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.     

Stimulation of 2',5'-oligoadenylate synthetase activity in sheep endometrium during pregnancy, by intrauterine infusion of ovine trophoblast protein-1, and by intramuscular administration of recombinant bovine interferon-alpha I1.

In Expt 1, activity of 2',5'-oligoadenylate (2',5'-A) synthetase in endometrium collected on Day 16 (oestrus is Day 0) from the uterine horn ipsilateral to the corpus luteum was greater (P less than 0.001) for pregnant (135.5 +/- 1.72 nmol/mg protein/h) than for cyclic ewes (58.5 +/- 0.99 nmol/mg protein/h). In pregnant ewes, activity of 2',5'-A synthetase in endometrium collected from the contralateral uterine horn (119.5 +/- 1.72 nmol/mg protein/h) did not differ from that of the ipsilateral horn. In Expt 2, three ovariectomized ewes were treated with progesterone for 10 days and then with oestrogen for 2 days. Activity of 2',5'-A synthetase on Day 13 was 18% greater (P less than 0.10) in endometrium collected from the uterine horn receiving infusions of 30 micrograms ovine trophoblast protein-1 (oTP-1) twice a day on Days 10, 11 and 12(57.7 +/- 0.22 nmol/mg protein/h) than from the uterine horn receiving control infusions of serum protein (SP; 48.8 +/- 0.22 nmol/mg protein/h). In Expt 3, activity of 2',5'-A synthetase on Day 15 was not significantly greater in endometrium collected from the uterine horn of cyclic ewes receiving infusions of 30 micrograms oTP-1 twice a day on Days 12, 13 and 14 (46.5 +/- 0.37 nmol/mg protein/h) than in endometrium from the uterine horn receiving infusions of SP (38.2 +/- 0.37 nmol/mg protein/h). When results of Expt 2 and Expt 3 were combined, intrauterine infusion of oTP-1 increased (P less than 0.05) activity of 2',5'-A synthetase in endometrium by 20%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Antiviral and antiproliferative activity of human interferons and their induction of 2',5'-oligoadenylate synthetase

Native preparations of alpha, beta and gamma-interferons as well as recombinant beta-interferon and purified leukocyte alpha-interferon and purified leukocyte alpha-interferon exert antiviral and antiproliferative activity in CaOv cells. Native interferon preparations were shown to be more antiproliferative than purified interferons per unit of antiviral activity (with EMC as well as with less susceptible VSV used as test viruses). It was shown that level of 2'5' oligoadenylatesynthetase activity induction in general correlates with antiproliferative and pronounced antiviral activity of interferons, besides that, the earlier (by 11 hours) induction of the enzyme activity by beta-interferon correlates with more rapid expression of antiproliferative effects by this interferon in comparison with that of alpha-interferon, the latter inducing the peak of enzyme activity by 24 hours.     

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.     

Chloroquine impairs the interferon-induced antiviral state without affecting the 2',5'-oligoadenylate synthetase

Chloroquine, a weak base which raises the pH in acidic cellular compartments such as lysosomes and endosomes, counteracts the induction by interferon of the antiviral state but not that of the 2',5'-oligoadenylate synthetase in three different types of cell lines (MDBK, WISH, and L929). Active interferon is recovered in crude extracts of cells which have been treated with interferon and chloroquine together, but not in extracts of cells treated with interferon alone, indicating that chloroquine has inhibited the intralysosomal proteolysis of interferon. A low pH-dependent event in the intracellular fate of interferon (perhaps its intralysosomal degradation) is, therefore, necessary for the establishment of the antiviral state but not for the induction of the 2',5'-oligoadenylate synthetase.     

Identification of bovine trophoblast protein-1, a secretory protein immunologically related to ovine trophoblast protein-1

This paper demonstrates that a group of proteins, representing a major secretory component of cattle conceptuses, is immunologically related to ovine trophoblast protein-1 (oTP-1), a principal product of culture Day 13 to 21 sheep conceptuses. Conceptuses from cows (Day 17-18) and ewes (Day 16-17) were cultured for 24 h in the presence of L-[3H]leucine. By using a rabbit antiserum to oTP-1 and Ouchterlony double-immunodiffusion analysis it was shown that material in the bovine conceptus culture medium was serologically related, but not identical, to oTP-1. A solid-phase radiobinding assay indicated that the cross-reacting bovine secretory component had an affinity for anti-oTP-1 antibody similar to that of oTP-1. Anti-oTP-1 antiserum specifically immunoprecipitated a group of 6-8 polypeptides from culture medium of cow conceptuses which, when analysed by two-dimensional gel electrophoresis, fell into two major molecular weight classes (22,000 and 24,000) with isoelectric points between 6.5 and 6.7. These immunoprecipitated polypeptides, defined as bTP-1, constituted the major secretory products of Day 16-25 cow conceptuses. They were larger and more basic than oTP-1 polypeptides (Mr about 18,000; pI 5.4-5.7). Anti-oTP-1 antiserum also recognized the major translation product of Day 17 bovine conceptus mRNA, a polypeptide significantly smaller (Mr approximately 18,000) than the secreted protein. It is suggested that oTP-1 and the homologous bovine protein may play similar roles in the phenomenon of maternal recognition of pregnancy in the two species.     

