Poly(rI) more important than poly(rC) in the interferon induction process by poly(rI)-poly(rC)

A significantly greater interferon production has been obtained in primary rabbit kidney cell cultures exposed to poly(rI) followed by poly(rC) than in cell cultures exposed to poly(rC) followed by poly(rI). The interferon response in cell cultures exposed to poly(rI) followed by poly(rC) was markedly more resistant to poly-l-lysine and pancreatic ribonuclease treatment than was the interferon response in cell cultures exposed to poly(rC) followed by poly(rI). In addition, poly-l-lysine treatment removed a substantially greater proportion of cell-associated radioactivity from cells exposed to [³H]poly(rC) followed by poly(rI) than from cells exposed to poly(rI) followed by [³H]poly(rC). These findings suggest that the poly(rI) · poly(rC) complex is more tightly and efficiently bound to the cell (surface) when the homopolymers are added in the order poly(rI), poly(rC) than when they are added in the order poly(rC), poly(rI) and that it is more effectively attached to the cell receptor site by its poly(rI) strand rather than by its poly(rC) strand.     

Interferon production by human-mouse hybrid cells: dominant mouse control of superinduction and priming

We have examined the production of interferon by a number of human-mouse hybrid clones in response to polyriboinosinic acid:polyribocytidylic acid copolymer [poly(rI).poly(rC)] all of which produced both human and mouse interferons when stimulated with a virus. Their capacity to be superinduced and primed for interferon production in response to poly(rI).poly(rC) was compared to that of the parental human and mouse cells. It was found that the hybrids responded in a way similar to their mouse cell parents, indicating dominant mouse control of both the priming and superinduction phenomena.     

Potentiating Effect of Freund's Adjuvant on Interferon Production by Endotoxin or Poly rI*Poly rC

Intraperitoneal injection of mice with mineral oil, incomplete (IFA) or complete Freund's adjuvant (CFA) increased the interferon response to endotoxin or (poly rI)•(poly rC) administered intravenously 2 days later. After endotoxin administration, circulating interferon titers were increased at several different times of sampling and with a variety of endotoxin dosages. When injection of endotoxin was delayed until 6 to 8 days after the administration of IFA or CFA, interferon production was markedly decreased. Mice treated with CFA and injected with endotoxin 2 days later became more resistant to intranasal vesicular stomatitis virus challenge than mice injected with endotoxin alone. Hyporeactivity to the interferon-inducing capacity of a second injection of endotoxin 2 days after the first injection could not be overcome by administering CFA simultaneously with the first dose. CFA treatment not only raised the serum interferon titers produced by endotoxin, but also increased the number of interferon-forming cells in the spleen after administration of endotoxin in vivo. In addition, CFA enhanced the intravascular clearance of (poly rI)•(poly rC). The possibility that Freund's adjuvant increased the interferon response to endotoxin and (poly rI)•(poly rC) by stimulating the uptake and processing of the interferon inducer by lymphoreticular cells is discussed.     

The toxic effect of double-stranded RNA for interferon-treated cells: evidence for a heterogeneous cellular response and the role of the cell nucleus.

When L929 cells were treated with interferon and subsequently with poly(rI).poly(rC), there was a pronounced toxic effect. Most of the cells lysed, but some survived and grew at the same rate as control cells to yield cells which were as sensitive to the effects of interferon and poly(rI).poly(rC) as the original population. The proportion of surviving cells did not vary with either the cell cycle or the cell density. The treated cells produced interferon and some of the interferon was produced by the resistant cells. Cells which had been X-irradiated before treatment with interferon and poly(rI).poly(rC) behaved similarly so cell division was not necessary for the development of toxicity. The toxic effect also developed when cells were enucleated with the aid of cytochalas in B after treatment with interferon, but not if they were enucleated before treatment. It is concluded that the nucleus is essential for interferon to exert its effect on the cells, but not for the development of cytotoxicity after the addition of poly(rI).poly(rC).     

