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Vesicular stomatitis virus (VSV) is a negative-strand RNA virus with intrinsic oncolytic specificity due to substantially attenuated antiviral responses in many tumors. We have recently reported that recombinant VSV vector can be used as an effective oncolytic agent to safely treat multifocal hepatocellular carcinoma (HCC) in the livers of immune-competent rats via hepatic artery infusion. When administered at doses above the maximum tolerated dose (MTD), however, the animals suffered from neurotoxicity and/or acute lethal hepatotoxicity. Since VSV is extremely sensitive to the antiviral actions of alpha/beta interferon (IFN-alpha/beta) in normal cells, we tested if prophylactic treatment with rat IFN-alpha would enhance VSV safety without compromising treatment efficacy in tumor-bearing rats. We found that VSV retained its replication potential in human and rat HCC cells after preincubation with relatively high doses of rat and human IFN-alpha in vitro, and its MTD in tumor-bearing rats treated systemically with rat IFN-alpha at 66 IU/g body weight (BW), equivalent to a human IFN-alpha dose that is currently prescribed for patients with viral hepatitis, was elevated by more than 1/2 log unit. Furthermore, we demonstrate that intratumoral replication of VSV was not attenuated by administration of 66 IU/g BW rat IFN-alpha, as tumor response and survival advantage in VSV-treated rats in the presence or absence of rat IFN-alpha were equivalent. The results suggest that prophylactic rat IFN-alpha treatment elevates the therapeutic index of hepatic arterial VSV therapy for multifocal HCC in rats. Since human IFN-alpha is currently in clinical use, its prophylactic application should be considered in future clinical translational protocols for VSV-mediated oncolytic virotherapy as a novel therapeutic modality in patients with advanced HCC, as well as other types of cancer.

Interferons (IFNs) in common with other cytokines activate Janus tyrosine kinases and latent STAT transcription factors upon binding to their cell surface receptor. Type I IFNs bind to a receptor composed of two transmembrane polypeptides, IFNAR1 and IFNAR2, which belong to the class II cytokine receptor family that also includes the cellular receptors for IFN-gamma, interleukin-10 and coagulation protease factor VII (tissue factor). The extracellular domain of the type I IFN receptor chain IFNAR1, has four fibronectin type-III sub-domains. Human IFNAR1 has intrinsic weak affinity for type I IFNs and plays an essential role in transmembrane signaling, formation of a high affinity complex with IFN and the modulation of ligand specificity. In order to characterise the ligand binding site on IFNAR1 we analysed the epitope recognized by the anti-IFNAR1 mAb, 64G12, which inhibits the binding and biological activities of both IFN-alpha and IFN-beta. The target peptide recognized by the 64G12 mAb was determined by screening a set of 48 overlapping peptides covering the first two subdomains (residues 23-229) of the extracellular region of IFNAR1. The results of this study show that the peptide (FSSLKLNVY), localized within the first sub-domain (residues 89-97) of IFNAR1, which is recognized by the 64G12 mAb, most likely overlaps a site to which both IFN-alpha and IFN-beta bind in the ligand-receptor complex. Thus, since the 64G12 mAb can neutralize the biological activities of all the type I IFNs tested, we suggest that the target peptide recognized by the 64G12 mAb, is a possible anchorage point on IFNAR1, common to binding of both IFN-alpha and IFN-beta.

To avoid possible uncertainty in comparing biological activities of interferon samples from different sources where interferon concentrations were determined independently, we prepared chromatographically pure preparations of consensus interferon and interferon-alpha-2b (one of the two commercially available recombinant alpha interferons). We revealed that consensus interferon has a stronger antiviral activity than interferon-alpha-2b, although the effects of these two recombinant interferons on the cellular macromolecule synthesis are at similar levels.

Interferon-tau (IFNtau) is the pregnancy recognition signal of bovine embryos, inhibiting luteolysis. We studied trophoblastic growth and IFNtau secretion of embryos with different developmental potential, i.e., in vivo derived and in vitro produced embryos, cloned embryos and demi-embryos, to evaluate if the ability of secreting IFNtau might be responsible for differences in pregnancy rates after transfer of these categories of embryos to recipients. Day 8 embryos of excellent quality were individually placed in microdrops of buffalo rat liver cell-conditioned medium and maintained for up to 23 days. Embryos were observed on Days 11, 15, 19 and 23, the mean diameter (2r) of attached and spherical embryos was measured, and their trophoblastic area was calculated as r2pi or 4r2pi, respectively. Simultaneously, medium was changed and the IFNtau levels of conditioned media were determined using a bioassay of antiviral activity. Trophoblastic area was smaller (P < 0.05) in demi-embryos than in all other groups, which exhibited similar trophoblastic growth until Day 19. However, on Day 23 trophoblastic area of in vivo derived embryos was more than twice (P < 0.05) as large as those of in vitro produced and nuclear transfer (NT) embryos. IFNtau levels increased only slowly with time in culture of demi-embryos. By contrast, the level of IFNtau doubled from Day 11 to Day 15 in conditioned media from all other groups of embryos. The linear increase in IFNtau production of vivo and in vitro derived embryos continued until the end of the culture period, whereas conditioned media from NT embryos contained significantly (P < 0.05) less IFNtau activity on Days 19 and 23 than those of the former two groups. Our results demonstrate different capabilities of secreting IFNtau for in vivo derived and in vitro produced embryos vs. NT and demi-embryos, which may--at least part--be responsible for the differences in pregnancy rates after transfer to recipients.

