Author index for volume 106, number 1

Treatment of mouse L929 cells with immune T (type II) interferon resulted in the induction of two double-stranded (ds) RNA-dependent enzymes which have been described previously in viral (type I) interferon-treated cells: pppA(2′p5′A)n synthetase and protein kinase(s). The induction of these enzymes by T interferon was blocked by anti-T but not by antiviral interferon serum. The kinetics of the development of the antiviral state along with a comparison of the levels of pppA(2′p5′A)n synthetase and protein kinase in T and viral interferon-treated cells were investigated. In addition, [³⁵S]methionine-labeled extracts from control, T, and viral inteferon-treated cells were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. Extracts from cells treated with either type of interferon were found to contain several identical proteins, of estimated molecular weights 60,000, 88,000, and 120,000, which were absent or found at much lower levels in preparations from control cells. The newly synthesized proteins can be partially purified by chromatography on columns of poly(I)·poly(C)-Sepharose, thus providing a convenient method for their further characterization. These results show that treatment of cells with T interferon results in the induction of intracellular events identical to those observed in viral interferon-treated cells.     

Interferon externalization by producing cell before induction of antiviral state.

The mechanism by which poly(I · C) induces the antiviral state in human fibroblast cells was studied using antisera which were selectively reactive with the inducer [poly(I · C)], the product (human fibroblast interferon), and the fibroblast cell surface. Anti-interferon serum completely neutralized the antiviral effect of human fibroblast interferon. Furthermore, when added to the medium of the cells in which interferon synthesis had already been induced by poly(I · C), interferon antiserum prevented the intracellular antiviral state from developing. Antibodies specific to double stranded RNA inhibited the antiviral activity of poly(I · C) whether it was in solution or bound to the cells, but the same antibodies were without effect when added after the induction of the antiviral state was initiated. Treatment with serum directed against the cell surface of human fibroblasts failed to inhibit the antiviral activity of poly (I · C) or of human interferon in these cells. These results indicate that the antiviral effect of poly(I · C) is interferon mediated, and that for the development of the antiviral state, interferon has to interact with the external part of the cellular membrane of the producing cell.     

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.     

Comparative analysis of interferon and antiviral protein messenger RNAs.

A comparative analysis of interferon and antiviral protein messenger RNAs was carried out. Differences in their biological activities and sedimentation coefficients were found. In RNA preparations from superinduced cells (cells treated with poly(I).poly(C) and antimetabolites) and from cells treated with interferon, messenger RNAs possesing interferon and antiviral activities were detected. The results suggest the existence of two types of mRNA (for interferon and antiviral protein, respectively) and support the hypothetic model of interferon action via an antiviral protein.     

A protein synthesizing system from interferon-treated cells that discriminates between cellular and viral messenger RNAs

The effect of treating Krebs II ascites tumor cells with interferon on the ability of extracts prepared from them to catalyze the translation of a variety of messenger RNAs was investigated. The following results were obtained: (1) Extracts of untreated cells translated endogenous mRNA, as well as exogenously added synthetic mRNA [poly(U)], cellular mRNA (Krebs cell and L cell), and viral mRNA (reovirus and vaccinia virus). By contrast, extracts of Krebs cells treated in the ascitic form with interferon translated endogenous mRNA, exogenously added cellular mRNA and poly(U) as efficiently as extracts of untreated cells, but they translated viral mRNAs very poorly (less than 10% as efficiently as extracts of untreated cells). Thus, the ability to discriminate between cellular and viral mRNAs that is characteristic of whole cells was also exhibited by these cell-free extracts. (2) Virus infection was not required for the development of the interferon-induced inhibition of viral mRNA translation. (3) Mixing experiments indicated that the inability of extracts of interferon-treated cells to catalyze the translation of viral mRNAs was due to the presence of an inhibitory factor (s) rather than to the absence of a required factor (s). (4) The inhibitory factor (s) was associated with ribosomes, and could be dissociated from them by washing with KC1. (5) Polyacrylamide gel electrophoresis revealed the presence of a 48,000 dalton polypeptide in the 0.3–0.6 M KC1 wash fraction of ribosomes prepared from interferon-treated cells that was not detectable in the corresponding wash of ribosomes prepared from untreated cells. This fraction inhibited the translation of viral mRNAs in cell-free extracts of untreated cells more than any other salt wash fraction.These results suggest that the antiviral activity of interferon is mediated, at least in part, by a ribosome-associated polypeptide that permits discrimination between cellular and viral mRNAs.     