Variation in antiviral 2',5'-oligoadenylate synthetase (2'5'AS) enzyme activity is controlled by a single-nucleotide polymorphism at a splice-acceptor site in the OAS1 gene

It is likely that human genetic differences mediate susceptibility to viral infection and virus-triggered disorders. OAS genes encoding the antiviral enzyme 2',5'-oligoadenylate synthetase (2'5'AS) are critical components of the innate immune response to viruses. This enzyme uses adenosine triphosphate in 2'-specific nucleotidyl transfer reactions to synthesize 2',5'-oligoadenylates, which activate latent ribonuclease, resulting in degradation of viral RNA and inhibition of virus replication. We showed elsewhere that constitutive (basal) activity of 2'5'AS is correlated with virus-stimulated activity. In the present study, we asked whether constitutive activity is genetically determined and, if so, by which variants. Analysis of 83 families containing two parents and two children demonstrated significant correlations between basal activity in parent-child pairs (P<.0001) and sibling pairs (P=.0044), but not spousal pairs, suggesting strong genetic control of basal activity. We next analyzed association between basal activity and 15 markers across the OAS gene cluster. Significant association was detected at multiple markers, the strongest being at an A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (AG or AA) of the OAS1 gene. At this unusual polymorphism, allele G had a higher gene frequency in persons with high enzyme activity than in those with low enzyme activity (0.44 vs. 0.20; P=3 x 10(-11)). Enzyme activity varied in a dose-dependent manner across the GG, GA, and AA genotypes (tested by analysis of variance; P=1 x 10(-14)). Allele G generates the previously described p46 enzyme isoform, whereas allele A ablates the splice site and generates a dual-function antiviral/proapoptotic p48 isoform and a novel p52 isoform. This genetic polymorphism makes OAS1 an excellent candidate for a human gene that influences host susceptibility to viral infection.     

Spectrophotometric pyrophosphate assay of 2',5'-oligoadenylate synthetase.

A nonradioactive multiwell spectrophotometric assay for the interferon-induced enzyme 2',5'-oligoadenylate synthetase measuring the inorganic pyrophosphate produced during oligoadenylate synthesis has been developed. A coupled enzymatic reaction results in a mole to mole formation of NADPH compared to the inorganic pyrophosphate through the use of the three enzymes UDP-Glc pyrophosphorylase (EC2.7.7.9), phosphoglucomutase (EC5.4.2.2), and glucose-6-phosphate dehydrogenase (EC1.1.1.49). The assay is at least as sensitive for measurements of 2',5'-oligoadenylate synthetase activity as the conventional assays using radioactive nucleotides as substrates. Even higher sensitivity of the assay can be obtained by taking advantage of the strong fluorescence of NADPH.     

Expression and characterization of recombinant 2',5'-oligoadenylate synthetase from the marine sponge Geodia cydonium

2',5'-oligoadenylate (2-5A) synthetases are known as components of the interferon-induced cellular defence mechanism in mammals. The existence of 2-5A synthetases in the evolutionarily lowest multicellular animals, the marine sponges, has been demonstrated and the respective candidate genes from Geodia cydonium and Suberites domuncula have been identified. In the present study, the putative 2-5A synthetase cDNA from G. cydonium was expressed in an Escherichia coli expression system to characterize the enzymatic activity of the recombinant polypeptide. Our studies reveal that, unlike the porcine recombinant 2-5A synthetase, the sponge recombinant protein associates strongly with RNA from E. coli, forming a heterogeneous set of complexes. No complete dissociation of the complex occurs during purification of the recombinant protein and the RNA constituent is partially protected from RNase degradation. We demonstrate that the sponge recombinant 2-5A synthetase in complex with E. coli RNA catalyzes the synthesis of 2',5'-phosphodiester-linked 5'-triphosphorylated oligoadenylates from ATP, although with a low specific activity. Poly(I).poly(C), an efficient artificial activator of the mammalian 2-5A synthetases, has only a minimal effect (an approximate two-fold increase) on the sponge recombinant 2-5A synthetase/bacterial RNA complex activity.     

