Enteroviral ribonucleic acid. I. Recovery from virus and assimilation by cells

Abstract

A relatively sensitive and adequately reproducible assay of infectious enteroviral RNA was obtained by exposing calf serum-grown HeLa cells to RNA suspended in 2 M magnesium sulfate solution. Highly purified enteroviral preparations yielded RNA exhibiting more than 0.1 per cent of the infectivity of whole original virus and infectivity regressed linearly with dilution. Radioisotope experiments with P(32)-labeled RNA and spectrophotometric studies demonstrated that Gierer-Schramm phenol extraction permits almost quantitative recovery of high molecular weight RNA from poliovirus. Intact chromatography-purified type 2 poliovirus in the analytical ultracentrifuge showed a sharp boundary and a sedimentation coefficient of S(20, w) = 147 +/- 5S. Phenol-extracted poliovirus RNA exhibited a heterodisperse sedimentation pattern with a large proportion of homogeneous, rapidly sedimenting material having a coefficient of S(20, w) = 37 +/- 2S. Although the bulk of extracted poliovirus RNA as measured by radiophosphorus labeling was not taken up by cells, the infectious fraction of RNA was adsorbed rapidly by HeLa-cell or L strain mouse fibroblast monolayers, indicating a possible dissimilarity between the bulk of extracted virus RNA and a relatively small fraction responsible for infectivity. Enhancement of poliovirus RNA infectivity for HeLa cells by high ionic-strength magnesium sulfate solution appeared due partly to an effect of hypertonicity on cells, and partly to an effect of high-concentration divalent cation on RNA itself, but not to enhancement of adsorption. Poliovirus RNA adsorbed by HeLa cells apparently was rapidly received within the cells since it became quickly insusceptible to ribonuclease. Heterologous nucleic acids and degradation products to the level of oligoribonucleotides inhibited infectivity of poliovirus RNA for HeLa cells. This inhibitory activity appeared due to intermolecular complexing, since exposure of cells to heterologous RNA immediately before or after exposure to virus RNA failed to reduce infectivity. Ultraviolet absorption spectra demonstrated that the RNA within intact poliovirus is more hypochromic (and thus more extensively hydrogen-bonded) than is the same RNA isolated by phenol extraction, and suspended in 0.02 M phosphate buffer.