Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either [³⁵S]methionine or a ¹⁴C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all [³⁵S]methionine-labeled proteins and 2.6% of all ¹⁴C-amino acid-labeled proteins released into the medium.     

Properties of nonglycosylated and glycosidase-treated mouse L cell interferon species

Properties of nonglycosylated interferon produced by tunicamycin-treated L cells (IFN-TM), as well as those of glycosidase-treated L cell α and β interferons were investigated. IFN-TM was found to consist solely of α antigenic molecules. It showed a similar cell growth-inhibiting activity to the normal glycosylated interferon. Interferons α and β showed widely different cross-species antiviral activities on human and hamster cells, β being virtually inactive, and IFN-TM was similar to α, suggesting that the protein, rather than the sugar moiety, is important in determining the species barriers. When treated with a glycosidase mixture from Diplococcus pneumoniae, α (24K) was reduced in size to that of IFN-TM (18K), whereas β (36K) was reduced by only 5 to 10K. The antigenicity of β was unchanged after the glycosidase treatment. IFN-TM is thus considered to represent the nonglycosylated form of α; for β, its nonglycosylated form could not definitely be identified, although the glycosidase-treated β might possibly be close to it. The reason why the β antigenic component disappeared from the IFN-TM preparations is not clear, but this finding strongly suggests that glycosylation is essential for β, in contrast to α, for its stability and/or synthesis-secretion.     

Antigenicity of mouse interferons: distinct antigenicity of the two L cell interferon species

Antibodies were raised in rabbits against Newcastle disease virus-induced L cell interferon of high purity, and against each of its two major species, F(24K) and S(36K) interferons. The two interferons were found antigenically distinct. Thus, the anti-F and anti-S sera failed to neutralize appreciably the antiviral, as well as the cell growth-inhibiting, activity of the heterologous interferon. Heterologous reactions were also undetectable in a modified neutralization test, in which secondary antibody against rabbit γ-globulin was used to remove, before the assay for the residual interferon, any interferon-antibody complexes that might remain biologically active. Affinity chromatography of interferons on immobilized antibodies also showed antigenic distinctness of F and S interferons. Poly (I) ·poly (C)-induced L cell interferon and Newcastle disease virus-induced C₂₄₃ cell interferon also consisted of two distinct species which are antigenically similar to F and S.     

Effect of 2'5'-oligoadenylic acid on a mouse cell line partially resistant to interferon

We have investigated the sensitivity of a mouse cell line, NIH 3T3 (clone 1), that was found to be resistant to the anticellular and certain antiviral activities of interferon, to 2′,5′-oligoadenylate (2′5′A). The 2′5′A was introduced into the cells by coprecipitation with calcium phosphate and by increasing cell permeability with lysolecithin. In both cases neither cellular protein nor DNA synthesis was inhibited by 2′5′A in the NIH 3T3 cells. In contrast, the synthesis of proteins and DNA in L cells, an IFN sensitive line, was inhibited by low concentrations of 2′5′A. Furthermore, treatment of the infected cell cultures with 2′5′A resulted in the inhibition of vesicular stomatitis virus (VSV) replication in L cells but not in NIH 3T3 cells. The production of MuLV in NIH 3T3 cells was also not affected by 2′5′A. These observations, together with our previous finding that NIH 3T3 (clone 1) cells are deficient in RNase F activity suggest that activation of the RNase F by 2′5′A is responsible for the inhibition of both protein and DNA synthesis in sensitive cells.     

Rapid increase in guanosine 3',5'-cyclic-monophosphate interferon treated mouse leukemia L1210 cells: relationship to the development of the antiviral state and inhibition of cell multiplication

Treatment of mouse leukemia L1210 cells with electrophoretically pure mouse interferon caused a rapid (1 to 5 min) and transitory (duration 5 to 10 min) increase in the intracellular concentration of guanosine 3′,5′-cyclic monophosphate (cyclic GMP). No significant increase in the intracellular concentration of adenosine 3′,5′-cyclic monophosphate (cyclic AMP) was observed until 2 to 3 hr after the addition of interferon. Electrophoretically pure mouse interferon caused a dose-dependent increase in the intracellular concentration of cyclic GMP at concentrations ranging from 1.0 to 10⁴ unite/ml. The interferon-induced increase in cyclic GMP was abrogated by specific antibody to mouse interferon. Furthermore, interferon had no effect on the intracellular concentration of cyclic GMP in a line of L1210 cells resistant to the action of interferon. The interferon-induced increase in cyclic GMP was calcium dependent and was inhibited by indomethacin and aspirin at concentrations of 10^?7 to 10^?6 M. Depletion of intracellular calcium prior to the addition of interferon abrogated the interferon-induced increase in cyclic GMP without preventing either the development of the antiviral state or the inhibition of cell multiplication in interferon-treated L1210 cells. These results suggest that cyclic GMP may not mediate these two biologic effects of interferon but may rather reflect rapid changes in interferon-treated cells.     

