Relation of the biological activity of double helical interferon inducers and their penetration into the cell and suppression of protein synthesis

The quantities of 125I-ds-inductors of interferon penetrating into the cells of transplantable cultures such as M19 (human fibroblast cells) and L-929 (mouse line) were not significant i.e. 10.5-4 per cent of the drug added. Under conditions of transfection with calcium phosphate and in complex with DEAE dextran the quantities of the inductors adhering to the cells and their contents in the cytoplasmic and nuclear fractions markedly increased. During the transfection with calcium phosphate up to 50 per cent of the applied inductor bound to the cells and its content in the cytoplasm and nuclei reached at least 10 per cent. After penetration into the cells poly I.poly C probably maintained its native structure and appeared to be firmly bound to the nuclear material. Preliminarily hydrolyzed inductors showed no such penetrating capacity. Contrary to the human fibroblast cells, in the mouse cells L-929 treated with the ds-inductors there was observed inhibition of the total protein synthesis which was probably due to activation of enzymes such as 2-5A-synthetase and proteinkinase. Increased penetration of the ds-inductors into the cells was accompanied by a marked (from 10- to 1000-fold) rise in their antiviral activity and a 2-4-fold rise in their interferon-inducing activity. It was concluded that there was immediate dependence of ds-inductor biological activity manifestation on the level of the inductor penetration into the cells.     

Translation of mouse interferon messenger RNA in a cell-free system

Crude mouse interferon mRNA extracted from poly (I) . poly (C)-induced L929 cells has been translated in a cell-free system from rabbit reticulocytes and in two-cell systems--L929 cells and chick embryo fibroblasts. Translational efficiency of interferon mRNA preparation was 100-fold higher in the cell-free system than in tissue culture cells. Interferon synthesized was species-specific and its antiviral activity was completely neutralized by mouse interferon antiserum.     

Induction of interferon-specific enzymes in cultures of cells sensitive and resistant to interferon

Induction of 2'-5'-oligoadenylatesynthetase (2-5A synthetase) by interferons and theophylline by means of activation of cAMP-system in interferon susceptible and resistant cell lines were studied. In interferon resistant cell lines the basal activity of 2-5A synthetase exceeded the level of the same enzyme in interferon susceptible cell lines. Activity of 2-5A synthetase is increased in interferon susceptible cell lines by interferon treatment, but the activity of the enzyme is not altered in interferon resistant cell lines. Among the studied cell lines the induction of 2-5A synthetase by theophylline was possible only in L929 cell line. The common mechanism for the absence of 2-5A synthetase induction by interferon and theophylline in interferon resistant cells is discussed.     

Effect of calcium chloride on interferon production and the biosynthesis of cellular macromolecules

The influence of calcium chloride in production of interferon and biosynthesis of cell proteins, RNA and DNA in the cultures of chick embryo fibroblast cells and murine cells L-929 in response to induction of the cells by poly(I).poly(C) was studied. It was shown that calcium ions in concentrations of 10 to 30 mM markedly increased formation of interferon in the cell cultures.     

Production of the hyporeactivity factor by mouse cells

The mouse cells L 929 experiencing the state of hyporeactivity to repeated interferon induction with the aid of poly (I) X poly (C) secrete to the incubation medium a factor which suppresses interferon production. The factor is not neutralized by antiserum against murine interferon. Apart from hyporeactivity factor, interferon itself may inhibit its own production.     

Induction of Interferon and Erythropoietic Differentiation in Cells Transformed by Friend Virus

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I) · poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefold increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I) · poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I) · poly(C) induction mechanisms.     

Induction of interferon and erythropoietic differentiation in cells transformed by Friend virus.

Two lines of Friend virus (FV)-transformed mouse spleen cells have been analyzed in respect to their interferon production capacity: neither F4 cells, which liberate infectious FV when kept under tissue culture conditions, nor the thymidine kinase-deficient B8 cells, which do not produce significant amounts of FV, release detectable amounts of autogenous interferon into cell supernatants. However, interferon is produced in these cells in amounts comparable to that in L-929 cells when interferon induction is initiated with UV-inactivated Newcastle disease virus. Conversely poly(I)-poly(C), a potent interferon inducer in L-929 cells, proved ineffective in this capacity in F4 or B8 cells. When erythropoietic differentiation is induced in these cells by dimethyl sulfoxide, no autogenous interferon production occurs, but with NDV-induction a four- to fivefode increase of interferon production is observed. A similar elevation of interferon production is achieved during 5-bromodeoxyuridine stimulation of differentiation in the thymidine kinase-deficient B8 cells. The refractiveness against poly(I)-poly(C) displayed in unstimulated cells is not overcome at any stage of differentiation, indicating major differences of Newcastle disease virus and poly(I)-poly(C) induction mechanisms.     