Interferon induces an antiviral state in ganglioside-deficient transformed mouse fibroblasts

The antiviral activity of mouse fibroblast interferon against vesicular stomatitis virus was investigated in L-929 mouse fibroblasts and the ganglioside-deficient L-929 mutant cells (ATCC clone NCTC 2071). Although it has been widely reported that gangliosides serve as primary receptors for interferon at the cellular membrane, only a small difference in interferon sensitivity was observed between the wild-type L-929 and the ganglioside-deficient NCTC 2071 cells. It was not possible, however, to overcome this difference by administration of exogenous gangliosides.     

p67K kinase in different tissues and plasma of control and interferon-treated mice

Interferon-treated mouse cells show an enhanced level of protein kinase activity which is manifested by the phosphorylation of an endogenous 67,000-molecular weight protein (p67K kinase). This kinase activity can be assayed efficiently after its partial purification on poly(I) · poly(C)-Sepharose. We have previously shown that the p67K kinase is present in the liver, spleen and plasma (heparinized) of mice with high levels of circulating interferon. Here we confirm these results by treatment of mice with interferon and furthermore show that besides the liver and spleen, the level of p67K kinase is enhanced in several other tissues such as thymus, brain, pancreas, heart, and lung. The action of interferon in mice was further monitored by the assay of pppA(2′p5′A)n synthetase (2–5A synthetase) in different tissues. The level of 2-5A synthetase was enhanced several fold in the following tissues: heart, pancreas, thymus, liver, and spleen. The detection of 2–5A synthetase and p67K kinase activities in the different tissues of mice provides suitable markers for the response of each individual tissue toward treatment with interferon. The phosphorylated 67K protein (pp67K) from control and interferon-treated mouse L-929 cells and from the plasma and different tissues of control and interferontreated mice was characterized by two-dimensional gel electrophoresis. The isoelectric point (pl) of pp67K from the different tissues and L-929 cells was 8 to 8.5. On the other hand the pI of pp67K from the plasma had a range of 7.5 to 8. These results indicated that the presence of p67K kinase in the plasma of mice is not due to lysis of tissue 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.     

Target-Organ Treatment of Neurotropic Virus Disease with Interferon Inducers

Interferon inducers were used against vaccinial encephalitis to study the target-organ treatment of neurotropic disease and to correlate interferon levels and the antiviral state following such treatment. A 45-mug amount of statolon, 30 mug of polyribinosinic-polyribocytidylic acid complex (poly I.poly C), or 0.0154 HA unit of Sendai virus given intracerebrally protected 100% of mice challenged the next day with 1,000 median lethal doses (LD(50)) of vaccinia virus. Significant protection against 1,000 LD(50) of vaccinia virus persisted for 1, 4, or 3 weeks after poly I.poly C, statolon, or Sendai virus (154 HA units), respectively. These doses of poly I.poly C and statolon were also used to study postinfection treatment. Mice challenged with 1, 10, 100, or 1,000 LD(50) were treated intracerebrally with poly I.poly C or statolon 24 or 48 hr later. Significant increases in survival time were seen in mice challenged with 1 to 100 LD(50) of vaccinia virus and treated 24 hr later. At challenges of 10 or 100 LD(50), statolon was more effective than poly I.poly C in increasing survival times. When treatment was delayed until 48 hr after infection, significant increases in survival time occurred only when the challenges were in the range of 1 to 10 LD(50), with poly I.poly C and statolon being equally effective. Interferon was measured by Finter's dye-uptake method, with L-929 cells and Semliki Forest virus. Poly I.poly C, statolon, or Sendai virus, given intracerebrally to mice, produced serum interferon peaks of 5,120 units/ml at 2 hr, 2,560 units/ml at 12 hr, or 320 units/ml at 18 hr, respectively. Corresponding brain interferon peaks were 640 units/g at 2 hr, 640 units/g at 4 to 24 hr, and 960 units/g at 72 hr.     