The expression of type I interferons (IFNs) in eukaryotic cells represents a first line of defense against viral infection. Cells pretreated by IFNs do not support viral replication and are protected from virus-induced cell destruction. A challenge of IFN-pretreated cells with vesicular stomatitis virus (VSV) is frequently used to quantitate this cytokine because, on the one hand, the replication of VSV is highly sensitive to IFNs and, on the other hand, in unprotected cells this virus induces a rapid cytopathic effect that can readily be quantified. However, as VSV may infect humans and is known to cause severe disease in a variety of animal species, this virus must be considered a biohazard. In this paper, we describe a bioassay for bovine IFN using Sendai virus, a paramyxovirus that grows readily in MDBK cells yet is released from these cells in a non-infectious form. The sensitivity and dynamic range of this assay are similar to those of the popular VSV-based IFN assay. We demonstrate that the Sendai-virus-based IFN assay permits rapid quantitation of recombinant bovine type I IFN, and also of native type I IFNs which are present in the supernatants of monocyte-derived macrophages infected with various pathogens. In view of the possible artifacts induced by viruses in samples to be assayed for IFN activity, we evaluated several methods of virus inactivation. Treatment with beta-propiolactone led to virus inactivation without affecting the bioactivity of IFNs as detected in the Sendai-virus-based assay.

Three murine monoclonal antibodies (mAb) directed to distinct epitopes on recombinant human interferon (IFN)-alpha 1, and three mAb recognizing distinct epitopes on recombinant human interferon (IFN) alpha 1, and three mAb recognizing distinct epitopes on recombinant human IFN-alpha sc, were studied by IFN-neutralizing assays. The efficacy of neutralization of the anti-viral and the anti-proliferative activities of IFN-alpha 1, or IFN-alpha 2c, by the specific antibodies used, individually or in combination, were evaluated. In comparison with single mAb, the mixtures of three mAb against IFN-alpha 1 or three mAb against IFN-alpha 2c were capable of neutralizing more than 10-times larger amounts of IFN-alpha 1 and alpha 2c, respectively. The strong potentiation of the neutralization efficacy resulting from mixing different mAb was demonstrated by neutralization of the anti-viral as well as the anti-proliferative activities of both recombinant IFN. The neutralization experiments support the interpretation that the observed potentiation results from simultaneous interaction of anti-IFN mAb with different epitope specificity.

Phospholipid/Ca2(+)-dependent protein kinase (protein kinase C; PKC) appears to be involved in the signal-transduction pathway mediated by human leukocyte interferon (IFN) in HeLa cells. IFN treatment results in a rapid increase in [3H]phorbol 12,13-dibutyrate binding to intact cells, indicating an activation of PKC. In addition, inhibitors of PKC (H7 and staurosporine) block the induction of antiviral activity by IFN against vesicular stomatitis virus. PKC inhibitors also block the accumulation of IFN-stimulated mRNAs in the cytoplasm of HeLa cells and suppress the transcriptional induction of IFN-stimulated genes. Activation of IFN-stimulated genes is mediated through a DNA response element that is necessary and sufficient for the transcriptional response to IFN. IFN treatment induces the appearance of several DNA-binding factors that specifically recognize the response element, and the appearance of these factors is suppressed by PKC inhibitors. This observation provides evidence that PKC activity is involved during IFN-stimulated signal transduction. Although activation of PKC appears to be required for the response to IFN, agonists of PKC activity alone do not turn on expression of IFN-stimulated genes.

The early events that occur after interferon binds to discrete cell surface receptors remain largely unknown. Human leukocyte interferon (interferon-alpha) rapidly increases the binding of [3H]phorbol dibutyrate to intact HeLa cells (ED50 = 100 units/ml), a measure of protein kinase C activation, and induces the selective translocation of the beta isoform of protein kinase C from the cytosol to the particulate fraction of HeLa cells. The subcellular distribution of the alpha and epsilon isoforms is unaffected by interferon-alpha treatment. Activation of protein kinase C by phorbol esters mimics the inhibitory action of interferon-alpha on HeLa cell proliferation and down-regulation of protein kinase C blocks the induction of antiviral activity by interferon-alpha in HeLa cells. Increased phosphatidylcholine hydrolysis and phosphorylcholine production is accompanied by diacylglycerol production in response to interferon. However, inositol phospholipid turnover and free intracellular calcium concentration are unaffected. These results suggest that the transient increase in diacylglycerol, resulting from phosphatidylcholine hydrolysis, may selectively activate the beta isoform of protein kinase C. Moreover, the activation of protein kinase C is a necessary element in interferon action on cells.

