Analysis of the Mechanism of Unfolding of Foot-and-Mouth Disease Virus-RNA During Heat and Chemical Denaturation

Two different approaches were used to examine the mechanism of unfolding of foot-and-mouth disease virus (FMDV) RNA. In the first procedure, we prepared single stranded 37S virion RNA by extracting purified virus with phenol and sedimenting the RNA through a 5 to 25% sucrose gradient. The purified RNA was then dissolved in NT buffer (10mM Tris-HCl, pH 7.4, 100 mM NaCl) containing either 1 mM EDTA or 1 mM Mg and absorbed onto Formvar-coated, carton shadowed, glow-discharged 200 mesh copper grids. In the second procedure, we heat denatured intact viral capsids in 0.05 M K2HPO4, 0.2 M KCl buffer (pH 7.0) in a thermostatically controlled Beckman spectrophotometer. We measured viral uncoating and the controlled release of the RNA by increased absorbancy at 260 nm. Samples taken at various temperatures were added to an equal volume of 20% formaldehyde and quick-frozen in covered BEEM capsules in liquid nitrogen. The heat-denatured formaldehyde-fixed virus was then spread onto grids using the BAC-formamide procedure of Vollenweider.