Chromatography of infective ribonucleic acid from foot-and-mouth disease virus on calcium phosphate.

Abstract

ALTHOUGH it is fairly generally accepted that the molecular weight of the infective unit of several ribonucleic acid-containing plant and animal viruses is approximately 2 × 106, there is still some objection to this conclusion. For example, Fraenkel-Conrat and his colleagues1 consider that the infective ribonucleic acid of tobacco mosaic virus may be an aggregate of smaller polynucleotide sub-units. Furthermore, Vizoso and Burness2, using the technique of chromatography on calcium phosphate, have shown that the infective material obtained by phenol extraction of Krebs-2 ascites tumour cells infected with mouse encephalomycarditis virus is eluted at phosphate molarities ranging from 0.01 to 0.21 M. As this method separates ribonucleic acids according to their molecular weights, the smaller molecules being eluted first3, it appears that the infectivity is associated with components of several molecular sizes, some with molecular weights considerably smaller than 106. This finding is of considerable importance. It seemed of interest, therefore, to apply the same method to the examination of the ribonucleic acid of another virus. Consequently, the chromatographic behaviour of the infective ribonucleic acid obtained by phenol extraction of both pig kidney tissue culture cells and the vesicular fluid from guinea pigs infected with the virus of foot-and-mouth disease has been investigated. Two strains of the virus have been used for the preparation of the infective ribonucleic acid: (a) strain 1 (type O) from the fluid from vesicular lesions of guinea pigs infected with the virus, and (b) strain 997 (type C) passaged more than 100 times in pig kidney cell monolayer cultures. Suspensions of each virus strain were extracted with phenol at 2° C in the usual manner and the infective extracts introduced on to 5 × 1 cm calcium phosphate columns (prepared according to the method described by Main and Cole4) in equilibrium with 0.01 M phosphate buffer, pH. 7.2. Gradient elution at 0° C was achieved by adding 0.5 M phosphate to a reservoir containing 0.01 M phosphate. Fractions (5 ml.) were collected and titrated in mice (for strain 1) or pig kidney cell monolayers (for strain 997). In each case the viral ribonucleic acid was mixed with cell ribonucleic acid (prepared by phenol extraction of uninfected pig kidney tissue culture cells) before addition to the column so that the behaviour of the viral and cell ribonucleic acids on the same column could be determined. Ribonucleic acid was estimated in each fraction by the orcinol method or by measuring the ultra-violet absorption at 260 mµ.