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.     

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).     

Constitutive and inducible expression of cloned human interferon-b1 gene in HeLa cells through an episomal eukaryotic vector

An EcoRI fragment (1.83 Kb) of human DNA containing B1-interferon coding sequences was cloned in the episomal eukaryotic vector pBK-TK. The recombinant plasmid was transfected into thymidine kinase-deficient HeLa cells. Thymidine kinase-positive transformants were obtained and the cells were found to express B-interferon both constitutively and after induction with Newcastle Disease Virus or poly(rI)-poly(rC). The recombinant plasmid was present in stably transformed HeLa cells in an episomal state in hundreds of copies per cell.     

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.     

Priming affects the activity of a specific region of the promoter of the human beta interferon gene.

Treatment of Daudi or HeLa cells with human interferon (IFN) alpha 8 before induction with either poly(I)-poly(C) or Sendai virus resulted in an 8- to 100-fold increase in IFN production. The extent of priming in Daudi cells paralleled the increase in the intracellular content of IFN-beta mRNA. IFN-alpha mRNA remained undetectable in poly(I)-poly(C)-treated Daudi cells either before or after priming. An IFN-resistant clone of Daudi cells was found to produce 4- to 20-fold more IFN after priming, indicating that priming was unrelated to the phenotype of IFN sensitivity. IFN treatment of either Daudi or HeLa cells transfected with the human IFN-beta promoter (-282 to -37) linked to the chloramphenicol acetyltransferase (CAT) gene resulted in an increase in CAT activity after induction with poly(I)-poly(C) or Sendai virus. A synthetic double-stranded oligonucleotide corresponding to an authentic 30-base-pair (bp) region of the human IFN-beta promoter between positions -91 and -62 was found to confer virus inducibility upon the reporter CAT gene in HeLa cells. IFN treatment of HeLa cells transfected with this 30-bp region of the IFN-beta promoter in either the correct or reversed orientation also increased CAT activity upon subsequent induction. IFN treatment alone had no detectable effect on the activity of either the 30-bp region or the complete human IFN promoter.     

Susceptibility of influenza viruses to interferon and to poly(I) . Poly(C) determined by the plaque reduction method

Susceptibility of eight strains of influenza A and B viruses to interferon and to poly(I) . poly(C) were determined by the plaque reduction method. All strains tested were slightly less susceptible than vesicular stomatitis virus (VSV) in an established line of canine kidney (MDCK) cells. The 50% plaque depression doses (PD50) of poly(I) . poly(C) for influenza A and B viruses were as high as 3.0- to 4.5-fold and 6- to 18-fold that for VSV, respectively. The amounts of interferon required to inhibit plaque formation of influenza A and B viruses by 50% were 3.0-6.2 and 7.3-15.2 units/ml, respectively. The ratio of PD50 of poly(I) . poly(C) for each strain of influenza viruses tested to that for VSV in chick embryo cells was almost the same as in MDCK cells. Furthermore, in chick embryo cells, the strains of influenza virus tested were demonstrated to be much more susceptible to poly(I) . poly(C) than both Newcastle disease virus and vaccinia virus. It is suggested that influenza viruses may be relatively susceptible to interferon and to poly(I) . poly(C).     

Interferon Induction in Rabbit Cells Irradiated with UV Light

UV irradiation of a continuous line of rabbit kidney cells (RK13) was used as a tool for the study of the mechanism of interferon induction. Irradiation of cells prior to their exposure to Newcastle disease virus (NDV) resulted in a dose-dependent decrease in interferon production. The inhibition of total cellular RNA synthesis by UV irradiation in uninduced cultures was similar to the inactivation curve of interferon production in NDV-induced cultures. In contrast, the production of interferon with polyinosinate-polycytidylate (poly[I].poly [C]) paradoxically was enhanced in cells irradiated with a wide range of doses of UV. However, in cells stimulated with poly(I).poly(C) and "superinduced" by the sequential addition of cycloheximide and actinomycin D, the rate of inactivation of interferon production by UV light was similar to that observed with NDV. These results are not inconsistent with the idea that both poly(I).poly(C) and NDV stimulate the same interferon gene(s), but indicate that the mechanism controlling its expression may be different for each inducer.     

Effect of modulation of the production of interferon by L-929 cells treated with theophylline

The specific inhibitor of cAMP phosphodiesterase theophylline has been shown to evoke in L929 cells 2.3-fold induction of 2-5A-synthetase activity and 3.5-fold superinduction of the same enzyme activity while acting in combination with actinomycin D. It has been shown also that temporal coincidence of 2-5A-synthetase induction with the active period of interferon production resulted in 8-16 times decrease in the level of interferon production. The result was supported by the experiments of superinduced cells (containing the high stable level of 2-5A-synthetase) fusion with monolayer of poly(I).poly(C)-induced L929 cells (taken at the start of interferon production). In this case the production of interferon was dramatically decreased in comparison with the control. Possible role of 2-5A-synthetase in regulation of interferon production is discussed.     

