Expression of antiviral activity and induction of 2',5'-oligoadenylate synthetase by conceptus secretory proteins enriched in bovine trophoblast protein-1

Establishment of pregnancy in cattle has been proposed to depend on production of a conceptus protein, bovine trophoblast protein-1 (bTP-1), which has a high degree of sequence homology with bovine interferon-alpha (bIFN-alpha), especially the alpha II subfamily. A preparation of bovine conceptus secretory proteins enriched for bTP-1 has antiviral and physico-chemical properties similar to other bIFN-alpha. Antiviral activity is initially detectable in uterine flushings on Day 14 of pregnancy, when the conceptus measures 4-5 mm in length, and increases as the conceptus elongates through Day 18. Day 17 conceptuses produce more than 10(6) U antiviral activity during 24 h of culture. All IFNs induce the enzyme 2',5'-oligoadenylate synthetase, which catalyzes production of 2',5'-oligo(A), which in turn is involved in antiviral and growth inhibitory effects of IFNs. This enzyme activity is induced in Madin-Darby bovine kidney cells by the partially purified bTP-1 preparation similarly to IFN-alpha, -beta, and -gamma. Likewise, the partially purified bTP-1 and bIFN-alpha 1 induce 2',5'-oligo(A) synthetase activity in monolayers of endometrial epithelial and stromal cells. Compared to epithelial cells, stromal cells have higher baseline activity of 2'-5'-oligo(A) synthetase activity (p less than 0.01) and show a greater degree of induction in the presence of either the partially purified bTP-1 or bIFN-alpha 1 (p less than 0.01). Also, 2',5'-oligo(A) synthetase of endometrial stromal cells is induced to a greater degree by our enriched bTP-1 preparation than by bIFN-alpha 1 (p less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)     

Porcine conceptus proteins: antiviral activity and effect on 2',5'-oligoadenylate synthetase.

Interferon-like proteins synthesized by conceptuses of domestic ruminants inhibit luteolysis during early pregnancy. Although pig conceptuses secrete trophoblast interferons during the period of CL maintenance, estrogen is involved with maintenance of the CL. The principal purposes of this work were to confirm production of trophoblast interferons by porcine conceptuses and to compare the effect of trophoblast interferons on endometrium of pigs and cattle. When measured using Madin-Darby bovine kidney (MDBK) cells challenged with vesicular stomatitis virus, antiviral activity in uterine flushings from cyclic gilts was not detectable throughout the estrous cycle; however, in pregnant gilts, antiviral activity increased from undetectable amounts to 4-11 x 10(3) U on Days 14, 16, and 18. Porcine embryos in culture produced 1,100 U/embryo/ml/24 h. Porcine conceptus secretory proteins induced 2',5'-oligo(A) synthetase in MDBK cells and in endometrial explants of cows but had no measurable effect on 2',5'-oligo(A) synthetase activity of endometrial explants of pigs. Similarly, endometrial 2',5'-oligo(A) synthetase of pregnant pigs was unaffected in vivo during the period of maximal synthesis of conceptus secretory proteins. Porcine conceptus secretory proteins produced no detectable increase in serum antiviral activity or 2',5'-oligo(A) synthetase activity of blood mononuclear leukocytes in utero-ovarian venous blood. These results suggest that conceptus interferons of pigs play different roles in the establishment of pregnancy compared to their roles in ruminants.     

Lectins modulate the internalization of recombinant interferon-alpha A and the induction of 2',5'-oligo(A) synthetase

After binding to specific cell surface receptors, interferon-alpha (IFN-alpha) along with its receptor is internalized by the cells. However, the physiological significance of the internalization of IFN is not known. We have found that the lectin concanavalin A (ConA), which does not inhibit the binding of 125I-rIFN-alpha A, inhibits both the internalization of 125I-rIFN-alpha A and the rIFN-alpha A-induced increase in the levels of 2',5'-oligo(A) synthetase mRNA and enzymatic activity in the B lymphoblastoid cell line Daudi. The reduced level of IFN-induced 2',5'-oligo(A) synthetase in ConA-treated cells was due neither to direct inhibition of the enzymatic activity nor to generalized inhibition of protein or RNA synthesis. The dose-response curves were similar for the effect of ConA to inhibit 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction. The correlation between the ConA-mediated inhibition of both 125I-rIFN-alpha A internalization and 2',5'-oligo(A) synthetase induction suggests that internalization of rIFN-alpha A plays a role in the responses to rIFN-alpha A. However, since ConA inhibits protein mobility in the plasma membrane, it is possible that ConA is also preventing aggregation of IFN receptors or interactions between IFN receptors and signal transducing proteins in the plasma membrane that may be necessary for responses to IFN.     