Theoretical and experimental data were obtained indicating a possible role of the amino acid sequence 69-80 of HuIFN-alpha for antiviral activity of interferon. In this paper, synthetic peptides (penta-, octa- and dodecapeptides), representing the sequence region of positions 69-80 of the primary structure of HuIFN-alpha-2 were investigated with regard to their ability to inhibit the cytopathic effect of VSV on MDBK cells and on embryonic human fibroblasts. The peptides themselves exhibit only a very weak antiviral activity; however, it was found that these peptides even increase the antiviral activity of HuIFN-alpha-2.

The antiviral activity of recombinant DNA-derived bovine alpha 1(-1) interferon on an established swine testicular cell line and primary testicular cell cultures derived from swine of various ages (2 days, 3 weeks, and 5 weeks) was determined. Bovine interferon induced a dose-dependent increase in 2-5A synthetase in testicular cells, regardless of the source of the cells. Furthermore, interferon inhibited replication of vesicular stomatitis virus to an equivalent extent in all testicular cell cultures. The results indicate that 2-5A synthetase is a reliable marker of interferon activity in swine testicular cell cultures and that the induction of 2-5A synthetase and antiviral effects of recombinant bovine interferon in primary testicular cell cultures are not dependent on the age of the donor animal.

Meclomen (sodium meclofenamate) is a nonsteroidal anti-inflammatory drug (NSAID) with dual inhibition of both cyclooxygenase and lipooxygenase enzymes involved in arachidonic acid metabolism. Meclomen at nontoxic levels (10(-5) to 10(-8) mol/l) caused a 2-6 fold increase in interferon (IFN) production in lymphocytes induced by Sendai virus. This enhancement was observed in the case of all blood donors tested. Meclomen at the same concentration range had no effect on the antiviral activity of human IFN alpha. However, meclomen did show some enhancement of antiproliferative activity of human IFN alpha against human adenocarcinoma lung cells. Meclomen (10(-5) mol/l) inhibited intracellular levles of prostaglandin E2 by over 90% within the first four hours. The results presented here strongly suggest that inhibition of intracellular levels of PGE2 has no adverse influence upon the antiviral and antiproliferative activities of human IFN alpha.

The interferon receptors of C3H/10T1/2 mouse cells respond differently to alpha and beta interferons under certain conditions. If C3H/10T1/2 cells which have been maintained in logarithmic growth phase are exposed to trypsin or Pronase immediately before they are treated with mouse interferons, they evince an antiviral response to alpha interferon but not to beta interferon. In contrast, contact-inhibited C3H/10T1/2 cells, L-929 mouse cells or human HEL cells lost the ability to respond to both alpha and beta interferons after treatment with trypsin or Pronase. When L-929 cells are incubated at 37 degrees C following exposure to these proteolytic enzymes, they completely regain their ability to respond to mouse beta interferon within 2 h. These observations suggest that the receptors for alpha and beta interferons are different in their topographical distribution in C3H/10T1/2 cells.

The interaction of the interferons (IFNs) that cooperate to potentiate the antiviral action of IFN was studied. Serial dilutions of MuIFN-gamma and MuIFN-alpha/beta were employed separately and in combination to block virus replication in one-step, virus yield reduction experiments. To calculate the potentiation of IFN activity, protection levels obtained for each combination of MuIFN-gamma and MuIFN-alpha/beta were compared with those obtained for the separate IFNs. Potentiation levels increased with increasing concentrations of each of the IFNs in a dose-dependent manner, suggesting that potentiation of IFN's antiviral activity was the result of the mutually synergistic interaction of the IFNs. Three challenge viruses were employed: Mengo virus (positive-strand RNA virus), vesicular stomatitis virus (negative-strand RNA virus), and vaccinia virus (DNA virus). Identical results were observed with the three different viruses, suggesting that mutual synergism was a basic feature of the potentiation of IFN's antiviral activity by combined preparations of MuIFN-gamma and MuIFN-alpha/beta.

Two cell lines derived from human choriocarcinomas (HCCM-5 and BeWo) are resistant to several biological effects of human interferon such as inhibition of VSV multiplication and inhibition of cell growth, but they develop a normal antiviral activity against EMCV. Nevertheless, in both cell lines, 2-5A synthetase and protein kinase are induced by IFN. 2-5A-dependent endonuclease can be measured by two independent methods and 2-5A itself is detected at least in poly(rI):poly(rC)- and IFN-treated BeWo cells. This is another example of two cell lines that are partially, with respect to the antiviral effect toward VSV, and totally, with respect to the anticellular effect, refractory to IFN treatment, although all the known elements of the 2-5A system are present.

A murine osteosarcoma (OGS) cell line was permanently infected with Newcastle disease virus (NDV). The presence of NDV in the subsequent passages was demonstrated by haemadsorption and by immunofluorescence. Compared to the uninfected OGS line the persistently infected cells had a slightly reduced growth rate and they had a reduced sensitivity to several viruses, shown by several different methods. Interferon sensitivity was considerably reduced in the OGS-NDV cells, both the antiviral activity and the cell multiplication inhibitory activity.