Induction of poly(I):poly(C)-binding 50 K protein and 2',5'-oligoadenylate synthetase in interferon-treated mouse L929 cells

A new protein retained by poly(I):poly(C)-Sepharose was induced together with dsRNA-dependent enzymatic activities, a protein kinase and 2',5'-oligoadenylate synthetase (2,5A synthetase), in interferon-treated mouse L929 cells; it had an apparent molecular weight of 50,000 (50 K) and was not phosphorylated by the protein kinase. The kinetics of the induction of the poly(I):poly(C)-binding 50 K protein were similar to those of dsRNA-dependent protein kinase and 2',5'-oligoadenylate synthetase, and their inductions were all dependent on the interferon dose added, though a relatively higher dose was required for the 50 K protein. When the interferon preparation was heated to 100 degrees C in the presence of sodium dodecyl sulfate, its effect on cells of inducing the activity of 2',5'-oligoadenylate synthetase was preserved completely, indicating that the interferon molecule itself is responsible for the induction of the synthetase. Since the induction of the enzymatic activity was inhibited by addition of either actinomycin D or cycloheximide, it may not be an activation of a latent enzyme but a de novo synthesis of the enzyme.     

Antibody targeted liposomes containing poly(rI).poly(rC) exert a specific antiviral and toxic effect on cells primed with interferons alpha/beta or gamma

Double-stranded RNA can stimulate interferon production and mediate an antiproliferative effect on certain cell types. We evaluated the possibility of specifically targeting to cells in vitro the RNA duplex poly(rI).poly(rC) in pharmacologically active form after its encapsulation in small, unilamellar liposomes, to which was covalently coupled protein A. These liposomes became bound to and were endocytosed by murine L929 cells in the presence of protein A-binding monoclonal antibodies specific for an expressed cell surface protein, the H-2K molecule. When L929 cells were preincubated in the presence of low doses of interferon alpha/beta or gamma, they could be activated to produce interferon following exposure to either free poly(rI).poly(rC), or specifically bound liposomes poly(rI).poly(rC), but not the same liposomes in the presence of non-cell binding control antibodies. Specifically bound liposome-encapsulated poly(rI).poly(rC) was toxic to L929 cells at dose levels at least three logs lower than free poly(rI).poly(rC). This toxicity was also dependent on pre-treatment with interferon. These results indicate that liposome-encapsulated poly(rI).poly(rC) can survive endocytosis and can be released in active form to specific cell populations, at concentrations much lower than that required for pharmacologic effects of the same molecule in free form. They suggest that introduction into cells of other nucleic acids might benefit from the antibody-targeted liposome technology described here.     

Inhibition of vesicular stomatitis virus replication in dexamethasone-treated L929 cells

We previously demonstrated that dexamethasone treatment of L929 cells inhibited plaque formation by vesicular stomatitis virus (VSV), encephalomyocarditis virus, or vaccinia virus. We now have characterized the antiviral effects of glucocorticoids in L929 cells. Dexamethasone did not directly inactivate VSV nor did steroid treatment of L929 cells affect virion adsorption or penetration. The VSV yield in L929 cells treated with dexamethasone for a period of only 4 or 8 hr was decreased by 50% when cells were infected the day following steroid treatment. Treating L929 cells with dexamethasone for a longer period resulted in greater inhibitions of virus synthesis. Interferon activity (less than 5 units/ml) was not detected in L929 cell culture fluids and cell sonicates from steroid-treated cells and the addition of antiserum to murine alpha/beta-interferon had no effect on the ability of dexamethasone to inhibit VSV replication. Dexamethasone treatment of L929 cells did not induce the production of double-stranded RNA-dependent protein kinase but did result in a slight elevation of 2-5A oligoadenylate synthetase activity, two enzymatic activities associated with the antiviral state induced by interferon. However, the elevated 2-5A synthetase activity was not associated with an inhibition of VSV RNA accumulation in dexamethasone-treated L929 cells. By contrast, the synthesis of all five VSV proteins was reduced by 50-75% in dexamethasone-treated L929 cells as early as 4 hr after infection. Thus, the dexamethasone-mediated inhibition of VSV replication in L929 cells is associated with decreased production of VSV structural proteins.     

