottle virus (CCMV), suggested that there were no interprotein subunit linkages of substance in CMV. This was in agreement with the fact that under no conditions was it possible to isolate or prepare CMV protein capsids. The dissociation behavior of the proteinRNA interactions in TYMV was difficult to analyze because the capsid screened all internal events from outside observation. Methods were devised either to: (1) perturb the capsid structure sufficiently (without breaking the interprotein subunit linkages) so that it allowed passage of dissociated RNA; or (2) destroy the interprotein subunit linkages and the protein-RNA linkages sequentially. Both methods showed that the protein-RNA interactions in TYMV were pH-sensitive. The linkages were intact below pH 6–7 but dissociated above this pH range. Because of the absence of interprotein subunit linkages in CMV, this virus derives its principal stabilization from protein-RNA interactions. These linkages can be dissociated by high concentrations of neutral salts and are restored by reducing the salt concentration. In contrast to the pH 6–7 sensitivity of the interaction in TYMV, the stabilizing interactions of protein-RNA linkages in CMV are affected only at pH values of about 10. In both viruses it is likely that the protein-RNA interactions are ionic or saltlike. In this type of interaction, the nucleic acid phosphate groups would function as polyanion, and the basic amino acid residues on the protein as polycation. On TYMV protein the histidinyl residues are the most likely groups to participate, whereas in CMV it is probably the lysyl and/or arginyl groups which are involved. Possible alternatives and complications in connection with these simplified models are discussed.