The inhibition of virus replication and the induction of protein phosphorylation were examined in human amnion U and human fibroblast GM2767A cells treated with highly purified cloned human leukocyte and immune interferons synthesized in Escherichia coli. Both leukocyte interferon (IFN-alpha A) and immune interferon (IFN-gamma) possessed antiviral activity as measured by the single cycle yield reduction of vesicular stomatitis virus (VSV) in the human U and GM2767A cell lines. By contrast, only IFN-gamma and not IFN-alpha A inhibited the single cycle replication of reovirus in U and GM2767A cells. IFN-alpha A, but not IFN-gamma, efficiently induced the double-stranded RNA-dependent phosphorylation of the ribosome-associated protein P1 and the alpha subunit of protein synthesis initiation factor eIF-2 in U cells. However, neither IFN-alpha A nor IFN-gamma induced the phosphorylation of P1 and eIF-2 alpha in GM2767A cells. The antiviral activities of IFN-alpha A and IFN-gamma were synergistic for the inhibition of VSV but not for the inhibition of reovirus or the induction of protein phosphorylation. These results suggest that human leukocyte and immune interferons differentially regulate the expression of certain genes and induce mechanistically distinct antiviral states in human cells.

Among the pleiotropic effects of human interferon are the inhibition of viral replication, the activation of natural killer cells, and the inhibition of cellular growth. Oxyphenbutazone, a nonsteroidal antiinflammatory agent, is a potent inhibitor of the antiviral activity of human alpha and beta interferons as determined by cytopathic effect and vesicular stomatitis virus synthesis and release in human foreskin fibroblasts. The inhibition of interferon activity is dose dependent with maximal inhibition at 25-50 microM and minimal inhibition at 1 microM. In contrast, oxyphenbutazone at concentrations as high as 100 microM has no effect on the activation of natural killer cells by human interferon. Similarly, oxyphenbutazone has no inhibitory effect on interferon-induced antigrowth activity in the human breast carcinoma cell line MDA-MB-231. This cell line is sensitive to oxyphenbutazone inhibition of interferon-induced antiviral activity in vitro. In another human cell line, the vulvar carcinoma A431, oxyphenbutazone apparently augments the antigrowth activity of interferon. Although oxyphenbutazone inhibits the fatty acid cyclooxygenase enzyme in these systems, other inhibitors of cyclooxygenase fail to inactivate the antiviral activity of human interferon. Thus, oxyphenbutazone appears to inhibit the interferon antiviral cascade at a site distinct from prostaglandin biosynthesis. Moreover, the failure to inhibit natural killer cell activation or cellular antigrowth effects of human interferon suggests a pathway different from that associated with the antiviral effect of human interferon.

The sensitivity of caprine synovial membrane cells to the antiviral effects of natural and recombinant DNA-derived human interferons (HuIFN) was compared with that of human foreskin fibroblast (FS7), ovine choroid plexus, and bovine turbinate cells. Caprine cells were found to be more sensitive (P less than 0.01) to natural HuIFN-alpha than human, ovine, and bovine cells. The sensitivity of caprine cells to recombinant DNA-derived HuIFN-alpha was equivalent to that of ovine cells, but greater than human or bovine cells. The sensitivity of caprine cells to natural and recombinant DNA-derived HuIFN-beta was equivalent to human cells, but less than that of ovine cells.

Vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) multiply in only a small percentage of peritoneal macrophages freshly explanted from 4- to 6-week-old male or female DBA/2, BALB/c, C3H, C57BL/6, or Swiss mice. However, when these mice were injected intraperitoneally with potent sheep (or goat) anti-mouse interferon alpha/beta globulin 4 days prior to harvesting peritoneal macrophages, the viruses multiplied to high titers and most of the cells were infected, as determined by total virus yield (VSV and EMCV), percentage of VSV antigen-positive cells (immunofluorescence), and determination of VSV infectious centers. This effect was not observed when mice were inoculated with other sheep hyperimmune or normal serum globulins. Anti-interferon globulin appeared to act in vivo because incubation of this globulin with peritoneal macrophages during the period of cell attachment or during the 18 hr after virus absorption did not render these cells permissive for VSV. Injection of mice with anti-interferon globulin did not affect the binding and uptake of labeled VSV by peritoneal macrophages. Although the underlying mechanism of this phenomenon is unknown, the results suggest that there may be low levels of endogenous interferon that contribute to host defense by maintaining some cells in an antiviral state.