Interferon induction with Newcastle disease virus in FS-4 cells: Effect of 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB)

Summary DRB is an inhibitor of heterogeneous nuclear RNA (hnRNA) and messenger RNA (mRNA) synthesis. The effect of DRB on interferon production stimulated by Newcastle disease virus (NDV) in the human FS-4 cells was studied. Interferon production in cells primed by treatment with interferon was markedly enhanced (superinduced) in the presence of DRB. This superinduction was essentially due to an inhibition of the rapid decline (shutoff) of interferon production observed in primed cells not treated with DRB. Continuous presence of DRB was required for maximal superinduction. In this and other respects the interferon response induced by NDV in primed cells resembled poly(I) · poly(C)-induced interferon production. In contrast interferon production in cells not primed with interferon was virtually abolished by DRB treatment. Since neither virus specific RNA synthesis nor virus replication were significantly affected by DRB, the inhibition of interferon production is likely to result from the inhibitory action of DRB on a cellular, rather than viral, function. Apparently some differences exist in the synthesis or processing of the mRNAs for interferons in primed and unprimed cells and these determine the different sensitivities of these two responses to DRB.With 2 Figures     

Studies on the Mechanism of the Priming Effect of Interferon on Interferon Production by Cell Cultures Exposed to Poly(rI) · Poly(rC)

Interferon induction by poly(rI).poly(rC) in primary rabbit kidney and mouse L-929 cell cultures was markedly increased if the cells were previously treated with homologous interferon. This priming effect has been established with different times of exposure of the cells to poly(rI).poly(rC), and was most pronounced for short pulses of contact of the polynucleotide with the cells (10 s, 1 min). Treatment of the cells with pancreatic ribonuclease immediately after their exposure to poly(rI).poly(rC) brought about a relatively greater reduction of the interferon response in interferon-primed cells than it did in unprimed cell cultures. Priming of the cells with interferon did not increase cell-binding of poly(rI).poly(rC), whether this cell-binding was measured quantitatively (by radioactivity, upon exposure of the cells to radiolabeled polymer) or qualitatively (by antiviral activity, by assaying the cell extract for virus plaque reduction). Similarly, interferon priming did not alter the sensitivity of cell-associated poly(rI).poly(rC) to extraneous ribonuclease treatment. Finally, priming with interferon did not decrease the rate of degradation of cell-bound poly(rI).poly(rC) by cellular nucleases nor did it increase the anti-nuclease potency of the cells. The exact mechanism by which previous exposure of the cells to interferon enhances subsequent interferon production, induced by either synthetic polynucleotides or viruses, has not yet been resolved.     

Nonspecificity of action of antiviral protein

The antiviral activity of antiviral protein messenger RNAs (AVP mRNA) isolated from cells after the superinduction of interferon in them was studied in order to ascertain the specificity of action of their translation product — AVP. After injection of AVP mRNA a marked decrease (from 1 to 5 log PFU/ml) in the infectious titers was observed in both homologous and heterologous cells. RNA preparations from control (uninduced) cells possessed weak (0.4–0.1 log PFU/ml) antiviral activity.D. I. Ivanovskii Institute of Virology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. M. Zhdanov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 85, No. 6, pp. 720–721, June, 1978.     

Toll-like receptor 3 agonist poly(I:C)-induced antiviral response in human corneal epithelial cells

The objective of this study was to examine the expression of Toll-like receptor 3 (TLR3) by human corneal epithelial cells (HCECs) and to determine whether exposure to the TLR3 agonist polyinosinic-polycytidylic acid [poly(I:C)] induces an antiviral response in these cells. Fluorescence-activated cell sorter (FACS) analysis revealed TLR3 to be constitutively expressed and distributed intracellularly in HCECs. Stimulation of HCECs with the TLR3 agonist poly(I:C) induced the activation of nuclear factor (NF)-kappaB and production of the proinflammatory cytokine interleukin (IL)-6 and the chemokine IL-8. Upon exposure to poly(I:C), HCECs initiated a potent antiviral response resulting in an increase of interferon (IFN)-beta mRNA expression (7-fold). Poly(I:C) stimulation also up-regulated mRNA expression of the antiviral chemokine IFN-gamma inducible protein 10 (IP10), myxovirus resistance gene A and 2',5'-oligoadenylate synthetase (5-, 10- and 9-fold, respectively), and secretion of IP10. These responses were also induced by exogenously added type 1 IFNs, but could not be blocked by pretreatment of the cells with anti-TLR3 monoclonal antibody, suggesting that the receptor was not expressed on the cell surface. Furthermore, incubation of HCECs with an endosomal acidification inhibitor, chloroquine, markedly inhibited poly(I:C)-mediated IFN-beta expression in HCECs. These results suggest that corneal epithelial cells are important sentinels of the corneal innate immune system against viral infection, and that stimulation of TLR3 can induce the expression of key proinflammatory cytokines and chemokines and antiviral genes that help in the defence of the cornea against viral infection.     