Interferon Production Linked to Toxicity of Polyriboinosinic Acid-Polyribocytidylic Acid

Different procedures have been used in attempts to increase the production of interferon by polyriboinosinic acid-polyribocytidylic acid (poly [rI].poly [rC]) in mice: simultaneous injection of lead acetate, cycloheximide, or actinomycin D and prior injection of Freund's adjuvant, chlorite-oxidized oxyamylose (COAM), endotoxin, or Brucella abortus. In the experimental conditions tested, lead acetate, cycloheximide, Freund's adjuvant, and COAM brought about a parallel increase in interferon production and toxicity (lethality) of poly (rI).poly (rC); actinomycin D, endotoxin, and B. abortus increased the lethality of poly (rI).poly (rC) without a concomitant raise of its interferon-inducing capacity. Our results indicate that no significant increase in interferon production (or antiviral activity, as far as the antiviral activity is accounted for by interferon production) without an accompanying increase in toxicity can be achieved with poly (rI).poly (rC) and that it might be impossible to increase its therapeutic ratio (ratio of maximum tolerated dose to minimum effective dose).     

Susceptibility of various cells treated with interferon to the toxic effect of poly(rI).poly(rC) treatment.

Various cells were treated with interferon and then exposed to polyriboinosinic-polyribocytidylic acid complex [poly(rI).poly(rC)]. With mouse L cells, there was a marked cytotoxic effect from low doses of interferon and poly(rI.poly(rC), whereas chick embryo cells showed an effect only after high doses. When primate cells (LLC.Mk2,BSC.B, Vero and human embryo cells) were treated with human or monkey interferon, poly(rI).poly(rC) was not cytotoxic.     

Effect of Separate and Combined Injections of Poly rI: Poly rC and Endotoxin on Reticulo-endothelial Activity, Interferon, and Antibody Production in the Mouse

The effect of repeated stimulation on both the interferon and antibody systems and on reticuloendothelial activity was studied by injection of endotoxin 2 days before injection of (poly rI):(poly rC). A single injection of endotoxin or (poly rI):(poly rC) increased or decreased the response in each system depending on the time of administration. If the injection of (poly rI):(poly rC) was preceded by an injection of endotoxin 2 days before, its activity was markedly reduced in all of the three systems studied. Although different doses of endotoxin were required to induce a state of hyporeactivity or tolerance to the effects of (poly rI):(poly rC) in either system, it is possible that a common mechanism underlies the hyporeactivity in all systems.     

Priming increases the amount of interferon mRNA in poly(rI).poly(rC)-treated L cells.

Priming by mouse interferon pre-treatment resulted in an accumulation of interferon mRNA in poly(rI).poly(rC)-treated L cells, starting early in the period of interferon synthesis. On electrophoresis, the priming activity of an interferon preparation co-migrated with the antiviral activity, which suggests identity of the functional principle(s) for these activities.     

The lact of antiviral effect of (polyinosinic acid): (polycytidylic acid) when attached to insoluble supports.

A local antiviral effect can be observed when (poly rI)-(poly rC), bound to Visking discs by u.v. irradiation, is incubated with monolayers of human foreskin fibroblast cells. Radioactive labelling of cytosine residues in (poly rI)-(poly rC) with -125I, has provided a much more sensitive method for determining the fate of the insoluble (poly rI)-(poly rC) than has been available hitherto. The antiviral effect is not related to the amount of (poly rI)-(POLY RC) present on the insoluble support but rather to the amount of polynucleotide lost from the support during incubation. Treatment of (poly rI)-(poly rC) which had been bound to cyanogen bromide-activated Sepharose with eigher dilute alkali or pancreatic ribonuclease released virtually all the polynucleotide. A small amount of (poly rI)-(poly rC) is released from the insoluble matrix in the presence of serum-free Minimum Eagle's Medium.     

Enhanced production of interferon from human lymphoblastoid (Namalwa) cells pre-treated with sodium butyrate

Pre-treatment of Namalwa cells with 1 mM-sodium butyrate enhanced interferon production induced by Sendai virus. This enhancement was reversed if the cells were incubated in butyrate-free medium. Butyrate treatment increased the rate of interferon production but not its duration. Yields of interferon from Namalwa cells were also enhanced in response to other inducers including the synthetic dsRNA, poly(rI) . poly(rC). Butyrate pre-treatment also increased interferon yields from cells of the vervet monkey kidney cell line, V3, but cells of the human diploid fibroblast line, MRC-5, did not respond.     

