Inducers of interferon and host resistance, IV. Double-stranded replicative form RNA (MS2-Ff-RNA) from E. coli infected with MS2 coliphage.

Abstract

Major effort has been given in our laboratories to finding a highly active inducer of interferon which would be worthy of clinical evaluation in man and domestic animals. Recent reports'-3 by our group recorded induction of interferon and resistance to viral infection in vitro and in vivo by complexed synthetic polynucleotides (I:C), by RNA derived from an extract of Penicillium funiculosum (Hel-RNA), and by RNA from the virion of reovirus type 3 (Reo 3-RNA). The essential property of RNA required for such induction was found to be doubleor multistranding and, in certain instances, freedom from inhibitory substances. The present report describes a double-stranded RNA extracted from E. coli infected with MS2 coliphage which was highly active as an inducer of interferon and resistance to viral infections. The substance, called MS2-RF-RNA, is of special promise as a practical source of double-stranded RNA for clinical purpose. Materials and Methods.-MS2 coliphage and E. coli strain Hfr 3000 were obtained from Dr. A. J. Clark of the Molecular Biology and Virology Laboratory, University of California, Berkeley. The phage was propagated and assayed quantitatively according to methods described by Loeb and Zinder.4 Preparation of double-stranded replicative form of MS2 coliphage RNA (MS2-RF-RNA): The methods described by Weissmann et al.5 and Billeter et al.6 wereused. E. coli cultures in exponential growth phase were infected with MS2 at an input multiplicity of about ten and incubated for one hour at 37°C with continuous aeration. The collected cells from one liter of culture medium were lysed using sodium dodecyl sulfate and the lysate was repeatedly extracted with phenol to remove protein. The aqueous phase was then treated with RNase and DNase, and the RNA was separated from smaller molecular weight substances by exclusion chromatography employing Sephadex G-200. The further purification including final removal of the bacterial substances is described in the text.Preparation of single-stranded MS2 virion form (VF) RNA (MS2-VF-RNA): The E. coli cultures were infected with MS2 phage and incubated overnight at 37°C with aeration. Cell lysis was aided by further incubation at 37°C for 30 minutes with lysozyme and ethylenediaminetetraacetate (EDTA) in final concentrations of 50 ,ug/ml and 0.1 M, respectively. The clarified lysate contained 3.8 X 1011 plaque-forming units of phage per milliliter. The virus was concentrated by the acid-precipitation method of Charney et al., I resuspended in phosphate-buffered saline, and purified further by freon extraction and density-gradient centrifugation in CsCl according to Strauss and Sinsheimer.8 The pooled virus from the gradient (1.8 X 1013 PFU/ml) was repeatedly dialyzed against 0.02 M phosphate buffer at pH 7.0. The final virus preparation showed a typical ultra-2102

RNA‐Induced Interference with Animal Virus Infection

“…Field et al [1] reported that doublestranded replicative form of phage RNA is an active inducer of interferon, but single-stranded phage RNA from the virions is not, in apparent disagreement with our results. The cause for this is not clear, but may be attributed to the different experimental systems used, and also to the use of protamine in our study, without which little interference is observed [4].…”

Relationship between Immunity and Interferon Production in Macrophages

“…For example, is it just the adsorption stage, as presented in this paper, or is it penetration, uncoating and release of viral nucleic acid [6], biosynthesis of the double-stranded. RNA such as the replicative form and replicative intermediate(s) [1,3,7,8], or the biosynthesis of viral coat protein? Studies and analysis of this problem using radioisotope-labeled virus particles are now in progress.…”

A 40-Year Journey in Search of Selective Antiviral Chemotherapy*

“…What attracted me personally to interferon was not the molecule itself, which for many years after its discovery in 1957 had remained an esoteric principle rather than a chemically identifiable entity, but the galvanizing observations made by Maurice Hilleman's group at Merck [published in a series of five consecutive PNAS papers (all sponsored by Max Tishler) (14)(15)(16)(17)(18)] that interferon could be induced by double-stranded RNAs from both viral and synthetic origins. I always viewed the discovery of interferon induction by double-stranded RNAs such as poly(I).poly(C) as the highlight of my early interferon days, and although Maurice Hilleman got his principal recognition for the many vaccines he developed (19), the importance of poly(I).poly(C) as an inducer of interferon should not go unnoticed.…”