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.     

Interferon response in normal and Aleutian disease virus-infected mink.

Studies were done to determine whether differences in interferon production are responsible for the resistance of pastel mink to Aleutian disease. The abilities of normal pastel and sapphire mink to produce interferon when inoculated with either Newcastle disease virus or a synthetic polyribonucleotide, poly (I):poly (C), were identical, even to the production of a novel, acid-labile interferon. The resistance of pastel mink to Aleutian disease did not correlate with interferon production, because neither sapphire nor pastel mink produced detectable amounts of interferon when infected with either the Pullman strain of Aleutian disease virus (ADV) or the highly virulent Utah I strain. Sapphire mink infected with the Pullman strain responded normally to poly (I):poly (C) early in the course of the disease, but interferon production was impaired late, when the mink were hypergammaglobulinemic and had renal, vascular, and hepatic lesions. These data suggest that ADV Pullman neither stimulates nor interferes with interferon production in infected mink and may represent a mechanism whereby ADV can more readily establish infection.     

Induction of interferon by polyribonucleotides containing thiopyrimidines.

The influence of thioketo substitution in pyrimidine bases of double-stranded polynucleotides on interferon induction was investigated. The stabilizing effect of 2-thioketo substitution was reflected in the increased interferon inducing activity of poly(A-s2U) over that of poly(A-U). Poly(A-s2U) and poly(I)-poly(s2C) were as effective as poly(I)-Poly(C) in rabbit cells. Poly(I)-poly(C) and poly(I)-poly(s2C) were compared in several animal species. No differences in biological effects were observed in rabbits and dogs. In rodents, poly(I)-poly(s2C) was less effective and less toxic.Poly(I)-poly(s2C) was highly resistant against degradation by human serum. Further investigations seem to be justified to elucidate whether this property offers any advantages for the potential clinical utilization of poly(I)-poly(s2C).     

Interferon production by primary culture of human endothelial cells

Endothelial cells of human umbilical vein are capable of producing interferon upon induction with Newcastle disease virus, influenza virus, and poly I: poly C, but not staphylococcal enterotoxin A. All the interferons produced belonged to the alpha-type. After treatment with influenza virus the endothelial cells produce two subtypes of alpha-interferon: acid-labile and acid-stable.     

8-Azaguanine resistant African green monkey kidney cell mutants (Vero 153): isolation and characterization

A clone of Vero cells resistant to up to 20 micrograms/ml 8-azaguanine was isolated. This clone (designated Vero 153) has a doubling rate of approximately 24 h and a maximum cell density of 10,000/mm2. Deficiency of the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) in Vero 153 was demonstrated by methods of radiochromatography. Vero 153 is susceptible to hypoxanthine-thymidine-aminopterin (HAT) medium and its resistance to 8-azaguanine seems to be nonreversible. Like parental cells, Vero 153 was also incapable of interferon production when challenged with Newcastle disease virus (NDV) or poly(inosinic acid) . poly(cytidylic acid) (poly I:C). Similar chromosome complements (majority range 56 to 57) and band patterns were observed in cells harvested at Passages 10, 20, and 50. The potential use of Vero 153 for somatic cell hybridization for purposes of gene mapping, virus rescue, and the control of interferon production is discussed.     

Interferon priming. Effects on interferon messenger RNA.

The effects of priming mouse cells with interferon on the production of interferon and its mRNA were investigated. Interferon-treated (primed) mouse L929 cells produce 3 to 10 times more interferon than do nonprimed cells following induction with Newcastle disease virus. Interferon appears 2 to 4 h sooner in the primed cultures than in nonprimed cultures and interferon production by primed cells becomes resistant to inhibition by actinomycin D about 4 h sooner than interferon production in nonprimed cells. Interferon mRNA is detected in primed-induced cells about 2 h earlier than in nonprimed-induced cells. It reaches peak levels about 2 to 4 earlier in primed cells, but it also disappears sooner in primed cells. The total amounts of interferon mRNA isolated from primed-induced cells and nonprimed-induced cells were indistinguishable, by the methods utilized. Therefore, although primed cells can produce significantly more interferon and make interferon mRNA sooner than nonprimed cells, the total amount of interferon mRNA produced is apparently not increased, nor is its half-life prolonged in primed cells. Thus, enhanced interferon production in primed cells may result from enhanced efficiency of translation of interferon mRNA in the primed cells.     