VIP induces in HT-29 cells 2'5'oligoadenylate synthetase and antiviral state via interferon beta/alpha synthesis.

Vasoactive intestinal peptide (VIP), composed of 28 amino acids, is a multifunctional neurotransmitter. We have demonstrated here that its action on human transformed colonic epithelial (HT-29) cells is mediated through the induction of interferon (IFN) synthesis. We have found that these cells have a functional receptor for IFN alpha 2; binding was specific to either IFN alpha 2 or IFN beta but not to IFN gamma. VIP induced the 2'5'oligoadenylate synthetase (2'5'A synthetase) and the antiviral state with the same efficiency as poly (I).poly (C). The induction of 2'5'A synthetase activity required cellular RNA and protein synthesis, and the maximum induction occurred with 10(-7) M VIP at 24 h. VIP, like some IFN inducers, induced the synthesis of the 70 hsp which, however, preceded the expression of 2'5'A synthetase. VIP treatment caused the induction and secretion of IFN, having a titer value of 32 international units/ml. This IFN has been identified as type beta/alpha, because both 2'5'A synthetase and the antiviral activities were abolished by anti-human IFN beta/alpha antibodies, but not by anti-IFN gamma antibodies. Thus the pathway of VIP action on HT-29 cells may be outlined as 1) binding of VIP, 2) synthesis of 70 hsp, 3) induction of IFN synthesis and its secretion, 4) binding of the secreted IFN to cell surface receptors and 5) turning on the induction of 2'5'A synthetase and antiviral activities.     

Antiviral activity of lambda-, beta-, and gamma-interferons in the presence of theophylline: involvement of 2',5'-oligo(A) synthetase

Theophylline, an inhibitor of cAMP phosphodiesterase, induces in human ovary carcinoma cells (CaOv) a 2-2.5-fold elevation of intracellular cAMP. This rise in the cAMP level is followed by an increase of the activity of 2',5'-oligo(A) synthetase in CaOv cells -insignificant (1.5-fold) after 16 hr incubation, and substantial (3.7-fold) after 30 hr incubation, as well as the development of antiviral resistance. Once CaOv cells have been incubated with the mixtures containing theophylline (2 mM) and lambda-, beta-, and gamma-interferon preparations (0.5-13 IU/ml), the total antiviral effect of the mixtures exceeds that generated by interferon or theophylline separately; the action of the above agents being additive. These data agree with the previously obtained results and support the suggestion that cAMP phosphodiesterase inhibitors partially mimic the antiviral action of interferon.     

Four different forms of interferon-induced 2',5'-oligo(A) synthetase identified by immunoblotting in human cells

Antibodies against synthetic peptides derived from the cDNA sequence of interferon-induced 2',5'-oligo(A) synthetase, and which immunoprecipitate the native enzyme activity, were found to detect multiple enzyme forms in denaturing electrophoretic immunoblots. In some human cell lines, four different interferon-induced proteins of 40, 46, 67, and 100 kDa were found to react with the same peptide antibodies. Each isolated form was shown to have 2',5'-oligo(A) synthetase activity, but the dependence on double-stranded RNA was markedly different for activation of the individual enzymes. The four enzyme forms also differ in their intracellular localization, on microsomes (100 kDa), in nuclei (67, 46, 40 kDa), and on membrane structures (67 kDa). Plasma membranes from interferon-treated Daudi lymphoblastoid cells are highly enriched in the 67-kDa 2',5'-oligo(A) synthetase form. The 2',5'-oligo(A) synthetase activity induced by interferons in human cells appears, therefore, as a complex multienzyme system.     