Four natural murine interferon-alpha genes (MuIFN-alpha 1, -alpha 2, -alpha 4 and -alpha 6) and four hybrid genes (alpha 1 alpha 4, alpha 2 alpha 4, alpha 4 alpha 1 and alpha 4 alpha 2) were transiently expressed in monkey COS cells under the transcriptional control of the simian virus 40 early promoter. The proteins were labelled with [35S]methionine during a 16 h incubation and proteins secreted by the cells during this period were separated by polyacrylamide gel electrophoresis and subsequently visualized by fluorography. Under the conditions used, the IFNs represented 5 to 10% of the total amount of secreted proteins. All genes were found to encode biologically active IFN subspecies, including alpha 4 which has a deletion of five amino acids. When the specific activities of the proteins were compared, it appeared that the specific antiviral activity of alpha 4 on mouse cells was three- to sixfold higher than the activities of the other natural IFN subspecies. The specific activities of the hybrid proteins were similar to those of the natural proteins, except for the alpha 2 alpha 4 hybrid which had a higher specific activity than the original proteins. The ability of the natural and hybrid subspecies to protect hamster cells against viral infection was determined using MuIFN-alpha 1 as a standard. Large differences in activity were found, with alpha 6 as the most and alpha 4 as the least active subspecies.

Three major subtypes of human interferon-alpha (IFN-alpha), isolated from virus-induced leukocytes, were compared for their antiviral and anticellular activities on one hand, and for their ability to induce (2'-5') oligoadenylate synthetase on the other hand. One subtype, IFN-alpha 1, was found to have low specific antiviral (6.10(6)-5.10(7) units/mg) and anticellular activities when measured on a variety of human cells. A second subtype, exhibiting an unusually high molecular weight (26,000) by SDS-polyacrylamide gel electrophoresis (IFN-alpha 26K), was found to have the highest known specific antiviral (8.10(8)-2.10(9) units/mg) and anticellular activities. Thus, these two subtypes of IFN-alpha differ by a factor of 330 and represent the two extremes in the antiviral scale on human cells. A third subtype, IFN-alpha 2, was tested as well and was found to have intermediate antiviral and anticellular activities. The ability of these three subtypes to induce (2'-5') oligoadenylate synthetase in human cells was then measured. It was found that on a weight basis, the three subtypes were equally effective in inducing the enzyme. Since the level of (2'-5') adenylate oligomers is affected also by the interferon-induced (2'-5') phosphodiesterase, the ability of these subtypes to induce this enzyme was compared as well and was found to be very similar. We therefore conclude that the differences in potency between these IFN-alpha subtypes are not related to their ability to induce (2'-5') oligoadenylate synthetase.

We evaluated 2'-5'Oligoadenylate (2-5 A) synthetase assay for pharmacokinetic study of human interferon (IFN) in cynomolgus monkeys. The enzyme was induced in primary cultures of cynomolgus monkey kidney (PMK) cells as well as in FL cells in response to human recombinant IFN-alpha A treatment. The enzyme activity increased with IFN dose and, in parallel with the enzyme elevation, developed the antiviral state of the cells. The enzyme activity induced in the peripheral blood lymphocytes peaked at 6 to 12 hr after iv or im administration. The peak level of the enzyme activity depended on the IFN concentration of the blood and the activity rapidly decreased as serum IFN was cleared from the blood. These results indicate that human recombinant IFN-alpha A induces 2-5 A synthetase in monkey cells both in vitro and in vivo, and that the enzyme assay can be used to quantitatively monitor the host response after IFN administration.

Interferon (IFN) level in the sera of normal humans was determined by their protective effect on a KB cell culture against the cytopathic effect (CPE) induced by vesicular stomatitis virus. The technique revealed all the three known types of IFN. In the investigated population 15 of 37 sera (40%) were found to protect against the CPE at 1/10 dilution, 11 of 37 (30%) still protect at 1/20 dilution, and 7 of 37 sera (19%) still protect at a dilution of 1/40. No serum was found to have detectable IFN levels at 1/80 dilution. We can thus consider that the normal serum level ranges up to a 1/20 dilution i.e., up to 20 IU/ml. The above titers are slightly higher than the normal IFN level reported by other investigators in other human populations, although no clear reasons can be given to account for the differences.

Recombinant bovine interferon-alpha and -gamma differ in their action against influenza virus on bovine cells. Bovine IFN-alpha severely impairs early protein synthesis and replication of influenza virus in bovine cells in contrast to bovine IFN-gamma which fails to induce an antiviral state against influenza virus. Otherwise the IFN system seems to function normally in bovine cells since both bovine IFN-alpha and -gamma induce an antiviral state against vesicular stomatitis virus. The establishment of the specific antiviral state against influenza virus correlates with the induction by bovine IFN-alpha, but not -gamma, of two cytoplasmic proteins related to the IFN-induced mouse protein Mx involved in the mechanism of resistance of mice to influenza virus infection. This study suggests that bovines possess a system for resistance to influenza virus similar to the mouse Mx system.