Properties of poly(I).poly(C)-induced mouse L cell interferon

Mouse interferon produced by L-929 cells after induction with poly(l) · poly(C) and DEAE-dextran can be separated into two molecular species, designated Mu IFN-α and Mu IFN-β on the basis of molecular weight and antigenic properties. We show here that Mu IFN-α and Mu IFN-β differ in antiviral activity on hamster and rat cells, stability, and pharmacokinetic character. The properties of both murine interferon species observed so far are in agreement with the idea that Mu IFN-α is the murine counterpart of human IFN-α and Mu IFN-β that of human IFN-β.     

Effect of tunicamycin on the physical properties and antiviral activities of murine L cell interferon

In the present study, we have examined the physicochemical, antigenic, and biological properties of murine L cell interferons produced in the presence of the glycosylation inhibitor, tunicamycin. Interferon produced by L cells treated with tunicamycin exhibited fourfold less antiviral activity on murine cells, but similar heterologous activity on human cultures when compared to interferon synthesized in the absence of the antibiotic. The antiviral activities of the interferons produced in the presence of tunicamycin were neutralized by specific anti-interferon sera to the same degree as control L cell interferon. A 17,000 molecular weight interferon was synthesized in the presence of tunicamycin, whereas two larger (43,000 and 26,000) interferon species were synthesized by untreated cultures. Isoelectric focusing of a control L cell interferon revealed considerable molecular microheterogeneity, whereas L cell interferon produced in the presence of tunicamycin focused as two discrete peaks (designated 1 and 2); only peak 2 was neutralized by antisera raised against human leukocyte interferon serum. These results suggest that two 17,000 molecular weight nonglycosylated interferon species were produced by murine L cells treated with tunicamycin, one of which possesses a common antigenic determinant(s) with interferon produced by human leukocytes.     

The mechanism of interferon induction in mouse spleen cells stimulated with HVJ.

This study showed that functional viral RNA and the penetration of virus into the cell are needed for interferon induction in L cells, while simple contact of the viral glycoprotein with the cell surface appears to be sufficient for interferon induction by the same HVJ in mouse spleen cells. Thirty minutes of uv irradiation resulted in complete loss of the interferon-inducing ability of HVJ in mouse L cells. In contrast to this result, HVJ irradiated for 2 hr could induce interferon in mouse spleen cells as efficiently as untreated HVJ. These findings showed that the actual inducer of interferon in mouse spleen cells was not viral nucleic acid, but some other viral component. When HVJ was treated with potassium periodate at 37° for 1 hr, infectivity for eggs and the hemolytic and neuraminidase activities of the virus were not detectable, but a considerable portion of its hemagglutinating activity was retained. The binding to erythrocytes of this inactivated HVJ, which showed no interferon-inducing ability in both L and mouse spleen cells, was restored in mouse spleen cells but not in L cells. The results indicated that hemolytic and neuraminidase activities are not essential for interferon induction in mouse spleen cells and that hemagglutinating activity might be closely related to interferon induction in the cells, although the presence of hemagglutinating activity alone is not sufficient for interferon induction in the cells. It seems that structural integrity of hemagglutinin on the erythrocyte surface may be important for interferon induction. HeLa cell-grown HVJ, which is characterized by its inability to penetrate into tissue culture cells, was found to stimulate interferon production in mouse spleen cells but not in L cells. This suggests that the process of virus penetration may be essential for induction of interferon in L cells. Interferon was produced in mouse spleen cells incubated with membranous particles with HVJ glycoproteins, but interferon activity could not be detected in the culture fluids of L cells. Aggregation of the glycoproteins by an antibody enhanced its interferon-inducing ability in mouse spleen cells. These results showed that the actual inducer of interferon in mouse spleen cells is not viral nucleic acid, but viral glycoproteins of HVJ, and that the size of its membranous structure is related to interferon inducibility. The mechanism of interferon induction by influenza virus in mouse spleen cells is similar to that of interferon induction by HVJ.     