Initial characterization of a spontaneous interferon secreted during growth and differentiation of Friend erythroleukemia cells

A gradual increase in the level of 2',5'-oligoadenylate synthetase takes place in Friend erythroleukemia cells after a shiftdown in the rate of cell growth. The increase is about 5-fold after entry of cells into the stationary phase of growth, but much higher (25-fold) when reduction in growth accompanies cell differentiation. In the latter case, the enzyme increase is similar to that which can be induced in these cells by exogenous interferon (IFN). The increase in 2',5'-oligoadenylate synthetase was shown to be due to a spontaneous secretion of IFN by the cells themselves: it is completely abolished if antiserum to murine type I IFN is added to the culture medium. In attempts to isolate some of this spontaneously secreted IFN, we show that it is stable at pH 2, not neutralized by antiserum to type II IFN, and that it also differs from the known IFN species induced by Sendai virus in Friend cells. The major component of this spontaneously secreted IFN is 20,000 M(r) and differs from the corresponding virus-induced 20,000-M(r) IFN by its lower affinity for antiserum to type I IFN and its antigenic characterization as beta-murine IFN. The major component of the spontaneous IFN also exhibits a higher ratio of antigrowth to antiviral activity than the Sendai-induced IFNs. We suggest that Friend cells produce this specific type of IFN for the regulation of their growth and differentiation.     

Ethanol induces 2',5'-oligoadenylate synthetase and antiviral activities through interferon-beta production.

We demonstrate here that ethanol, in contrast to heat shock (Chousterman, S., Chelbi-Alix, M.K., and Thang, M.N. (1987) J. Biol. Chem. 262, 4806-4811), induces interferon (IFN) synthesis and its related activities in Madin-Darby bovine kidney (MDBK) cells. The induced IFN is secreted maximally at 6 h, whereas the induction of 2',5'-oligoadenylate synthetase mRNA peaks between 9 and 12 h and its activity at 15 h. The appearance of both 2',5'-oligoadenylate synthetase activity and the antiviral state upon ethanol treatment is prevented by anti-bovine recombinant IFN-beta antibodies. Bovine diarrhea virus infection-free MDBK cells cultured in medium supplemented with serum substitute also gave similar results, thus indicating that IFN synthesis induced by ethanol is not mediated by the activation of bovine diarrhea virus. Together, these results show that: 1) ethanol induces the 2',5'-oligoadenylate synthetase and antiviral activities through IFN-beta production; and 2) the IFN produced does not act directly from inside the cells, but has to be first secreted to bind to its receptor. In MDBK cells, ethanol induces the synthesis of the 70-kDa protein, which precedes the expression of 2',5'-oligoadenylate synthetase; moreover, the transient nature of the synthesis of the hsp 70 in these cells is similar after both heat shock and ethanol treatment.     

Cyclosporin A inhibits the production of gamma interferon (IFN gamma), but does not inhibit production of virus-induced IFN alpha/beta

The effect of cyclosporin A (CsA) on the production of gamma interferon (IFN gamma) versus IFN alpha/beta was studied using mouse and human lymphocytes and fibroblasts. Spleen cells from C57Bl/6 mice produced low but significant levels (40-60 U/ml) of IFN gamma after 2 to 3 days of culture with irradiated DBA spleen cells. The addition of CsA at concentrations as low as 0.1 microgram/ml completely inhibited (less than 10 U/ml) IFN gamma production in these cultures. High levels of IFN gamma (170-1200 U/ml) were produced when either C57Bl/6 spleen cells or Ficoll-Hypaque-purified human peripheral blood lymphocytes (PBL) were cultured with the T-cell mitogen staphylococcal enterotoxin A (SEA). The addition of CsA (0.1 microgram/ml) to these cultures also completely inhibited (less than 10 U/ml) IFN gamma production. This inhibition was shown not to be due to a change in the kinetics of IFN gamma production or to a change in the amount of SEA required for stimulation. IFN gamma production in SEA-stimulated mouse spleen cells was inhibited at 3 days of culture even when CsA was added at 24 or 48 hr postculture initiation. Thus, CsA inhibits IFN gamma production even when early events associated with lymphocyte activation have been allowed to take place. In contrast to IFN gamma production, IFN alpha/beta production by Newcastle disease virus (NDV)-infected mouse and human lymphocytes or fibroblasts was not inhibited by the addition of CsA (1 microgram/ml). CsA also did not block the action of IFN gamma or IFN alpha/beta since addition of CsA (1 microgram/ml) to reference IFN standards had no effect on their antiviral activity. Thus, CsA inhibits the production of IFN gamma by T cells but appears to have no effect on the production of IFN alpha/beta by virus-infected cells or on the antiviral action of already produced IFN gamma and IFN alpha/beta.     