The effects of human alpha and beta interferons (IFN) on the production of HBsAG by PLC/PRF/5 cells, an HBsAg-producing human hepatoma cell line, were studied in the exponential and stationary phases of cell growth. When exponential phase cells were treated with 100 or 1,000 U of IFN per ml for 48 hr. the amount of HBsAg in the culture medium decreased. The number of cells and the synthesis of DNA and proteins were also reduced by the IFN treatment. These results suggested that IFN did not affect the production of HBsAg specifically in exponential phase cells. When cells in the stationary phase were similarly treated with IFN, HbsAg production was not inhibited nor did the number of cells decrease. To examine the antiviral state induced by IFN in PLC/PRF/5, induction of 2'5'-oligo (A) synthetase and susceptibility to two kinds of viruses were examined. The 2'5'-oligo (A) synthetase activity was increased in an IFN-dose dependent manner. Susceptibility to vesicular stomatitis virus (VSV) and encephalomyocarditis virus (EMCV) was decreased by treatment with 10 and 100 U of IFN per ml for 20 hr. It was concluded that IFN-alpha and IFN-beta induce 2'5'-oligo (A) synthetase and the antiviral state, but do not inhibit HBsAg production by PLC/PRF/5 cells.

Vesicular stomatitis virus and encephalomyocarditis virus do not multiply in the majority of peritoneal macrophages freshly explanted from 4- to 8-week-old male or female mice. However, when peritoneal macrophages were cultivated in vitro for 3 to 5 days, these cells became permissive for both viruses. The loss of antiviral state in "aged" macrophages paralleled a significant decrease in the intracellular levels of (2'-5')oligo-adenylate synthetase activity. Although biologically active interferon was not detected in the nutrient medium of macrophage cultures, freshly harvested peritoneal cells could confer an antiviral state on monolayer cultures of mouse cells (aged macrophages, embryonic fibroblasts, and L cells) but not on heterologous chicken embryo, rabbit kidney, or human cells infected with vesicular stomatitis virus or encephalomyocarditis virus. The conferred antiviral state required at least 7 h to develop in target cells and was totally inhibited by the presence of antibody to mouse interferon alpha/beta but not to interferon gamma in the cocultures. Heterologous guinea pig and rabbit peritoneal cells could not transfer an antiviral state to target mouse cells. Donor peritoneal cells from mice preinjected with antibody to interferon alpha/beta could not transfer an antiviral state to target mouse cells. This ensemble of results indicating that freshly harvested peritoneal cells transfer interferon (which is responsible for inducing an antiviral state in susceptible mouse target cells) adds further experimental evidence that interferon is spontaneously expressed in normal mice and plays an important role in maintaining some host cells in an antiviral state.

Treatment of HeLa cells with human lymphoblastoid interferon (IFN-alpha) does not inhibit reovirus type 3 protein synthesis during virus infection. In contrast, reovirus translation is blocked by treatment of L cells with mouse IFN-alpha. The (2'-5')A synthetase activity is induced in HeLa cells by IFN-alpha treatment and is activated after reovirus infection, since cell lysates from these cells synthesize in vitro (2'-5')A oligonucleotides. The IFN-induced protein kinase activity is also triggered in those lysates upon dsRNA addition. Thus, contrary to DNA-containing viruses, such as vaccinia virus or adenovirus, reovirus infection does not destroy or reverse the IFN-induced antiviral state. In support of this conclusion, superinfection with poliovirus or vesicular stomatitis virus of reovirus-infected HeLa cells treated with IFN leads only to a blockade of translation of the former viruses. These results provide a remarkable example where in the same cells doubly infected with two different viruses, the antiviral state induced by IFN-alpha is manifested by selectively inhibiting translation of one kind of virus (poliovirus or vesicular stomatitis virus) without affecting the translation of reovirus type 3. In addition, these results indicate that the resistance of reovirus translation to inhibition by IFN is different from the mechanism of resistance induced by DNA-containing viruses.

Human beta-interferon (HuIFN-beta) exhibits antiproliferative and antiviral properties. Successful clinical application of this drug depends on knowledge of the thermal stability of these activities under physiological conditions. In the present study, both the antiproliferative and antiviral activities were stabilized by the addition of very small quantities of serum proteins. This supplement was sufficient to mask the slightly higher thermosensitivity of the antiviral activity. In the absence of serum proteins, the values of both the half-life and the energy of activation were higher for the antiproliferative activity than for the antiviral function. Each had a half-life of at least 24 h and identical values for the energy of activation in the presence of proteins furnished by 1% fetal bovine serum. This study provides additional evidence to support a thesis recently advanced by Carter et al. that the antiproliferative domain of glycosylated beta interferon may be separable from the antiviral domain. It is concluded that the efficacy of HuIFN-beta, under clinical conditions, will not be seriously impaired by thermal inactivation. Antiviral assays of serum may be freely substituted for antiproliferative assays during pharmacological studies.

