Biophysical studies on the RNA cores of satellite tobacco mosaic virus

Satellite tobacco mosaic virus (STMV) was probed using a variety of proteas es. Consequences of the degradation were analyzed using gel electrophoresis , quasi-elastic light scattering (QELS), and atomic force microscopy (AFM). Proteolysis rates of 30 minutes for complete degradation of the protein ca psid, up to many hours, were investigated. With each protease, degradation of virions 17 nm in diameter was shown by QELS to result in particles of 10 nm diameter, which is that of the RNA core observed in the virion by x-ray diffraction analysis. This was verified by direct visualization with atomi c force microscopy. Using QELS, it was further shown that freshly prepared RNA cores remain as individual, stable, 10-nm condensed particles for 12 to 24 h. Clusters of particles then formed, followed by very large aggregates of 500 to 1000 nm diameter. AFM showed that the aggregates were composed o f groups of the condensed RNA cores and were not due to unfolding of the nu cleic acid. No unfolding of the core particles into extended conformation w as seen by AFM until the samples were heated well beyond 90 degreesC. Mass spectrometry of RNA core particles revealed the presence of a major polypep tide whose amino acid sequence corresponded to residues 2 through 25 of the coat protein. Amino acids 13 through 25 were previously observed to be in direct contact with the RNA and are presumably protected from protease dige stion. Low resolution difference Fourier analyses indicated the courses of the remainders of the amino terminal strands (amino acids 2-12) in intact v irions. Any individual strand appears to have several choices of path, whic h accounts for the observed disorder at high resolution. These positively c harged strands, serving as virtual polyamines, engage the helical segments of RNA. The intimate association of amino acid residues 2 through 25 with R NA likely contributes to the stability of the condensed conformation of the nucleic acid cores.