Effect of serum on the antiviral and anticellular activities of mouse interferon

Summary Fetal bovine serum markedly decreased the ability of mouse L-929 interferon preparations to inhibit the formation of L-929 clones, but did not affect their ability to inhibit vesicular stomatitis virus (VSV) plaque formation in these cells. This dissociation of effects by interferon preparations indicates that: 1. the mechanism of action of interferon for its anticlonal and antiviral activities is different; or 2. the molecule responsible for the anticlonal activity is a separate growth inhibitory factor.With 2 Figures     

Interferon Induction by Poly(I):Poly(C) Enclosed in Phospholipid Particles

Liposomes were prepared with phospholipids (sphingomyelin, lecithin, and phosphatidylethanolamine) in combination with cholesterol and charged lipids (dicetyl phosphate and stearylamine) and contained either poly(I):poly(C) or poly(I). Neutral and positively charged liposomes attached much better to L-929 cells in tissue culture than did negatively charged particles. Liposomes were toxic to L cells at relatively low concentrations, making the determination of antiviral activity induced by particles containing poly(I):poly(C) difficult to measure by the plaque reduction assay. When injected into mice, all of the liposomes containing poly(I):poly(C), except phosphatidylethanolamine liposomes, greatly potentiated and extended the serum interferon response of poly(I):poly(C). Lecithin and sphingomyelin liposomes given intravenously were ten times more effective than free poly(I):poly(C) in stimulating production of serum interferon. Sphingomyelin liposomes containing [(14)C]poly(I):poly(C) were 88% cleared from the bloodstream of mice by 3 min after intravenous injection. Most of the radioactivity (70%) was captured by the liver and remained there for at least 4 h. By 2 h, 7% of the radioactivity could be found in the spleen. Five percent of the radioactivity was found in the lungs at 30 min, with decreasing amounts thereafter. Small amounts of radioactivity were found in the muscle and kidneys. The spleen was shown to contain appreciable levels of interferon at 4 h, and low levels were found in the liver. Radioactivity accumulated slowly in the liver following an intraperitoneal injection of sphingomyelin liposomes containing [(14)C]poly(I):poly(C). By 4 h, 26% of the dose was recovered from the liver and 4.9% from the spleen, with small amounts in the lung, kidney, and omentum.     

Virus-inhibitory effect of a yeast RNA-tilorone molecular complex in cell cultures.

The virus-inhibitory activity of a molecular complex (MC) of tilorone and yeast RNA was studied in vitro on three virus-cell systems: vesicular stomatitis virus (VSV) - murine fibroblast L929 cells, Venezuelan equine encephalittis virus (VEEV) - swine embryo kidney (SEK) cells and encephalomyocarditis virus (EMCV) - established piglet testicular (EPT) cells. In all these systems the MC exerted an antiviral effect similar to that of polynucleotide interferon (IFN) inducers such as poly(I)-poly(C), larifan and ridostin. The antiviral effect of the MC was similar when the compound was applied before or after virus adsorption to cells. The MC may be regarded as a perspective antiviral agent of common use.     

Comparison of the effects of rabies virus infection and of combined interferon and poly(I).poly(C) treatment on the levels of 2',5'-adenyladenosine oligonucleotides in different organs of mice