Antiviral activity of polynucleotides: copolymers of inosinic acid and N2-dimethylguanylic of 2-methylthioinosinic acid.

Complexes of poly(C) with copolymers of inosinic acid containing various amounts of mismatched bases (see journal for formula) have been examined for direct resistance to virus infection, interferon induction and toxicity in two different cell cultures (primary rabbit kidney cells and mouse L-929 cells). Complexes in which 20% of the hypoxanthine bases were replaced by (see journal for formula) or ms-2I were partially active whereas complexes in which 40% or more of the hypoxanthine bases were replaced by the odd bases were entirely inactive. The decrease in biological activity observed upon intrusion of (see journal for formula) or ms-2I in the poly(I) strand of poly(I) with poly(C) closely paralleled the amount of odd bases introduced irrespective of the system employed to assess the biological activity (resistance to virus infection, interferon induction or toxicity).     

Specific residues within an amino-terminal domain of 35 residues of interferon alpha are responsible for recognition of the human interferon alpha cell receptor and for triggering biological effects

Bovine interferon alpha C (IFN-alpha C) manifest at least 10(5)-fold lower antiviral activity on human cells than on bovine cells (Velan, B., Cohen, S., Grosfeld, H., Leitner, M., and Shafferman, A. (1985) J. Biol. Chem. 260, 5498-5504). By oligonucleotide site-directed mutagenesis within the coding region for the NH2-terminal 44-residue domain of BoIFN-alpha C, we replaced up to 18 residues by the corresponding HuIFN-alpha J1 residues. (HuIFN-alpha J1 is less than 60% homologous in sequence to BoIFN-alpha C.) The nine different bovine-human-IFN alpha hybrids obtained were compared to BoIFN-alpha C and HuIFN-alpha J1 with respect to their potential to induce an antiviral state, synthesis of 2-5A-synthetase, and their specific binding to human and bovine cells. Relative to BoIFN-alpha C, a gradual increase in biological activities (antiviral or 2-5A-synthetase) of approximately 10-, 10(2)-, 10(3)-, and approximately 10(4)-fold is obtained, depending on the number and positions of the residues substituted. A direct correlation exists between biological response and ability of IFN alpha to bind specifically to human cells. A BoIFN alpha molecule mutated in the 10-44 NH2-terminal domain was obtained which is 15, 8, and 35% as active as HuIFN-alpha J1 on human cells in specific binding, induction of antiviral, and 2-5A-synthetase activities, respectively. We concluded that at least 5 of the 12 residues at positions 10; 21, 22, 24; 27; 31, 34, 35, 37, 40; 42, 43 in the 10-44 NH2-terminal domain are critical for recognition of the human IFN-alpha cell receptor and for biological activity. These residues are found among 10 strictly conserved residues in all reported mammalian IFN alpha S, and they act in a cooperative manner to induce a biological response in human cells. The gap between the extent of improvement in binding capacity of the BoIFN alpha mutants on human cells and the corresponding biological response suggests that the primary signal of binding to the cell receptor is amplified within the cell. On bovine cells, HuIFN-alpha J1 and BoIFN-alpha C also compete for the same receptor, and it seems that at least part of the 10-44 NH2-terminal domain on IFN alpha is also involved in interaction with the bovine IFN alpha cell receptor.     

The state of hyporeactivity in interferon production: localization and partial purification of the factor repressing the interferon production

The hyporeactivity factor in interferon production by L-929 cells designated IRP (interferon repressing protein) has been studied. In particular, its localization and methods of its purification have been studied. The kinetics of IRP accumulation by producing cells correlate with the development of hyporeactivity condition. Most of IRP is localized in cell sap and in ribosomal fraction in evidence to regulatory role of repressor at the level of interferon mRNA translation. A 100-fold increase in repressor activity was achieved by IRP concentration by ammonium sulfate precipitation. IRP as well as interferon have been shown to possess high affinity to polyU sepharose. The preparations of IRP and interferon concentrated by ammonium sulfate precipitation were subsequently purified by fractioning in a polyI sepharose column. A 10,000-fold (6 x 10(4) U/mg) purification was achieved for IRP and 250-fold (10(4) U/mg) for interferon.     

Analogue inhibitor of 2-5A action: effect on the interferon-mediated inhibition of encephalomyocarditis virus replication.