Differential effect of poly rI.rC and Newcastle disease virus on the expression of interferon and cellular genes in mouse cells

The expression of type I murine interferon (MuIFN) genes and several other cellular genes was examined in poly rI.rC induced and Newcastle disease virus (NDV) infected mouse cells. Northern analysis of RNA from induced L cells revealed that the MuIFN-alpha s are expressed efficiently in NDV infected cells but only at low levels in poly rI.rC induced cells. MuIFN-beta 1, however, is expressed equally well in cells treated with poly rI.rC or infected with NDV. As shown by the use of a probe specific for poly rI.rC, interferon induction correlates with the cellular uptake of poly rI.rC into the cells. The relative levels of alpha and beta 1 mRNAs in the cells reached a maximum at 10 hr after the induction which indicates coordinate expression of alpha and beta 1 interferon genes. The effect of viral infection on the expression of two murine genes coinduced with interferon (pMIF20/11 and pMIF3/10) and several cellular genes was also examined. While pMIF20/11 is an inducible gene, the pMIF3/10 gene is expressed constitutively in mouse L cells. Viral infection, but not poly rI.rC treatment, enhanced the expression of the pMIF3/10 gene, as well as two other cellular genes; H-2 and c-myc, however, the expression of beta-actin gene was unaltered. These data indicate that enhancement of gene expression in virus infected cells in not limited to the interferon system.     

Regulation of the interferon system: evidence that Vero cells have a genetic defect in interferon production.

A clone of Vero cells was isolated and shown to be totally unable to synthesize interferon and insensitive to the toxic effect of poly(rI).poly(rC) treatment. Cells of this clone and mouse L cells were fused by treatment with polyethylene glycol or Sendai virus. Hybrid cell clones were isolated following selection in medium containing hypoxanthine, thymidine and ouabain. The hybrids were sensitive to the antiviral effect of poly(rI).poly(rC) and synthesized mouse, but not primate, interferon. It is proposed that in Vero cells, the gene for interferon synthesis is defective or absent.     

The production and action of interferon in Chinese hamster cells.

The interferon system has been investigated in primary cell cultures established from Chinese hamster embryos and new born pups. Interferon synthesis was induced with Sindbis virus, ultraviolet inrradiated Newcastle disease virus (u.v.-NDV) and with polyriboinosine acid-polyribocytidylic acid complex [poly (rI). poly (rC)]. Only u.v.-NDV induced significant production of interferon, maximum amounts being produced in 'aged' cells. Its apparent mol. wt. was 25000. CHO-KI cells, an established line of Chinese hamster cells, did not synthesize interferon in response to viruses, but were sensitive to its action.     

Identification of a cell membrane receptor for interferon induction by poly rI:rC.

PR-RK, a cell line derived from rabbit kidney cells (RK-13), was insensitive to the cytotoxic effect and interferon (IFN) inducing activity of the copolymer of riboinosinic and ribocytidylic acid (poly rI:rC). However, PR-RK was sensitive to the cytotoxic effect of the copolymer of riboadenylic and ribouridylic acid (poly rA:rU). Comparison of PR-RK cells and RK-13 cells by cytofluorometric analysis revealed that the binding of poly rI:rC was considerably reduced on PR-RK cells. These results suggested that the receptor for poly rI:rC might be different from the receptor for poly rA:rU, and this difference could provide a basis for the identification of the dsRNA receptor on cell surface. Western blot analysis of the components of cell membrane fraction prepared from RK-13 cells was performed by using a monoclonal antibody, which binds to cell membrane of RK-13 cells but not to PR-RK cells, and which blocks IFN induction by poly rI:rC in RK-13 cells. The 60K protein was identified as one of the poly rI:rC receptor protein.     

The effect of combination of different inducers on the refractory state in interferon production.

A second injection of 100 mug poly (rI) poly (rC) per mouse at 6 and 24 hours after the first injection stimulated additional peaks of interferon production. The dynamics of the process of accumulation and disappearance of interferon was similar to that after a single injection of poly (rI). poly (rC). Injection of the above dose 12 hours after the first injection induced no interferon production as it apparently coincided with the refractory state in interferon production. After pretreatment of poly (rI) poly (rC) with DEAE-dextran, the refractory phase occurred in 6 hours. Inoculation of Venezuelan equine encephalomyelitis virus as a second interferon inducer resulted in a repeated stimulation of interferon production both in animals and in tissue culture; however, interferon titres in this case were low. The use of an inactivated virus as a second interferon inducer stimulated interferon production to higher titres (5120 IU/ml) than a single injection of DEAE-dextran-treated poly (rI). poly (rC). It is possible that a combined use of poly (rI). poly (rC) and noninfectious virus as a second interferon inducer eliminates the development of the refractory state.     