Intracellular levels of 2',5'-adenyladenosine oligonucleotides were analyzed in different organs of mice during the course of a rabies virus infection. Phosphorylated and nonphosphorylated 2',5'-adenyladenosine oligonucleotides were measured by radioimmunoassay and analyzed further by HPLC. As the infection progressed, concentrations of phosphorylated 2',5'-adenyladenosine oligonucleotides increased strongly, reaching their maxima late in the infection. In contrast, concentrations of the nonphosphorylated 2',5'-adenyladenosine oligonucleotides decreased. A similar phenomenon was observed in spleens analyzed at intervals after treatment of noninfected mice with interferon and poly(I).poly(C) and to a lesser extent after treatment of noninfected mice with interferon and poly(I).poly(C) and to a lesser extent after treatment with poly(I).poly(C) alone, but not after treatment with interferon alone. The products which accumulated during virus infection were primarily phosphorylated dimers whereas during combined interferon and poly(I).poly(C) treatment, the entire range of phosphorylated molecules from dimer to pentamer was present. These data show that infection of mice with rabies virus provokes both the induction and the activation of 2-5A synthetase, as does interferon and poly(I).poly(C) treatment. However, our data indicate that the intracellular products are different in the two situations: the species active on the nuclease were only detected in interferon- and poly(I).poly(C)-treated mice. The absence of molecules able to activate the 2-5A-dependent nuclease in virus-infected mice might well be one of the reasons why the interferon system is ineffective in rabies virus infection.     

Combined action of a specific vaccine, exogenous interferon and an interferon inducer on the development of acute experimental encephalomyelitis

Comparative studies of the effectiveness of specific vaccine, exogenous interferon, and interferon inducer poly I:C in mice experimentally infected with human acute encephalomyelitis (HAE) virus showed the interferon inducer poly(I) X poly(C) to exert the highest protective activity (up to 72.5% protection). Irrespective of the inoculation route (intraperitoneal, intramuscular, intravenous), the vaccine in combination with interferon or interferon inducer poly(I) X poly(C) produced a considerably higher level of protection of mice (78.5%) against fatal infection with HAE virus than each of the preparations used alone. Studies of the humoral and cellular immunity reactions showed that increased protection levels after the combined use of interferon inducer and vaccine were due to interferon production and activation of the mechanisms of cellular antiviral immunity.     

Effect of amphotericin B on the activity of synthetic polyribonucleotide interferon inducers

The authors investigated the intensity of interferon production and the degree of the associated antiviral resistance induced by double-stranded synthetic polyribonucleotide interferon inducers poly(G) . poly(C), poly(G, I) . poly(C) and poly(G, A) . poly(C) in chick embryo cell cultures, continuous diploid human fibroblasts and in mice in the presence of amphotericin B, a macrolide polyene antibiotic enhancing the permeability of plasma membranes for macromolecules. Amphotericin B was found to increase considerably the interferon-inducing and antiviral activity of the above polyribonucleotide complexes in those cell systems where they induced interferon production and antiviral resistance alone, without the antibiotic, but to a lower degree. Amphotericin B did not contribute to the activity of the complexes in those cell systems where they were inert alone. The importance of permeability of the plasma membrane for interferon induction is discussed, and a conclusion is reached that the inertness of the complexes under study in one cell system and their activity in the others are not associated with differences in the permeability of the plasma membranes of different cells for polyribonucleotide interferon inducers.     

Biological actions and therapeutic perspectives of double stranded polyribonucleotides: a reappraisal]

Double-stranded polynucleotides, which are composed of two complementary homopolyribonucleotides containing no genetic information, are synthetic molecules capable of mimicking the action of natural double-stranded RNA or viral RNA on cells. Double-stranded polyribonucleotides act as an alarm system alerting the cell to the presence of an external aggression, e.g. a viral attack. In addition, polyribonucleotides have a more active function in that they trigger cell defense processes through activation of a family of genes, of which some encode cytokines, activation of cytoplasmic enzymes involved in antiviral mechanisms or signal transduction, and activation of nonspecific immune responses. Double-stranded polyribonucleotides containing one mismatched base pair per helix have been found to be especially interesting. The best known example is poly(I).poly(C12U), also called ampligen. Poly(I).poly(C12U) is capable, in experimental models, of limiting the development of viruses (including HIV), reducing tumor growth, eliminating metastases, and, according to one report, preventing steady declines in T-cell counts in HIV-positive patients. Therapeutic doses used in the USA as an experimental drug induced little toxicity. In vitro, poly(I).poly(C12U) acts synergistically with interferon, interleukin 2 or AZT, suggesting that these latter drugs may be effective in lower, less toxic doses when used in combination with poly(I).poly(C12U). The therapeutic activity of poly(I).poly(C12U) holds promise. More extensive prospective studies of this agent are warranted.