Chemically synthesised CH3Sp(A2'p)2A2'pp3'OCH3 has been used to assess the importance of the ppp(A2'p)nA (n greater than or equal to 2: 2-5A) system in the antiviral action of interferon against encephalomyocarditis virus (EMC). It inhibits activation of the 2-5A-dependent RNase by 2-5A in intact mouse L929 cells and cell-free systems. In interferon-treated, EMC-infected L929 cells it inhibits 2-5A-mediated rRNA cleavage and partially restores EMC RNA synthesis and virus yield. Activation of the 2-5A-dependent RNase must, therefore, play some part in interferon action against the growth of EMC virus in such cells.     

Characterization of 69- and 100-kDa forms of 2-5A-synthetase from interferon-treated human cells

The existence of distinct 69- and 100-kDa forms of 2-5A-synthetase in addition to the smaller (40 and 46 kDa) forms has recently been established. Using specific monoclonal antibodies we investigated the induction, synthesis, and activity of 69- and 100-kDa 2',5'-oligoadenylate (2-5A) synthetases in interferon-treated human Daudi cells. Although induction of these synthetases is detectable in cells treated with as little as 1-5 units/ml of human alpha-interferon, higher concentrations are required for maximum synthesis of the 100 kDa than the 69-kDa protein. At 5 units/ml of interferon, enhanced synthesis of both proteins is detectable at 4 h with maximum synthesis occurring between 8 to 12 and 12 to 16 h for 69- and 100-kDa 2-5A-synthetases, respectively. At 24 h after addition of interferon, synthesis of these synthetases declines due to a decrease of active interferon in the culture medium. The synthesis of both synthetases is blocked by actinomycin D, and the half-life of these proteins is estimated to be 8 h. The activities of immunoaffinity purified 69- and 100-kDa synthetases are dependent on double-stranded (ds)RNA but show different requirements for optimum concentration of dsRNA and pH of the reaction. The apparent Km of 69- and 100-kDa synthetases for ATP is 1.7 X 10(-3) M and 3.6 X 10(-3) M, respectively. At optimum conditions for the activity of these enzymes, the pattern of 2',5'-linked oligoadenylates synthesized are different, the 69-kDa protein synthesizing higher oligomers than the 100-kDa species. Taken together, these results indicate that the 69- and 100-kDa 2-5A-synthetases are distinct proteins each with specific characteristics of induction and enzymatic activity.     

Effect of Platelet-activating Factor on in vitro and in vivo Interleukin-6 Production

The aim of the present study was to investigate the possible effect of platelet-activating factor (PAF), by comparison with interleukin-1beta and polyriboinositic/polyribocytidylic (poly I-C) acid, on IL-6 production by L 929 mouse fibroblasts. At concentrations above 1 muM PAF, the production of IL-6 by mouse fibroblasts was enhanced in a dose dependent fashion. At 5 muM PAF, the peak increase (60.1 +/- 19.4 U/ml) was similar to that induced by 50 mug/ml poly I-C (60.0 +/- 35.0 U/ml) and higher than the one evoked by 100 U/ml IL-1beta (3.8 +/- 1.8 U/ml). The increase of 11-6 activity induced by 5 muM PAF was maximal after a 22 h incubation period with L 929 cells. Lyso-PAF (5 muM) also increased IL-6 activity from fibroblasts to a similar extent compared with 5 muM PAF. In addition, the IL-6 activity induced by 5 muM PAF was still observed when the specific PAF antagonist, BN 52021 (10 muM), was added to the incubation medium of L 929 cells. The result suggests that the production of IL-6 by L 929 cells evoked by PAF in vitro is not receptor mediated. The in vivo effect of PAF on IL-6 production was also investigated in the rat. Two hours after intravenous injection of PAF (2 to 4 mug/kg), a dramatic increase of IL-6 activity in rat serum was observed, this effect being dose dependent. The increase of IL-6 induced by 3 mug/kg PAF was not observed when the animals were treated with the PAF antagonist, BN 52021 (1 to 60 mg/kg0. These results demonstrate that PAF modulates IL-6 production and that the in vivo effect is receptor mediated.     

Effect of nucleosides on interferon production and development of antiviral state induced by poly I.poly C.