The antiviral activity of certain thiophosphate and 2'-chloro substituted polynucleotide homopolymer duplexes

Poly(rI)·poly(rsC) (polyriboinosinic acid) (polythiophosphate ribocytidylic acid) was found to be a slightly less potent interferon inducer than poly(rI)·poly(rC). The rate of degradation for poly(rI)·poly(rsC) and poly(rI)·poly(rC) by serum nucleases also proved to be very similar. In contrast, substitution of the 2′-hydroxyl by a chlorine atom in poly(rU) (polyribouridylic acid) and poly(rC) greatly increased their resistance to serum nucleases. Poly(rA)·poly(2′-ClU) (polyriboadenylic acid) (poly2′-chlorouridylic acid) and poly(rI)·poly(2′-ClC) were incapable of producing interferon although they possessed as great or greater thermal stability and much greater nucleolytic resistance than the unsubstituted interferon producing duplexes. This lack of interferon production by the 2′-Cl duplexes could not be explained by the failure of human foreskin fibroblasts (HFF) to take up these polymers. In addition, poly(rI)·poly(2′-ClC) did not compete with the interfering activity or the cellular association of poly(rI)·poly(rC). Substitution of the 2′OH by a 2′NH₂ in poly(rU) also produced a polymer incapable of producing interferon.     

Binding of polyriboinosinic-polyribocytidylic acid with cultured cells

Summary Homogenates prepared from polyriboinosinic-polyribocytidylic acid copolymer [poly(rI) · poly(rC)]-treated cells exhibited antiviral activity in chick embryo, L and rabbit kidney cells. The antiviral activity in the homogenate co-sedimented with cellular membrane material and was shown to be poly(rI)·poly(rC) by a hybridization competition test with immobilized polyribocytidylic acid. The results indicate that poly(rI)·poly(rC) binds firmly to cellular membrane. These studies, however, could not differentiate between specific binding leading to the interferon induction and non-specific binding possibly unrelated to the induction of interferon.With 6 Figures     

Interferon production-stimulating factor

A factor stimulating interferon production (FSIP) has been isolated from cells treated once or twice (at an interval of 6-8 hours) with poly(rI).poly(rC) followed by actinomycin D (2 microgram/ml) or dehydrorifampicin (50 microgram/ml). In a dilution 1:512 the preparation stimulated interferon production 4-fold. In higher concentrations, the preparation induced interferon yields of 1280-2560 IE50/ml, i.e. exceeded the control values 16-fold. The factor does not affect the antiviral activity of interferon and has the tissue specificity. Its stimulating effect was not manifested with inducers of other nature. All the properties of FSIP studied show it to be similar with homologous interferon and repressor of its production but, unlike the latter, it was thermolabile and sensitive to acid pH.     

Induction of Viral Interference: Effects of Poly rI·rC and Diethylaminoethyl-Dextran on the Activity of the Antiviral Protein

The activity of the antiviral protein induced by various ratios of poly rI.rC and diethylaminoethyl (DEAE)-dextran was studied. It was found that, when large doses of poly rI.rC were used, very little viral interference was observed. This effect was initially attributed to the cells being refractory for production of antiviral protein. Subsequent experiments offered alternative explanations suggesting that, at any given dosage of poly rI.rC, an excess of DEAE-dextran is necessary for the production of viral interference. It is suggested that DEAE-dextran acts by exposing a cell receptor site for poly rI.rC.     

Interferon induction by viruses and polynucleotides: a differential effect of comptothecin.

The plant alkaloid comptothecin inhibits interferon production induced by Newcastle disease virus (NDV) or ultraviolet-irradiated NDV in chick and human cells, and by Sindbis virus in chick cells. It has no effect on interferon production induced by poly (rI).poly(rC) in chick and human cells. No effect of comptothecin could be detected on the multiplication of NDV, and it is concluded that the inhibition reflects a difference between interferon induction by viruses and by polynucleotides.