Mechanism of interferon action. Kinetics of interferon action in mouse L929 cells: phosphorylation of protein synthesis initiation factor elF-2 and ribosome-associated protein P1.

Mice of different inbred strains varied in their immune response to endogenous leukemia viruses (MuLV). The ability of mice to produce antibodies against the gp70 proteins of xenotropic and ecotropic MuLV was to some extent dependent upon the level to which these mice naturally expressed their endogenous leukemia viruses. Three patterns of response were observed upon immunization with AKR leukemia cells: (1) Mice of low leukemic strains, which did not contain inducible ecotropic or xenotropic MuLV, produced antibodies that reacted with the gp70 proteins of both xenotropic and ecotropic MuLV; (2) mice of low leukemic strains, which contained low levels of ecotropic and inducible xenotropic MuLV, produced antibodies that reacted primarily with the gp70 proteins of ecotropic MuLV; and, (3) mice of high leukemic strains, which contained high levels of ecotropic and inducible xenotropic MuLV, failed to produce antibodies that reacted with the gp70 proteins of either xenotropic or ecotropic MuLV. Antibodies induced by immunization with leukemia cells reacted with type-specific antigenic determinants of the gp70 protein; by absorption analysis distinctive antigenic determinants could be identified on the gp70 proteins of BALB xenotropic, NZB xenotropic, and AKR ecotropic MuLV.     

Enzymatic activities induced by interferon in human fibroblast cell lines differing in their sensitivity to the anticellular activity of interferon

IF^r, a subline of transformed human fibroblast cells, which is sensitive to the antiviral but resistant to the anticellular activity of interferon, was found to be equally well inducible as its parental cell line RSa for the two major interferon-mediated double-stranded RNA-dependent enzymatic activities, 2–5A synthetase and 73K phosphoprotein kinase. The induction of 2-5A synthetase as a function of interferon dose, the specific activity of the 2-5A synthetase, the nature of the 2-5A oligonucleotide products, and the activity of the 2-5A-activated endonuclease were essentially the same for both cell lines. The 73K phosphoprotein kinase was induced at a similar rate of activity, whether detected in solution or after immobilization on poly(I)·poly(C)-Sepharose. Our observations thus suggest that the induction of these two enzymatic activities are not sufficient for the anticellular activity of interferon.     

Activation of endogenous type C virus by amino acid alcohols.

Amino acid alcohols were examined for their ability to inhibit protein synthesis and induce endogenous type C virus from Kirsten sarcoma virus (KiSV)-transformed Balb/c mouse cells. Histidinol and tyrosinol, amino alcohols of histidine and tyrosine, were very effective short-term activators of virus. Both inducers were very efficient inhibitors of protein synthesis reducing [³H]leucine incorporation by more than 90% in 1 hr. Two other alcohols, valinol and methioninol, also reduced protein synthesis but gave low activation. The activated virus had the host range of the xenotropic Balb virus:2, and following removal of inducer, the activated state decayed rapidly.     

Characterization of a subspecies of mouse interferon cross-reactive on human cells and antigenically related to human leukocyte interferon

Mouse interferon preparations from various virus-induced cell cultures exhibited some antiviral activity (about 0.1% of homologous cell titers) on human cells. When analyzed by SDS-polyacrylamide gel electrophoresis the activity of mouse interferon on mouse cells migrated as two bands with activity peaks at 38,000 and 22,000 daltons, while the activity on human cells was detectable only in the faster migrating portion of the lower molecular weight band, at about 21,000 daltons. When the mouse interferon preparation was chromatographed on a column of immobilized anti-human leukocyte interferon antibodies, the specifically retained interferon was found to migrate in SDS-polyacrylamide gels as a 21,000-dalton species which exhibited similar degrees of activity on both mouse and human cells. Thus, mouse interferon preparations contain a low molecular weight interferon species which is both active on mouse cells and is responsible for all the heterologous antiviral activity on human cells and is antigenically related to human leukocyte interferon. These data suggest significant structural similarities between the active cores of certain interferons from phylogenetically diverse animal species.     

Reactivation of DNA synthesis in aging diploid human skin fibroblasts by fusion with mouse L karyoplasts,cytoplasts and whole L cells

Diploid human skin fibroblasts derived from an 82-year-old donor with a 21–28 cell population doubling (CPD) range (where 28 CPD marked the end of the in vitro life span of the cells) were fused with whole L cells, L karyoplasts and L cytoplasts.The proportion of human nuclei incorporating tritiated thymidine after fusion was measured autoradiographically. Statistically significant increases in the labeling indices were found in the human nuclei in hybrid, heterodikaryon and cybrid cells when compared to control unfused human cells. Fusion of human diploid fibroblasts with human cytoplasts derived from cells of the same CPD showed no significant changes in the labeling indices of the human nuclei.     