Effect of nucleosides both on induction of antiviral state in chick embryo cells (CEC) or rabbit kidney cells (RK13) and on interferon production in RK13 or mouse fibroblast cells (L cells) by polyriboinosinic-polyribocytidylic acid (poly I.poly C) was studied. Addition of inosine or a fifty-fifty mixture of inosine and uridine at a final concentration of 0.1 mM to 10 mM to a growth medium enhanced development of antiviral state in CEC. The nucleoside effect was also observed in RK13 at 0.1 mM but not at a concentration higher than 1 mM. Interferon production in RK13 by superinduction (sequential treatment with metabolic inhibitors after exposure to poly I.poly C) was enhanced 1.5- to 4.0-fold by addition of the nucleoside mixture to the growth medium. When RK13 was pretreated with 10 units per ml of interferon and then superinduced by inhibitors, the enhancing effect of nucleosides on interferon production was not observed. Interferon production in L cells was potentiated a little by addition of 1 mM of the nucleoside mixture to the growth medium. The effect of nucleoside was not observed when the nucleosides were added after exposure to poly I.poly C. The nucleoside effect may be applicable for production of high titered interferon.     

Chloroquine impairs the interferon-induced antiviral state without affecting the 2',5'-oligoadenylate synthetase

Chloroquine, a weak base which raises the pH in acidic cellular compartments such as lysosomes and endosomes, counteracts the induction by interferon of the antiviral state but not that of the 2',5'-oligoadenylate synthetase in three different types of cell lines (MDBK, WISH, and L929). Active interferon is recovered in crude extracts of cells which have been treated with interferon and chloroquine together, but not in extracts of cells treated with interferon alone, indicating that chloroquine has inhibited the intralysosomal proteolysis of interferon. A low pH-dependent event in the intracellular fate of interferon (perhaps its intralysosomal degradation) is, therefore, necessary for the establishment of the antiviral state but not for the induction of the 2',5'-oligoadenylate synthetase.     

Purification of mouse cell interferon induced by isotopically labelled double-stranded f2 phage RNA

The dynamics of interferon formation by an established cell line of mouse fibroblast (L cells) and by mouse peritoneal leukocytes induced by double-stranded RNA extracted from E. coli f2 phage is described. The L cells produced interferon at a lower rate, the maximum values were obtained at 12 to 20 hours after induction, and the production was ultimately dependent on the established cell line used and on the presence of DEAE-dextran during induction. The mouse peritoneal leukocytes (MPL), on the other hand, did not require DEAE-dextran and the maximum of interferon production was reached between 6 and 12 hours after induction. Both the L cell- and the MPL-interferons were purified and concentrated so that the final specific biologic activity was 100-to 300-fold higher than that of the initial preparations (1 to 5 X 10(6) interferon units per mg protein). Polyacrylamide gel electrophoresis showed similar migration profiles for the preparations of both interferons. The smaller part of the activity was situated in a broader, slow-moving peak and the greater part formed a sharp, high and fast-moving peak. Using 3H uridine-labelled f2 ds-RNA for induction of interferon it was found that one of the radioactivity zones coincided with the fast-moving activity peak of the purified and concentrated interferon.     

Interferon: a mediator of indoleamine 2,3-dioxygenase induction by lipopolysaccharide, poly(I) X poly(C), and pokeweed mitogen in mouse lung

When C3H/He mice were treated with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, the serum interferon titer increased almost instantaneously (100-2000 units/ml), and then the pulmonary indoleamine 2,3-dioxygenase was induced 50- to 140-fold. The peaks corresponding to interferon induction always preceded (approximately 24 h) those corresponding to dioxygenase induction. In C3H/HeJ (lipopolysaccharide-nonresponder) mice, however, lipopolysaccharide was totally inert in induction of both interferon and dioxygenase, although treatment with poly(I) X poly(C) and pokeweed mitogen led to a remarkable increase in the serum interferon titer and the enzyme activity. When lymphocytes of C3H/HeJ mice were inactivated by X irradiation and then reconstituted by the transfer of spleen cells from C3H/He mice, both enzyme and interferon from C3H/HeJ mice thus treated were induced almost normally after the lipopolysaccharide treatment. In addition, murine interferon alpha/beta, which was injected intravenously in C3H/He or C3H/HeJ mice, almost instantaneously and dose-dependently induced the pulmonary enzyme, and at a dose of 10(5) units per mouse the enzyme activity was enhanced 20- to 26-fold in these two strains of mice. These results suggest that interferon, which is generated by the interaction of lymphocytes with lipopolysaccharide, poly(I) X poly(C), or pokeweed mitogen, is a mediator of indoleamine 2,3-dioxygenase induction in the mouse lung by these agents.