Polypeptides of hepatitis B virus surface antigen produced by a hepatoma cell line.

The PLC/PRF/5 cell line derived from a human hepatoma produces hepatitis B surface antigen (HBsAg) in 22-nm particles of the same buoyant density as those found in the serum of infected patients. The HBsAg particles from this cell line were labeled with [35S]methionine and purified, and the polypeptides were compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with those of serum-derived particles. The two major polypeptides of serum-derived HBsAg particles (p20 and p23) were found in the same relative amounts in the particles from the cell line. The three smallest of the five minor components observed in HBsAg particles from serum were present in particles from the cell line. These polypeptides (p31, p36, and p43), as well as p20 and p23, were precipitated with anti-HBs-containing serum. The two largest polypeptides of serum particles (p49 and p66) were not detected in particles from these cells. When the PLC/PRF/5 HBsAg particles were radiolabeled with tritiated sugars, p23, and not p20, was found to contain radioactivity, indicating that the pattern of polypeptide glycosylation is similar to that of serum HBsAg. None of the other possible gene products of hepatitis B virus was detected in the PLC/PRF/5-derived HBsAg particles, in the cells, or in the cell supernatants.     

Vasoactive intestinal peptide induces 2',5'-oligoadenylate synthetase and antiviral state in cells of HT-29 colonic cancer]

Vasoactive Intestinal Peptide (VIP) is able at the concentration 10 to 100 nM to induce in HT-29 cells 2'5' oligoadenylate (2'5' A) synthetase activity. The kinetics of this induction show that the maximal effect is attained after 24 hrs. VIP induces 2'5' A synthetase parallel to inhibition of vesicular stomatitis virus growth. The levels of these two induced activities after VIP treatment are comparable to those induced by the poly (I).poly (C), an inducer of IFN beta/alpha in mammalian cells. Moreover the anti-IFN beta/alpha antibodies abolish the VIP-induced 2'5' A synthetase whereas anti-IFN gamma antibodies are ineffective. The fact that VIP establishes an antiviral state in HT-29 cells potentiates new pharmaceutical applications for this neuropeptide.     

Interaction of interferon-alpha with interleukin-1 beta or tumor necrosis factor-alpha on hepatitis B virus enhancer activity.

The interaction of IFN-alpha with IL-1 beta or TNF-alpha on hepatitis B surface antigen (HBsAg) expression was analysed in hepatitis B virus (HBV)-DNA integrated PLC/PRF/5 and non-integrated HuH-7 human hepatoma cells. Secretion of HBsAg in PLC/PRF/5 cells was reduced by IFN-alpha, IL-1 beta or TNF-alpha, and synergistically depressed when IFN-alpha was used in combination with IL-1 beta or TNF-alpha. By Northern blot analysis, the levels of HBsAg mRNA were suppressed by IFN-alpha in combination with IL-1 beta or TNF-alpha. In the chloramphenicol acetyltransferase plasmid transfection assay, IFN-alpha in combination with IL-1 beta or TNF-alpha caused a much greater suppression of HBV enhancer activity than IFN-alpha, IL-1 beta or TNF-alpha alone in both hepatoma cells. These findings suggest that the interaction of IFN-alpha with IL-1 beta or TNF-alpha synergistically represses HBV enhancer activity, resulting in depressed expression of HBsAg.     