Treatment of mice with polyinosinic-polycytidilic polyribonucleotide reduces T-cell involvement in a localized inflammatory response to vaccinia virus challenge.

Mice inoculated intracerebrally with 10(3) PFU of vaccinia virus developed a nonfatal meningitis which was maximal 7 days after challenge. Intravenous administration of an interferon (IFN) inducer, polyinosinic-polycytidilic polyribonucleotide [poly(I)-poly(C)], on days 4 and 6 postinjection was associated with a three- to fourfold decrease in the number of T lymphocytes present in cerebrospinal fluid, reflected primarily by a decreased number of vaccinia virus-specific cytotoxic T-lymphocyte precursors. The lack of a concomitant reduction in the overall cytotoxic activity of cerebrospinal fluid cells directed against virus-infected target cells seemed to be largely due to an increase in natural killer cell activity. IFN was implicated as mediating the effect of poly(I)-poly(C) because high systemic levels of IFN were evident after injection, and neither the magnitude of the inflammatory response nor the T-cell levels were affected when poly(I)-poly(C)-treated mice were also given anti-IFN antiserum. However, the poly(I)-poly(C)-induced IFN did not seem to reduce the localized inflammatory response by affecting viral replication in brain tissue because the vaccinia virus titers present on days 6 through 8 of infection were similar to the titers in phosphate-buffered saline controls. These findings are consistent with either an effect of IFN on T-cell recruitment to the central nervous system or an inhibition of proliferation of cells participating in the response. These findings suggest that there is a potential source of complications for clinical protocols that use IFN or inducers to enhance T-cell function in various disease situations, and this effect of IFN may be a contributing factor to the immunosuppression often associated with many viral infections.     

Determination of the species specificity of interferons in the translation of the their mRNA from various cell cultures

Interferons obtained on induction of human lymphocytes with Newcastle viruses and staphylococcal enterotoxin A and diploid fibroblast cells of human embryos with poly (I).poly (C), as well as translation products of interferon mRNA obtained from these cells were analysed serologically. It was shown that the main type of interferon produced by the cells depended on the cell culture and inductor nature. It was defined at the level of the respective gene depression. Effective translation of mRNA of the interferons of the 3 types makes possible production of cDNA and creation of bacterial plasmids coding the genetic information for the synthesis of human interferon.     

Inhibition of herpes simplex virus type 2-induced biochemical transformation by interferon.

The effect of interferon on the biochemical transformation of thymidine kinase-deficient cells by UV-inactivated herpes simplex virus type 2 has been studied. Transformation was much less sensitive to the action of interferon than virus multiplication. However, the continuous presence of a high dose of interferon (2,000 U) inhibited transformation almost completely. Although we could not differentiate between the effect of interferon on fixation and expression of the virus thymidine kinase gene, data suggest that the inhibitory effect of interferon on transformation might be partially due to the suppression of virus thymidine kinase expression.     

Control of Interferon Synthesis: Effect of Diethyl-aminoethyl-Dextran on Induction by Polyinosinic-Polycytidylic Acid

Interferon production in cultures of rabbit kidney cells (RKC) stimulated with 10 to 250 mug of polyinosinic-polycytidylic acid (poly I.poly C) per ml peaked at 3 to 4 hr after the exposure of cells to inducer and rapidly declined thereafter. On the other hand, RKC stimulated with poly I.poly C (10 or 2 mug/ml) in the presence of diethylaminoethyl (DEAE)-dextran (100 or 20 mug/ml, respectively) produced a protracted interferon response, with the release of interferon continuing for over 24 hr. The kinetics of interferon production in RKC stimulated with lower concentrations of the mixture of poly I.poly C and DEAE-dextran were similar to the response produced by poly I.poly C alone (10 to 250 mug/ml). Only the responses that terminated early were paradoxically enhanced by treatment with low doses of actinomycin D or with cycloheximide. Cells stimulated with 50 mug of poly I.poly C/ml showed hyporesponsiveness to a second interferon induction with poly I.poly C when restimulated 7 hr after primary induction. This hyporesponsiveness could be overcome by restimulating with higher concentrations of the poly I.poly C-DEAE-dextran complex. The results are compatible with the hypothesis that the early termination of interferon production and hyporesponsiveness to repeated induction with poly I.poly C are due to a cellular repressor exerting negative control on interferon synthesis, and that the increased cellular uptake of poly I.poly C in the presence of DEAE-dextran may effectively neutralize the repressor. These results also suggested that the often observed different kinetics and the varied effects of inhibitors of ribonucleic acid or protein synthesis on interferon responses in various cells and in cells stimulated with different inducers (such as with viruses as compared with polynucleotides) need not imply the existence of fundamentally different mechanisms of interferon production.