Studies on the stabilizing forces of simple RNA viruses. II. Stability, dissociation and reassembly of cucumber mosaic virus.

Abstract The stability properties of cucumber mosaic virus were investigated in relation to those of two other, well-described, icosahedral RNA viruses of similar geometry; the cowpea chlorotic mottle virus and the turnip yellow mosaic virus. High concentrations of neutral salts caused the dissociation of cucumber mosaic virus into its constituent RNA and protein subunits irrespective of the pH of the solution. At low ionic strength the effect of pH on the infectivity and the sedimentation behavior of the virus was tested between pH 4.0 and 8.5. No effect was noticed in this range, but significant change became evident at pH 9.8 and was complete at pH 10.45. The products of this alkaline treatment were a mixture of slower sedimenting nucleoproteins. The RNA inside cucumber mosaic virus was accessible to pancreatic ribonuclease. There was little or no pH-dependence of the ribonuclease susceptibility. Under no circumstances were protein capsids of cucumber mosaic virus ever obtained, neither by degradation of the virion, reassembly of the protein subunits, nor directly from the infected plant. These stability properties of cucumber mosaic virus are strikingly different from those of cowpea chlorotic mottle virus and turnip yellow mosaic virus, as reported in the literature, and indicate the possession of only weak inter-protein subunit linkages, or their total absence. Cucumber mosaic virus could be reversibly dissociated by varying the concentration of lithium chloride in its solutions. The reassembled virus was shown to be identical to cucumber mosaic virus in its physical properties, in its reaction with cucumber mosaic virus antiserum, and in the degree of its sensitivity to ribonuclease. The infectivity of the reassembled virus was almost as high as the infectivity of the untreated virus. Cucumber mosaic virus protein combined with great efficiency with turnip yellow mosaic virus RNA of different molecular conformations and size. Fragmented turnip yellow mosaic virus RNA (molecular weight 60,000) combined to yield particles with the sedimentation properties of cucumber mosaic virus ( S 20 , w about 93 s); full size turnip yellow mosaic virus RNA gave particles with sedimentation rates of about 130 s. The presence of RNA was mandatory for the reassembly of cucumber mosaic virus protein subunits. No reaggregation of alkali-converted cucumber mosaic virus could be achieved. Salt-dissociated cucumber mosaic virus failed to reassemble in a normal fashion below pH 5, where insoluble nucleoprotein precipitates were formed, or above pH 10, where slower sedimenting nucleoproteins were obtained. These nucleoproteins also could not be assembled further into virus-like particles, and might represent minimum free-energy structures resulting from a less specific interaction of the different sized RNA species of cucumber mosaic virus with protein subunits. Cucumber mosaic virus probably derives its stability from relatively strong ionic linkages between nucleic acid-phosphate residues functioning as a polyanion, and basic amino-acid residues of the protein which function as a polycation.