Intracellular inhibition of hepatitis B virus S gene expression by chimeric DNA-RNA phosphorothioate minimized ribozyme

Chronic hepatitis B virus (HBV) infection is a major problem in Asia. Current therapies for chronic hepatitis B have limited efficacy. The successful use of ribozymes for intracellular inhibition of HBV gene expression was recently reported. As an alternative to ribozymes, the use of DNA-containing, phosphorothioate-modified, minimized hammerhead ribozymes (minizymes) to inhibit hepatitis B surface antigen (HBsAg) expression and viral replication was investigated. Such molecules can be synthesized and supplied exogenously. Two conserved sites within the HBsAg open reading frame (ORF) were targeted. PLC/PRF5 cells or 2.2.15 cells were treated with minizymes or antisense oligomers to assess the effects on cell viability, HBsAg expression, and viral DNA production. Treatment with the minizyme, MZPS1, resulted in >80% inhibition of HBsAg expression in PLC/PRF5 cells. MZPS1 had more inhibitory effect than the antisense oligonucletoide target at the same region, whereas the control minizyme had little effect. Another gene-specific minizyme, MZPS2, did not show any effect. Treated cells remained fully viable. Treatment of 2.2.15 cells with MZPS1 also led to decreased HBsAg expression. In addition, a 2.3-fold decrease in viral production was observed. Our data showed that minizymes can inhibit HBV gene expression and may potentially be useful for clinical therapy against chronic HBV infection.     

Augmentation of Interferon Production after Cell-Differentiation of U937 Cells by TPA

Persistent infections with mumps virus were established in several human lymphoid cells of T-cell origin (Molt-4, TALL-1, and CCRF-CEM) and human monocyte cells (U937 and THP-1). 2′,5′-Oligoadenylate synthetase (2–5AS) activity was demonstrated to be only slightly induced by interferon (IFN) or TPA (12-O-tetradecanoyl-phorbol-13-acetate) treatment in these cells. Treatment of the persistently infected cells with IFN or TPA did not stimulate an increase in the amount of synthetase mRNA. Induction of cell differentiation and augmentation of IFN production by TPA were demonstrated in U937 cells persistently infected with mumps virus (U937-MP). Similar results for IFN production were obtained from differentiated U937 cells. It is suggested that cell differentiation of U937 cells might be associated with the development of IFN inducibility.     

Identification of upregulated genes in scrapie-infected brain tissue

The pathogenesis of scrapie, and of neurodegenerative diseases in general, is still insufficiently understood and is therefore being intensely researched. There is abundant evidence that the activation of glial cells precedes neurodegeneration and may thus play an important role in disease development and progression. The identification of genes with altered expression patterns in the diseased brain may provide insight on the molecular level into the process which ultimately leads to neuronal loss. Differentially expressed genes in scrapie-infected brain tissue were enriched by the suppression subtractive hybridization technique, molecularly cloned, and further characterized. Northern blotting and nucleotide sequencing confirmed the identities of 19 upregulated genes, 11 of which were unknown to be affected by scrapie. A considerable number of these 19 genes, namely those encoding interferon-inducible protein 10 (IP-10), 2',5'-oligo(A) synthetase, Mx protein, IIGP protein, major histocompatibility complex classes I and II, complement, and beta(2)-microglobulin, were inducible by interferons (IFNs), suggesting that an IFN response is a possible mechanism of gene activation in scrapie. Among the newly found genes, that coding for 2',5'-oligo(A) synthetase is of special interest because it could contribute to the apoptotic loss of neuronal cells via RNase L activation. In addition, upregulation of the chemokine IP-10 and B-lymphocyte chemoattractant mRNAs was seen at relatively early stages of the disease and was sustained throughout disease development.     

Interferon-responsive regulatory elements in the promoter of the human 2'',5''-oligo(A) synthetase gene.