High affinity binding of 125I-Labeled mouse interferon to a specific cell surface receptor. II. Analysis of binding properties

Direct ligand-binding studies with highly purified ¹²⁵I-labeled virus-induced mouse interferon on mouse lymphoma L 1210 cells revealed a direct correlation of specific high-affinity binding with the biologic response to interferon. Neutralization of the antiviral effect by anti-interferon gamma globulin occurred at the same antibody concentration as the inhibition of specific binding. These results suggest that specific high-affinity binding of ¹²⁵I-interferon occurred at a biologically functional interferon receptor. Competitive inhibition experiments using ¹²⁵I- and ¹²⁵I-labeled interferon provided strong evidence that the fraction of ¹²⁵I-interferon inactivated upon labeling did not bind specifically. Scatchard analysis of the binding data yielded linear plots and thus suggested that interferon binds to homogeneous noncooperative receptor sites. In contrast to a characteristic property of several peptide hormone systems, binding of ¹²⁵I-interferon to its specific receptor did not induce subsequent ligand degradation. At 37° bound interferon was rapidly released in a biologically active form without evidence for molecular degradation. The expression of interferon receptors was not modified by treatment with interferon. Trypsin treatment of target cells and inhibition of protein synthesis abolished the specific binding of ¹²⁵I-interferon. Three major molecular weight species of Newcastle disease virus-induced mouse C 243 cell interferon were isolated separately and identified as mouse α and β interferons. These interferons were shown to inhibit competitively the specific binding of the highly purified labeled starting material which contained all three size species, thus providing evidence for a common receptor site for mouse α and β interferon.     

Complete amino acid sequence of the basic nucleic acid binding protein of feline leukemia virus

The complete amino acid sequence of the nucleic acid binding protein p10 of the Rickard strain of feline leukemia virus (FeLV) has been determined. Fragments obtained by enzymatic digestion were purified by reverse-phase liquid chromatography and subjected to semiautomated Edman degradation. FeLV p10 is a basic polypeptide composed of 57 amino acids with Mr = 6604. The structure of p10 is compared to the structures of other retroviral nucleic acid binding proteins, and an analysis of a highly conserved region, the putative binding domain, is presented.     

Low interferon binding activity of two human cell lines which respond poorly to the antiviral and antiproliferative activity of interferon

Two human cell lines (HT1080 and MRC5) were found to be resistant to the antiproliferative effects of human interferon α. These cells showed also a reduced antiviral response, when virus yield was measured by a plaque assay. The binding of ¹²⁵I-labeled α interferon to HT1080 and MRC5 cells was greatly reduced, relative to another human cell line (A549) which responded well to the antiproliferative and antiviral activities of interferon. These results suggested that the poor response of HT1080 and MRC5 cells was due to the presence of fewer interferon receptors than in A459 cells. Some functional receptors, however, appeared to be present in HT1080 and MRC5 cells, since treatment with interferon resulted in a slight reduction in virus yield and in a modest increase in 2′,5′-oligo(A) polymerase activity.     

Specific high-affinity binding of 125I-labeled mouse interferon to interfevon resistant embryonal carcinoma cells in vitro

Multipotential mouse embryonal carcinoma cells are resistant to several biologic effects of mouse interferon: inhibition of viral multiplication and inhibition of cell division. Nevertheless using ¹²⁵I-labeled highly purified mouse interferon we have shown that these embryonal carcinoma cells express specific interferon receptors in similar number and of the same affinity as interferon sensitive differentiated embryonal carcinoma cells. Thus, mechanisms of interferon resistance are probably multiple: some cells are resistant because they lack specific binding sites for interferon (interferon resistant mouse L1210 cells) and other cells, as shown herein, are resistant to some of the effects of interferon despite binding of interferon to specific receptor sites. Furthermore, these binding sites may be considered functional, since interferon does induce an increase in 2-5A synthetase in these cells.     