Human adenovirus type 5 (Ad5) is a DNA virus which replicates as efficiently in human A549 cells treated with human interferon-alpha 2 (IFN) as in untreated cells. Vesicular stomatitis virus (VSV), on the other hand, is a negative-strand RNA virus which is very sensitive to the effects of IFN treatment in A549 cells. The IFN-mediated inhibition of VSV replication was not observed in cells coinfected with Ad5. Abrogation of IFN-mediated antiviral activity was maximal when Ad5 infection preceded VSV infection by at least 36 h, but did not require adenovirus DNA synthesis for manifestation. Coinfection experiments with VSV and deletion variants of adenovirus demonstrated that neither virus-associated RNA synthesis nor expression of adenovirus early regions E1B, E2A, E3, or E4 are required for abrogation of IFN-mediated inhibition of VSV replication. However, expression of early region E1A was essential, suggesting that E1A products can modulate, either directly or indirectly, IFN activity in adenovirus-infected cells.

An in vitro microtitration system of the antiproliferative effect of human leucocyte interferon (IFN-alpha(Le)) was investigated. The preliminary experiments suggested that the antiproliferative effect of IFN-alpha(Le) was increased by prolonging the incubation period, by reducing the target cell concentration, and might be prescribed by the total IFN amounts in the challenge medium. By employing a system consisted of 1.5 X 10(4) Daudi cells in 0.2 ml of medium containing IFN dilutions and an incubation period of 3 days at 37 degrees C, the antiproliferative effects of twenty-one lots of partially purified IFN-alpha(Le) (PIF-alpha(Le)) preparations were titrated. The interassay variations in the antiproliferative titers of three PIF-alpha(Le) established in the present system were found to be in the same range as those in the antiviral titers estimated by a standard macroplaque reduction assay. The each titration curve was parallel, and the antiproliferative titers, assessed by the reciprocals of the IFN dilutions which suppress the cell growth in 50%, were significantly (p less than 0.01) correlated to the antiviral international units of them.

The allele Mx regulates the extent to which interferon alpha/beta inhibits the growth of influenza viruses in mouse cells such as peritoneal macrophages. The time course of induction of the antiviral state against an influenza A virus is comparable in macrophages with and without Mx and is similar to that found with vesicular stomatitis virus. In contrast, the decay of the antiviral state against influenza virus is markedly slower in Mx-positive cells and slower than that against vesicular stomatitis virus observed in either Mx-positive or Mx-negative cells. Thus, after removal of interferon alpha/beta, Mx-positive cells remain protected against influenza virus at times when they have lost protection against vesicular stomatitis virus. These results suggest that interferon alpha/beta treatment activates different antiviral mechanisms, each acting against distinct groups of viruses and each independently controlled by host genes.

Human interferon-gamma was more effective than interferon-beta or -alpha in stimulating production of immunoassociated antigens; HLA-A, -B, and -C; and beta(2)-microglobulin in human M14 and Namalva cells. The comparison was made on the basis of antiviral units, and the stimulation could be abolished by treatment of the interferon-gamma preparation with pH 2 or anti-interferon-gamma serum.

Interferon (alpha, beta and gamma) susceptibility was tested in a human cell line, UVr-1, a u.v. light-resistant variant of RSa cells; the latter have high sensitivity to both u.v. lethality and the cell proliferation inhibition (anticellular) effect of human interferon (HuIFN) preparations. UVr-1 cells were less sensitive than the parental RSa cells to the inhibitory effects of HuIFN preparations, as measured by cell proliferation and the incorporation of [3H]deoxythymidine and [3H]deoxyadenosine into acid-insoluble cellular material. Nevertheless, UVr-1 cells exposed to HuIFN showed almost the same enhanced levels of antiviral activity and pppA(2'p5'A)n synthetase activity as similarly treated RSa cells. Further, UVr-1 cells had much the same binding capacity for 125I-labelled HuIFN-alpha A. Thus, it seems likely that the variant has an increased resistance to the anticellular effect but not to the antiviral effect of HuIFN preparations. UVr-1 cells showed no significant difference from RSa cells in u.v.-induced DNA repair synthesis. However, when a comparison was made between the susceptibility of normal fibroblasts and fibroblasts from patients with Cockayne's syndrome, characterized by an altered u.v. sensitivity but no alteration of DNA repair replication synthesis, the Cockayne's syndrome fibroblasts, CCK-3 and CCK-4, were more susceptible to HuIFN-beta as judged by cell proliferation and deoxythymidine incorporation tests.

We showed previously that the mouse fibroblastoid cell line Ltk-aprt- is resistant to the antiviral effects of beta interferon. This lack of response reflects a partial sensitivity to the interferon that is accompanied by a failure to activate expression of several interferon-regulated genes, although certain other genes respond in a normal manner. We show here that Ltk-aprt- cells were also unable to establish an antiviral state and to activate expression of 2,5-oligo(A) synthetase when treated with gamma interferon. Strikingly, however, treatment with a combination of beta interferon and gamma interferon provided complete protection against viral replication. Although the cells were completely insensitive to up to 250 U of the interferons per ml added singly, essentially complete protection from viral cytopathic effects was achieved when as little as 10 U of each of the interferons per ml were combined. Expression of 2,5-oligo(A) synthetase was also sensitive to this synergistic effect. Activation of an antiviral state could also be achieved by sequential treatment, first with gamma interferon and then with beta interferon. Partial protection against viral replication could be achieved by pretreatment with gamma interferon for as little as 1 h before incubation with beta interferon and could be blocked by the addition of specific antibodies or by cycloheximide, indicating that gamma interferon induces the synthesis of a protein which can act synergistically with a signal produced by the beta-interferon receptor. We suggest that Ltk-aprt- cells suffer from defects in one or more components of the gene activation pathways for both type I and type II interferons. Nonetheless, gamma interferon is able to activate the expression of a gene encoding a protein required for signal transduction. This protein acts synergistically with a transient signal produced in response to beta interferon, thereby activating the expression of a further group of genes.

