Studies in our laboratories during the past several years have been directed toward finding a highly active interferon inducer which would be worthy of clinical evaluation. It was found in these studies that capacity for inducing interferon by ribonucleic acids depends on (a) multistrandedness of RNA and (b) in certain instances, freedom from inhibitory proteins.The present paper reports the discovery that certain multistranded polynucleotide complexes are highly active in microgram amounts in inducing interferon and host resistance in vivo arid in vitro. Other reports inl the present series describe the isolation and demonstration of similar activity of double-stranded RNA from Penicillium funiculosurn (HeJ-RNA) I and from reovirus type 3.2 Materials and Methods.-(1) l'olynucleotides were purchased from Mann. Research Laboratories, New York, New York; Miles Laboratories, Elkhart. Indiana; and Sigma Chemical Co., St. Louis, Missouri. The designated polymers were mixed in equimolar concentration in phosphate-buffered saline, pH 7.0 (0.006 M sodium phosphate, 0.15 M NaCl), and complex formation, as determined by hypochromic effect, developed within the first minutes after mixing.3' 4Homopolymer solutions were stored at -20'C. In this paper the nomenclature of Inman and Baldwin will be followed.5 Thus, I, C, A, U, X, G, and DHU will signify homopolymers of hypoxanthine (for which the ribonucleoside is inosine), cytosine, adenine, uracil, xanthine, guanine, and dihydrouracil. CU, IU, AC, GU, and AG will signify copolymers of C and U, I and U, A and C, G and U, and A and G. Octanucleotides of A and U are signified by SA and 8U. A dephosphorylated trinucleotide of I is indicated by 31. CpA, CpG, GpU, and CpC signify dephosphorylated dinucleotides of C and A, C arid G, G and U, and C and C. A colon between polynucleotides signifies that complex formation has occurred, as shown by hypochromic effect. A mixed solution of polymers showing no hypochromic effect is indicated by (+) between polynucleotides. (2) Assays for interferon induction in rabbits, for interferon characterization, viz., species-specificity, trypsin sensitivity, molecular weight, and isoelectric point, and for induction of host resistance in mice were described previously.1 Other pertinent methods are presented in the text.Results.-(1) Induction of interferon in rabbits by complexed polynucleotides: (a) Induction: Table 1 shows that I: C induced interferon in rabbits when as little as a 0.5-,ug dose was given intravenously per rabbit. A: U and I+CpC were less active and induced interferon less consistently in repeat tests. The following were found to be inactive at doses between 50 and 200 ,ug per rabbit:

Since interferon' per se shows little promise as a prophylactic or therapeutic agent, interest has shifted to a search for acceptable inducers by which the body might be stimulated to make its own interferon.Shope2 demonstrated that a substance which was derived from Penicilliumfuniculosum and which was called helenine induced resistance to Semliki Forest and to Columbia SK virus infections in mice. Lewis et al.3 fractionated helenine and prepared a factor active in the mouse assay which exhibited properties of a nucleoprotein. Rytel, Shope, and Kilbourne4 demonstrated that helenine elicited a viral inhibitor in cell cultures and in mice which exhibited properties similar to those of interferon. In commenting, they stated that "the active principle in helenine is still a matter of conjecture since it has not been obtained in a sufficiently pure form to permit definite identification." Studies in our laboratories during the past several years have been directed toward the discovery of an interferon inducer which would be worthy of clinical evaluation. It was found that certain ribonucleic acids were highly active in inducing interferon and host resistance but were dependent upon (a) freedom from inhibitory protein and (b) multistrandedness of the RNA. Ribonucleoproteins and single-stranded nucleic acids were inactive.This series of papers describes the purification or synthesis and characterization of three kinds of multistranded RNA active in inducing interferon and host resistance. The present report describes the isolation and characterization of a double-stranded RNA from extracts of Penicillium funiculosum (helenine) which, when freed of protein, is a highly active inducer of interferon and host resistance. The substance is referred to as HeI-RNA. Subsequent papers will present data showing high level interferon and host resistance-inducing activities of multistranded complexes of synthetic polynucleotides5 and of double-stranded RNA of reovirus origin.6 Materials and Methods.-(1) Extract of Penicitlium funiculosum:7 P. funiculosum was grown as described elsewhere.3 The mycelium was extracted with 0.033 M sodium phosphate buffer, pH 8.0, by rapid stirring for 1 hr at room temperature. The clarified extract was treated as shown in steps A-C of Table 1. The supernate served as starting material for fractionation and purification purposes.(2) Assay for interferon induction in rabbits: Young adult New Zealand white rabbits weighing 4-5 lb were injected intravenously with 0.5 ml of the material for assay. Blood samples were taken 2 hr later. The sera were assayed for interferon content in primary rabbit renal cell cultures in roller tubes or in plaque assay flasks by published procedures8 9 using vesicular. stomatitis virus for challenge. The titer of interferon was the highest initial dilution of rabbit serum which showed 100% suppression of viral cytopathic effect in 50(% or more of the tube cultures, or which effected at least 50% reduction in plaques compared with controls in the plaque assay. The plaque assay...