Augmented glucose uptake and amino acid release by muscle underlying the burn wound and their moderation by ketone bodies

Glucose uptake and amino acid release by skeletal muscle from the burned and unburned regions of the body were compared in the presence or absence of ketone bodies. Rats were scalded on one hind limb and, 3 days later, soleus muscles from the burned and unburned limbs of burned rats as well as from controls were incubated in Krebs-Ringer bicarbonate medium with 10 mM glucose. Acetoacetate (5 mM) or β-hydroxybutyrate (10 mM) was included as necessary. In the absence of ketones, burned limb muscles took up, on the average, 85% (P < 0.002) more glucose and released 110% (P < 0.001) more alanine, 67% (P < 0.002) more glutamate, and 54% (P < 0.002) more glutamine than controls. The unburned limb muscles of burned rats usually did not differ from controls. Acetoacetate depressed glucose uptake in all groups of muscles by a comparable absolute amount, which represented a drop of 33% (P < 0.02) in burned limb muscles and 64% (P < 0.04) in both types of uninjured muscles. Alanine release was depressed about 15% (P < 0.03) in all muscle groups. β-Hydroxybutyrate produced depressions in glucose uptake and alanine release by both types of uninjured muscles but similar effects on burned limb muscles were not statistically significant. Neither ketone had any appreciable effect on glutamate or glutamine release by any muscle group. It is concluded that glucose uptake and the release of alanine, glutamate, and glutamine are chronically increased in skeletal muscle from the burned but not the unburned region of the rat. Both acetoacetate and β-hydroxybutyrate depress glucose uptake and alanine release by muscle remote from the injured region but only acetoacetate exhibited this effect in metabolically altered muscle underlying the burn wound.     

Mechanism of interferon action. Species specificity of interferon and of the interferon-mediated inhibitor of translation from mouse, monkey, and human cells.

The species specificity of interferon and of the interferon-mediated ribosome-associated inhibitor of translation from mouse fibroblast, African green monkey kidney, and human amnion cells was investigated. The following results were obtained: (1) The antiviral activity of mouse fibroblast, monkey kidney, and human leukocyte interferons was maximal on the homologous species of cells. Mouse interferon was not active on heterologous monkey cells, and monkey interferon was not active on mouse cells. Monkey and human interferons were almost equally active on heterologous human and monkey cells, respectively; mouse and human interferons were about 10 and 125 times more active, respectively, on their homologous cell species than on heterologous human and mouse cells. (2) Ribosomal salt-wash fractions prepared from interferon-treated mouse fibroblast, monkey kidney, and human amnion cells inhibited the translation of reovirus mRNA catalyzed by the mouse ascites cell-free protein-synthesizing system prepared from untreated ascites cells. The corresponding fractions prepared from untreated mouse and human cells either did not affect or stimulated the translation of reovirus mRNA. Ribosomal washes from untreated monkey kidney cells were inhibitory, but not as inhibitory as washes from interferon-treated cells. (3) The concentration of KCl required to release the interferon-mediated ribosome-associated inhibitor from ribosomes of mouse fibroblast cells was higher than that required for ribosomes from human amnion and monkey kidney cells. (4) The translation of both methylated and unmethylated reovirus mRNA catalyzed by the mouse ascites cell-free system was inhibited by the mouse fibroblast ribosome-associated interferon-mediated factor. These results suggest that the interferon-mediated ribosome-associated antiviral factor(s) is not species specific, even though certain interferons are relatively species specific.     

Induction of interferon in L cells by defective-interfering (DI) particles of vesicular stomatitis virus: Lack of correlation with content of [±] snapback RNA

The ability of defective-interfering (DI) particles of vesicular stomatitis virus (VSV) to induce interferon was studied in relation to the amount of snapback [±] double-stranded sequences in their RNA. Five DI particles propagated in BHK-21 cells were analyzed: two DI particles generated by undiluted passages of cloned wild-type VSV (Indiana); two DI particles generated by serial undiluted passages of culture fluid from L cells persistently infected with VSV; and DI-011, a DI particle with [±] snapback RNA, which is known to be a potent inducer of interferon. Induction of interferon in L cells by these DI particles was not proportional to the amount of [±] sequences in their RNA. DI-011 (26 to 37% [±] RNA sequences) induced a significant amount of interferon at a multiplicity of infection of one DI particle per cell. In contrast, the two DI particles from wild-type VSV (43 to 54% [±] RNA sequences) were 20- to 30-fold less efficient inducers of interferon than DI-011. Furthermore, the two DI particles (1 to 4% [±] RNA sequences) generated from L cell carrier cultures were only slightly less efficient inducers of interferon than the wild-type DI particles. The data also indicate that a population of DI particles which contains [±] RNA is not selected in L cells persistently infected with VSV.