Inhibition of uterine prostaglandin-F2 alpha production by bovine conceptus secretory proteins

The effect of bovine conceptus secretory proteins (CSP) on uterine prostaglandin (PG)-F2 alpha production was evaluated in dairy cattle following injection of estradiol-17 beta. Intrauterine injections of dialyzed serum proteins (Control, n = 5) or CSP (n = 5) were administered from days 15 through 18 post-estrus. Following intrauterine treatments on day 18, all cows were injected with E2 (3 mg) to stimulate uterine PGF2 alpha production. Plasma concentrations of progesterone (P4) and 15-keto-13,14-dihydro-PGF2 alpha (PGFM) were determined by RIA. The PGFM responses following E2 challenge were decreased (p less than 0.01) for cows receiving CSP versus serum proteins into the uterine lumen. Individual PGFM, P4 and cycle length responses are discussed. Data suggest that proteins secreted by the bovine conceptus suppress uterine PGF2 alpha production during pregnancy recognition in the cow.     

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.     

Differential induction of 2',5'-oligoadenylate synthetase by IFN-beta ser and IFN-alpha 2 in serum-supplemented and serum-free HL-60 leukemic cell cultures

The influence of cell culture conditions on the induction of 2',5'-oligoadenylate (2-5A) synthetase by recombinant interferons IFN-beta ser and IFN-alpha 2 has been investigated in human HL-60 leukemic cells. Cells maintained either in the fetal bovine serum-supplemented medium (FBS-SM) or in a serum-free, chemically defined Nutridoma-supplemented medium (SFN-SM) are treated with different concentrations of the two types of IFN and the extent of 2-5A synthetase induction compared. While cells in FBS-SM show a substantially greater increase in 2-5A synthetase by IFN-beta ser than cells in SFN-SM, the latter culture condition is significantly more effective in elevating synthetase activity with the addition of IFN-alpha 2. These data suggest that there are growth modulators and other "factors" in the fetal bovine serum which may interact specifically with each type of IFN to coordinate the optimal expression of the 2-5A synthetase protein.     

2',5'-Oligoadenylates and related 2',5'-oligonucleotide analogues. 1. Substrate specificity of the interferon-induced murine 2',5'-oligoadenylate synthetase and enzymatic synthesis of oligomers

The substrate specificity of the interferon-induced mouse L-cell enzyme, 2',5'-oligoadenylate synthetase, was determined with a number of nucleoside 5'-triphosphate analogues. Selected nucleoside 5'-triphosphates were converted to 2',5'-oligonucleotides with the following order of efficiency for the nucleoside: 8-azaadenosine greater than adenosine = 2-chloroadenosine greater than sangivamycin greater than toyocamycin greater than formycin greater than 3-ribosyladenine greater than ribavirin greater than tubercidin greater than adenosine 1-oxide greater than 2-beta-D-ribofuranosylthiazole-4-carboxamide greater than inosine = 1,N6-ethenoadenosine greater than guanosine greater than 8-bromoadenosine = uridine greater than cytidine. Adenosine 5'-((beta, gamma-imidotriphosphate) did not seem to be a recognizable substrate since no detectable product resulted. Either the 2',5'-oligoadenylate synthetase is not as specific as had been previously thought, or there may be more than one 2',5'-oligonucleotide synthetase. The 2',5'-oligonucleotide analogue products in which the adenosine of ppp(A2'P5')nA was replaced by the various nucleoside analogues were separated by DEAE-cellulose column chromatography and the chain length and number of 5'-phosphate residues analyzed by a rapid, efficient high-performance liquid chromatographic (HPLC) system involving ion-pairing C18 reversed-phase column chromatography. Separation of the 5'-mono-, 5'-di-, and 5'-triphosphorylated forms of the 2',5'-oligonucleotide analogue dimers, trimers, tetramers, and pentamers was readily achieved by this useful HPLC system. No 5'-nonphosphorylated forms were detected for any of the 2',5'-oligonucleotide analogue products.     

2',5'-Oligoadenylate and 2',5'-oligoadenylate phosphodiesterase in human plasma

2',5'-Oligoadenylate and 2',5'-oligoadenylate phosphodiesterase activity were detected in the human plasma and serum by sensitive radioimmuno assays. The phosphodiesterase in the serum degraded 20 nM of added 2',5'-oligoadenylate in less than 1 hr. Addition of EDTA in the blood sample inhibited the phosphodiesterase activity completely and allowed the measurement of low levels of 2',5'-oligoadenylate. The concentration in the plasma from healty people was in the range of 0.03 to 0.3 nM.     