Two human recombinant lymphoblastoid interferon-alpha subtypes, LyIFN-B (alpha 8) and LyIFN-D (alpha 1), and 10 hybrids generated therefrom were produced in Escherichia coli and purified. The antiviral and antiproliferative activities and the induction of (2'-5')oligoadenylate synthetase were compared to their receptor binding affinities. The IFN subtypes and their hybrids had similar specific antiviral activities on bovine cells. On human cells both the specific antiviral and antiproliferative activities of LyIFN-B were about 30-fold higher than those of LyIFN-D. This difference in activity could be attributed partly to the N-terminal amino acids 1 to 60 and partly to amino acids 61 to 92. A third domain affecting the biological activities was found within the carboxy-proximal segment from amino acids 93 to 150. The differences in these activities were found to correlate with their ability to bind the receptor, suggesting that the differences in activity might be due to altered binding of the IFNs to the cellular receptors. In contrast, the induction of (2'-5')oligoadenylate synthetase did not follow the same activity profile. On mouse cells, the efficiency of the hybrids was affected by at least four sites on the IFN protein. A hybrid with the N-terminal segment 1 to 60 from IFN-B and amino acids 61 to 166 from IFN-D had a specific antiviral activity on mouse cells as high as on human cells corresponding to a 500- and 5000-fold increase in specific activity compared to IFN-D and IFN-B, respectively. We suggest that on mouse cells the IFN activity may be more dependent on conformational differences than on human cells, which in turn might reflect a less precise fit to the mouse receptor than to the human receptor.

Interferon (IFN) treatment inhibited the replication of vesicular stomatitis virus (VSV) in human GM2767 and mouse JLSV-11 cells. The replication of this virus in either human RD-114 or mouse A402 cells was insensitive to IFN treatment. We analyzed various steps in the VSV life cycle as they occurred under different conditions of IFN treatment to identify the point(s) at which IFN was exerting its inhibitory effect. IFN treatment led to strong inhibition of viral protein synthesis and accumulation of viral RNA in both lines of IFN-sensitive cells. No such effect was observed in the IFN-resistant cells. Using a temperature-sensitive mutant (tsG41) and wild-type VSV that were not undergoing protein synthesis, we determined that the major site of action of IFN against VSV replication in JLSV-11 and GM2767 cells was at the level of primary viral transcription. The accumulation of primary viral transcripts was strongly inhibited in these cells by IFN treatment. This effect was not a consequence of any effect of IFN on virus entry and uncoating. Thus, it appears that IFN exerts a direct effect on the VSV transcriptional process in GM2767 and JLSV-11 cells.

Interferons, in addition to their antiviral activity, induce a multiplicity of effects on different cell types. Interferon (IFN)-gamma exerts a unique regulatory effect on cells of the mononuclear phagocyte lineage. To investigate whether the antiviral and antiproliferative effects of IFN-gamma in macrophages can be genetically dissociated, and whether IFN-alpha and IFN-gamma use the same cellular signals and/or effector mechanisms to achieve their biologic effects, we have derived a series of somatic cell genetic variants resistant to the antiproliferative and/or antiviral activities of IFN-gamma. Two different classes of variants were found: those resistant to the antiproliferative and antiviral effects of IFN-gamma against vesicular stomatitis virus (VSV) and those resistant to the antiproliferative effect, but protected against VSV and encephalomyocarditis virus (EMCV) lysis by IFN-gamma. In addition, a third class of mutants was obtained that was susceptible to the growth inhibitory activity, but resistant to the antiviral activity of IFN-gamma. Analysis of these mutants has provided several insights regarding the regulatory mechanisms of IFN-gamma and IFN-alpha on the murine macrophage cell lines. The antiproliferative activity of IFN-gamma on these cells, in contrast to that of IFN-alpha, is mediated by a cAMP-independent pathway. The antiproliferative and antiviral activities of IFN-gamma were genetically dissociated. Variants were obtained that are growth resistant but antivirally protected, or are growth inhibited but not antivirally protected against VSV or EMCV. The genetic analysis indicated that IFN-alpha and IFN-gamma regulate the induction of the dsRNA-dependent P1/eIF-2 alpha protein kinase and 2',5'-oligoadenylate synthetase enzymatic activities via different pathways. Finally, a unique macrophage mutant was obtained that was protected by IFN-gamma against infection by VSV, but not EMCV, suggesting that antiviral mechanisms involved in protection against these different types of RNA viruses must be distinct at some level.