468PURIFICATION AND CHARACTERIZAT'ION OF INTERFERON sorption of HC03-ions per se, but rather is the secretion of H+ ions which react with filtered HC03-to generate H2C03. Second, the accumulation of excess H2C03 in tubular fluid disproves the previously suggested hypothesis (3,7) that endogenous carbonic anhydrase might be located in the luminal membrane of renal tubular cells, thereby exerting a catalytic action on dehydration of excess H2C03 in tubular fluid.Finally, the calculated pH gradient (pHB-pHTE') between blood and tubular fluid was 1.07. For a pH gradient of this magnitude to result from passive distribution of H+ ions along an electrochemical gradient (8,9), it can be calculated from the Nernst equation [ET = -61.5 (pHB-pHTF)] that a transtubular potential difference ( ET) greater than 66 mV, lumen negative to blood, would be required. Previous measurement of proximal ET under conditions of NaHC03 diuresis gave an average value of only -20 mV( 10). Therefore, it is concluded that H+ ions are secreted into the lumen against an electrochemical gradient by an active transport process.Various virus-host animal or virus-cell culture systems have been shown to elaborate a "soluble" substance called interferon ( 1 ) , which interferes with the propagation of viruses in related host or host cell systems. Interferon appears to be an active principle in the virus interference phenomenon. Interferon, as first described by Isaacs and Lindenmann( l ) ? was prepared in fragments of chick chorioallantoic membrane incubated with hea t-inac tivated influenza virus. Wagner ( 2 ) demonstrated the presence of an interferon in the allantoic fluid of embryonated eggs infected with influenza virus and other workers have demonstrated interferons in other systems. Several groups of workers (2-6) have described the biological and biophysical properties of crude or semi-purified interferons. The present report describes the biological and biophysical qualities of a highly purified interferon derived from the allantoic fluid of embryonated hens' eggs infected with the WS strain of influenza virus.Materials and methods. Preparation of crude interferon. Nine-or 10-day-old embryonated eggs were inoculated by the allantoic route with lW5 ID50 of WS influenza strain RIE 5372 virus obtained from Dr. I. Tamm. The eggs were incubated at 36°C for 84-96 hours after which they were chilled a t 4OC and the allantoic fluid containing the interferon was harvested. The crude fluids were centrifuged in the cold for 20 minutes at 1500 rpm and the supernates were stored at 4°C for periods up to 14 days prior to processing for purification. In vitro assay for interferon activity. Serial 2-fold dilutions of the interferon sample were diluted in mixture 199 containing 1% calf serum and antibiotics, and one ml amounts were added in by guest on August 11, 2015 ebm.sagepub.com Downloaded from

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Previous reports1-6 from this laboratory recorded that double-stranded ribonucleic acid molecules from numerous sources are efficient inducers of interferon and of resistance to viral infection in vivo and in vitro. The present report describes the induction of resistance against several viruses in a variety of cell cultures by the complex of polyriboinosinic and polyribocytidylic acids (rI: rC) and presents evidence to associate such resistance with interferon induction. The inactivity of the individual homopolymers, rI and rC, is reaffirmed, and interferon induction by rI: rC in vitro is shown to be inhibited by exposure of cells to actinomycin D.Materials and Methods.-(I) Polyrzboinosinic acid (rI) and polyribocytidylic acid (rC) were purchased from Miles Laboratories, Elkhart, Indiana. The rI:rC complex was prepared by mixing rI and rC in equimolar concentration in phosphate-buffered saline (0.006 M sodium phosphate 0.15 M NaCl pH 7.0). (2) Cell cultures: The various primary, cell strain, and line cell cultures listed in Table 1 were prepared and maintained by ordinary procedures. The RK13 culture is a stable line of rabbit kidney cells, and the WI-38 and HFL cultures are diploid cell strains of human embryonic lung. The RK13 and WI-38 cultures are well documented and the HFL cell strain was developed by Dr. C. Baugh in these laboratories. Rabbit spleen cell suspensions were prepared according to Field et al.2 (3) The viruses used were prepared from the seed stocks of this laboratory. The rhinovirus serotypes were designated according to the number system of Hamparian et al.7 (4) Induction of resistance to viral infection was measured after overnight treatment of cell cultures with rI:rC. Following removal of inducer, interference with virus replication was measured by the plaque-reduction assay.1 (5) Interferon induction in primary rabbit kidney cells or in rabbit spleen cell suspensions by rI: rC was assayed by transfer of serial dilutions of the cell supernatant fluids to RK13 cultures in Falcon flasks. This was followed by overnight incubation prior to removal and challenge with vesicular stomatitis virus (VSV) with observation for reduction in plaque formation. RK13 cells were used for assay since they were relatively insensitive to induction of resistance to VSV by rI: rC, requiring more than 1 yg/ml. Hence, these cells were unaffected by the small residual amount of rI :rC in the interferon samples. The interferon titer was the highest dilution of sample which caused at least 50% reduction in plaque formation. (6) Other pertinent methods are described in the text.Results.-(1) Induction of resistance in vitro to viral infection by rI: rC: (a) Antiviral activity in cell cultures: Cell cultures varied in their sensitivity to rI: rC with respect to species of origin and as to whether they were primary, cell strain, or line cells. As shown in Table 1, many primary cell cultures were sensitive to rI: rC, although mouse embryo cells showed variable response. The HFL diploid human cell strain was sensitive, w...