Assay of 2',5'-oligoadenylate phosphodiesterase activity in mouse L-cell extracts

A rapid and convenient assay for adenylyl(2' leads to 5')adenosine(A2'p5'A) or adenylyl(3' leads to 5')adenosine(A3'p5'A) phosphodiesterase activities is described. The dinucleotides A3'p5'A and A2'p5'A were labeled to a high specific activity by means of a catalytic-exchange procedure. Degradation studies of each of these labeled dinucleotides showed an asymmetrical distribution of label between the two adenine bases. Enzymatic degradation of [3H]A3'p5'A or [3H]A2'p5'A could be quantitated by first digesting the reaction products with bacterial alkaline phosphatase and then adding a slurry of DEAE-Sephadex. Under conditions described, adenosine did not adsorb to the resin, whereas dinucleotides as well as AMP did adsorb. As a consequence, when liquid scintillation fluid was added to the DEAE-Sephadex reaction mixture slurry, the radioactivity of the dinucleotides and AMP was severely quenched. This permitted a direct estimation of the amount of adenosine liberated during the phosphodiesterase degradation and subsequent alkaline phosphatase digestion. This method was applied to the measurement of A2'p5'A degrading activities in extracts of mouse L cells. Extracts from control mouse L cells were as active in degrading A2'p5'A as extracts from interferon pretreated cells.     

Induction and function of 2',5'-oligoadenylate synthetase in differentiation of mouse myeloid leukemia cells

The activity of 2′,5′-oligoadenylate synthetase (2-5A synthetase), known to be induced by interferon, was detected in mouse myeloid leukemic M1 cells only when they differentiated to phagocytic cells after incubation with conditioned medium (CM) from rat embryo cells. However, no interferon activity occurred in culture fluids of CM-treated M1 cells, although some activity was detected in the cell extracts. When anti-interferon serum was added to M1 cell cultures, the induction of 2-5A synthetase by CM was suppressed. These results suggest that CM stimulated the M1 cells to produce a minute amount of interferon, which was reponsible for induction of the 2-5A synthetase activity. On the other hand, development of the phagocytic activity of M1 cells could not be influenced by addition of antiserum. Interferon added exogenously per se neither induced phagocytic activity of M1 cells, nor did it enhance the CM-induced differentiation of the cells. Moreover, dexamethasone, which induced differentiation of M1 cells, was not capable of inducing 2-5A synthetase. These results indicate that interferon and/or 2-5A synthetase plays no essential part in the differentiation of M1 cells.     

Protein synthesis-dependent and -independent induction of p69 2'-5'-oligoadenylate synthetase by interferon-alpha.

The 2'-5'-oligoadenylate (2-5A) synthetases are a family of interferon-alpha (IFN-alpha) inducible enzymes that block viral replication by activating a latent endonuclease during viral infections. In Ramos cells, induction of mRNAs for the intermediate isoform of 2-5A synthetase (p69) requires five-fold higher IFN-alpha than is required for induction of the small isoform (p40). The p40 and p69 isoforms are similarly induced between 1 and 24 h with maximal induction at 8 h. At 48 h, however, p69 is more strongly induced than p40. Induction of p69 and p40 between 1 and 24 h is protein synthesis independent whereas at 48 h, p69 induction becomes dependent on protein synthesis. Initial induction of both isoforms requires tyrosine kinase activation and is enhanced by activation of a separate signalling pathway by the tumour promoter, 12-0-tetradecanoyl phorbol-13-acetate (TPA). These data suggest that induction of the p40 is predominantly protein synthesis independent, whereas p69 induction occurs in two phases, an initial protein synthesis independent phase and a delayed protein synthesis dependent phase.     

Inhibition of 2',5'-oligoadenylate synthetase expression and function by the human cytomegalovirus ORF94 gene product

The human cytomegalovirus (HCMV) ORF94 gene product has been reported to be expressed during both productive and latent phases of infection, although its function is unknown. We report that expression of pORF94 leads to decreased 2',5'-oligoadenylate synthetase (OAS) expression in transfected cells with and without interferon stimulation. Furthermore, the functional activity of OAS was inhibited by pORF94. Finally, we present evidence of OAS modulation by pORF94 during productive HCMV infection of human fibroblasts. This study provides the first identification of a function for pORF94 and identifies an additional means by which HCMV may limit a critical host cell antiviral response.