The interferon (IFN)-activated human 2',5'-oligo(A) synthetase E gene contains 11 RNA starts and lacks TATA and CAAT signals. DNA sequences around the promoter make the expression of the chloramphenicol acetyltransferase gene (CAT) inducible over 20-fold by IFN. A 72-base-pair segment (E-IRS) immediately upstream of the RNA starts was defined as being required for IFN-activated expression of the E-gene promoter-CAT constructs and acts in a position-independent manner. It also confers IFN-activated enhancement to the herpes simplex virus thymidine kinase promoter. On this promoter, the 5' part of the E-IRS functions as a constitutive enhancer, while the last 16 base pairs of the E-IRS is sufficient to give IFN-induced expression. On the E-gene promoter, the constitutive enhancer and the IFN-activated sequence are both needed but can be separated. In addition, promoter competition experiments indicate a third regulatory region which helps to repress expression of the E gene in uninduced cells.     

Autogenous production of interferon-beta switches on HLA genes during differentiation of histiocytic lymphoma U937 cells

The expression of class I HLA genes was measured during the in vitro differentiation of human U937 lymphoma cells towards macrophages. Following the onset of differentiation by phorbol myristate acetate the levels of cytoplasmic mRNA that hybridized with a [32P]HLA-B cDNA probe increased by a factor of nine. Elevation in HLA mRNA accumulation was followed by an increase in the rate of synthesis of HLA proteins and also by a dramatic increase in class I HLA cell surface antigen expression, as shown by cytofluorimetric analysis. The elevation in HLA mRNA and surface antigens could be prevented by adding antibodies against human interferon-beta (IFN-beta) to the culture medium at the onset of differentiation. Interferon antiviral activity was detected in the medium of differentiated U937 cells. The same anti-IFN-beta antibodies prevented the increase in (2'-5')oligo(A) synthetase activity which also takes place in differentiating U937 cells. Accumulation of the IFN-induced (2'-5')oligo(A) synthetase in U937 cells is preceded by an increase in its specific 1.6-kb mRNA as shown by hybridization to cloned (2'-5')-oligo(A) synthetase cDNA. The enzyme was preferentially found in the nuclear fraction of differentiating U937 cells. We suggest that an autogenous production of interferon-beta by the differentiating cells, switches on expression of the class I HLA genes as well as that of the (2'-5')oligo(A) synthetase.     

Hepatitis a virus infection of HBsAg-producing hepatocellular carcinomas in athymic mice

Summary Two human hepatocellular carcinoma cell lines were grown in athymic mice. Morphologically, the tumors resembled hepatocellular carcinomas. HBsAg, anti-HBs, and -fetoprotein were detected in sera of mice bearing tumors of PLC/PRF/5 or Hep 3B cells; their amounts correlated with tumor weight. Tumors of both cell lines were infected with cell culture-adapted hepatitis A virus (HAV); HAV antigen and infectious virus were recovered from infected tumors of PLC/PRF/5 and Hep 3B cells. Our results indicate that HAV-infected tumors may be useful in studying the production in vivo of hepatitis A virus antigen.     

Supernatant derived from a human hepatocellular carcinoma cell line (PLC/PRF/5) activates a population of T-suppressor cells

Harvest fluid derived from a primary hepatocellular carcinoma cell line (PLC/PRF/5) inhibited the incorporation of 3H-thymidine into PHA-activated human lymphocytes. A similar effect was observed when lymphocytes were pre-incubated with the tumour supernatant and washed prior to mitogen activation. Not only did the tumour supernatant inhibit 3H-thymidine incorporation by mitogen-activated lymphocytes, but it also inhibited production of the lymphokine leucocyte inhibitory factor (LIF). In experiments designed to establish whether a component of the tumour harvest fluid was activating a population of suppressor cells, normal mononuclear (MN) cells were treated with the PLC/PRF/5 or embryonic fibroblast supernatant for 48 h, after which they were washed and added to normal mitogen-activated lymphocyte cultures. Only cells pretreated with the PLC/PRF/5 supernatant suppressed mitogenesis. The cell responsible for the suppressor effect was a T cell, which after a further 24 h in culture liberated a suppressor factor responsible for inhibiting lymphocyte function. Although the nature of the factor/s in the PLC/PRF/5 supernatant responsible for activation of the T-suppressor cell population is unknown, it is suggested that this mechanism may be important in protecting the tumour from the immune response.