Inducers of interferon and host resistance. 3. Double-stranded RNA from reovirus type 3 virions (reo 3-RNA).

Abstract

Studies in our laboratories have shown that the essential quality of RNA necessary for induction of interferon and of resistance to viruses in vivo and in vitro is doubleor multistranding of the individual polynucleotides and, in certain instances, freedom from inihibitory proteins.1 2 The present paper shows that the uinique double-stranded RNA isolated from virions of type 3 reovirus is a highly active inducer of interferon in rabbits and of resistance to virus infection in vitro. This RNA is referred to as Reo 3-RNA. Materials and Methods.--(1) Reovirus: Dearing strain reovirus type 3 was grown in primnary cell cultures of grivet monkey kidney and harvested after three to four days' incubationl at 35?C. (2) Preparation of reovirus RNA (Reo 3-RNA): The reovirus type 3 in the cell culture fluid was concentrated by the acid precipitation method described by Charney et al.3 The precipitate was collected and resuspended in 0.1 Il/f sodium phosphate buffer, pH 8, equivalent to a 50-fold concentrate of the original virus pool and was clarified by centrifuging for 10 minutes at 3000 rpm. The supernate was then centrifuged at 78,000 X g for 3 hours. The pellet which contained the virus was resuspended in sodium phosphate buffered saline, pH 7.0, containing 0.005 M magnesium chloride to give a 500-fold concentrate of starting material. Further purification of the virus and extraction and purification of the Reo 3-RNA was by the procedure of Gomatos and Tamm.4 (3) Assays: Assays for interferon induction in rabbits, for interferon, and for induction of resistance in cell culture have been described previously.1, 2 (4) Characterization of the interferon isiduced by Reo 3-RNA, viz., specificity, trypsin sensitivity, molecular weight, and isoelectric point, was carried out as previously presented.1 (5) The methods employed in characterizing RNA were outlined earlier.1 Results.-(1) Induction of interferon in rabbits: (a) In a dose as small as 0.5 ig per rabbit, Reo 3-RNA was highly active in inducing interferon in rabbits. (b) Figure 1 shows the kinetics for interferon inductioni in rabbits by 'Reo 3-RNA aIld by whole ilifectious reovirus type 3. The whole virus dose was 8 X 1010 virioils per 0.5 ml based on particle counts by electron microscopy. The 8S-pg Reo 3-RNA dose was the equivalent amount of RNA obtainied from 8 X 1010 virions. The Reo 3-RNA was not infectious. The RNA was noninfectious based on tests for infectivity in susceptible mornkey renal cells and in LJ cells. Lack of infectiousness of such RNA has been repeatedly shown.5 A high level of interferon appeared withini one hour after inijectioni of the Reo 3-RNA, reachled a peak by two hours, ancd deelined slowvly to less thain 1'/l the peak level four hours later. Inlfectious reovirus 3 virions did not iniduce a significant anmount of inLterf eron before five hours avid the maximum level was rno mnore tharn 1/06 that induced by the IRNA. (2) Characterization of interferon induced in rabbits by Reo 3-RNA: Identifica-