Effect of interferon on the production of HBsAg and induction of an antiviral state in human hepatoma cell line PLC/PRF/5

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.     

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.     

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.     

Presence of 2',5'-oligo(A) and of enzymes that synthesize, bind, and degrade 2',5'-oligo(A) in HeLa cell nuclei

Nuclei prepared from HeLa cells by lysis with nonionic detergents or by a nonaqueous fractionation procedure were assayed for enzymatic activities which synthesize, bind, and degrade 2',5'-oligo(A). Isolated nuclei synthesized micromolar concentrations of 2',5'-oligo(A) when incubated with poly(inosinic) . poly(cytidylic) acid. The products of nuclear synthesis were identified with authentic 2',5'-oligo(A) by several criteria. The nuclei synthesized nanomolar amounts of 2',5'-oligo(A) even when incubated without added double-stranded RNA. These oligonucleotides were identified by their pattern of degradation with different nucleases and by a specific competition-binding assay. This assay revealed the presence in nuclei of an activity which binds 2',5'-oligo(A) with an affinity constant similar to that of the cytoplasmic binding activity previously identified with the 2',5'-oligo(A)-dependent endoribonuclease (Nilsen, T. W., Wood, D. L., and Baglioni, C. (1981) J. Biol. Chem. 256, 10751-10754). The nuclei had also an activity which degraded 2',5'-oligo(A). Finally, unincubated nuclei isolated by the nonaqueous fractionation procedure contained detectable concentrations of 2',5'-oligo(A). These results show that an activator of the enzyme which synthesize 2',5'-oligo(A) is present in nuclei and that these oligonucleotides are normally formed in HeLa cells, and suggest a possible role for the 2',5'-oligo(A)-activated endoribonuclease in nuclear RNA metabolism.     

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)     

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.     

Regulation of protein synthesis in lymphoblastoid cells during inhibition of cell proliferation by human interferons

Treatment of human lymphoblastoid (Daudi) cells with interferons inhibits cell proliferation in culture within 24 h. The failure of cell growth has been shown to be associated with impaired processing and decreased stability of newly replicated DNA. Because there is a close relationship between DNA replication and protein synthesis we have measured protein synthesis in intact Daudi cells. Protein synthesis declined steadily between 24 and 96 h after interferon treatment to a value which is only 20-30% of the rate in control cells. The enzyme 2',5'-oligo(A) synthetase is induced but our data do not support a role for the 2',5'-oligo(A)-activated ribonuclease in the control of translation in this system.     

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.     

Interferon regulatory factor 4 contributes to transformation of v-Rel-expressing fibroblasts

The avian homologue of the interferon regulatory factor 4 (IRF-4) and a novel splice variant lacking exon 6, IRF-4DeltaE6, were isolated and characterized. Chicken IRF-4 is expressed in lymphoid organs, less in small intestine, and lungs. IRF-4DeltaE6 mRNA, though less abundant than full-length IRF-4, was detected in lymphoid tissues, with the highest levels observed in thymic cells. IRF-4 is highly expressed in v-Rel-transformed lymphocytes, and the expression of IRF-4 is increased in v-Rel- and c-Rel-transformed fibroblasts relative to control cells. The expression of IRF-4 from retrovirus vectors morphologically transformed primary fibroblasts, increased their saturation density, proliferation, and life span, and promoted their growth in soft agar. IRF-4 and v-Rel cooperated synergistically to transform fibroblasts. The expression of IRF-4 antisense RNA eliminated formation of soft agar colonies by v-Rel and reduced the proliferation of v-Rel-transformed cells. v-Rel-transformed fibroblasts produced interferon 1 (IFN1), which inhibits fibroblast proliferation. Infection of fibroblasts with retroviruses expressing v-Rel resulted in an increase in the mRNA levels of IFN1, the IFN receptor, STAT1, JAK1, and 2',5'-oligo(A) synthetase. The exogenous expression of IRF-4 in v-Rel-transformed fibroblasts decreased the production of IFN1 and suppressed the expression of several genes in the IFN transduction pathway. These results suggest that induction of IRF-4 expression by v-Rel likely facilitates transformation of fibroblasts by decreasing the induction of this antiproliferative pathway.     

Synthesis of 2',5'-oligoadenylate by rat liver nuclear matrix protein

Nuclear matrix was prepared from unstimulated rat liver by treatment of nuclei with DNAse and 0.4 M NaCl and was further extracted with 2.0 M NaCl. Proteins were bound to poly(rI):(rC)-agarose, incubated with (alpha-32P) adenosine 5'-triphosphate and 2',5'-linked oligoadenylate was isolated from the supernatant. The substance inhibited amino acid incorporation in a reticulocyte translation system and was identified after enzymatic treatment followed by thin-layer chromatography on PEI-cellulose. The possible function of 2',5'-oligo(A) synthetase in the maturation of pre-mRNA associated with nuclear matrix is discussed.     

Isolation and characterization of an interferon-resistant cell line deficient in the induction of (2''-5'')oligoadenylate synthetase activity.

To screen for cells with different sensitivities to interferon (IFN), NIH 3T3 mouse fibroblasts were subcloned and examined for their response to IFN treatment. Of 30 clones tested, 2 appeared to be relatively resistant to IFN, since the replication of both vesicular stomatitis virus and mengovirus was not inhibited, even in the presence of 1,000 U of IFN per ml. One resistant (A10) and one sensitive (A5) clone were further analyzed. In both clones, murine leukemia virus replication was equally inhibited by IFN, indicating the presence of functional receptors for IFN in the resistant clone. Using the (2'-5')oligoadenylate (2-5A) radiobinding assay, we could demonstrate that both clones contained the RNase L protein. Furthermore, this enzyme appears to be active, since a similar reduction in the rate of protein synthesis was evident after the introduction of exogenous 2-5A to the cells. We also analyzed the activity of another enzyme in the 2-5A pathway, namely, 2-5A synthetase. In the sensitive cells (A5), the induction of enzyme activity was proportional to the IFN concentration used, reaching a maximum of more than a 10-fold increase over the background of untreated cells. However, little if any induction over the basal activity was observed in the resistant cells (A10) when similar doses of IFN were used. It is thus probable that the lack of induction of 2-5A synthetase activity by IFN in A10 cells is at least partly responsible for their relative resistance to IFN treatment.     

Reversibility of the antiproliferative effect of interferon

The reversibility of the antiproliferative effect of interferon (IFN) and its correlations to the induction of (2',5') oligoadenylate synthetase (2-5A synthetase) activity was studied on NIH/3T3 cells transformed by Moloney murine sarcoma virus. The cells were treated with various doses of mouse beta-IFN. At 72 h after treatment, the cultures were subdivided. While half received fresh doses of IFN, the second half received no IFN. Reversibility of the IFN effect was then followed. Three different parameters as indicators for cell proliferation were used: cell growth, protein synthesis and cloning efficiency. In parallel, the IFN-induced activity of 2-5A synthetase was determined. The data obtained led to the following conclusions. (1) The antiproliferative effect of IFN increases with increased IFN concentration (90-1,800 IU/ml) and with time of treatment, up to 72 h after treatment. (2) The induced activity of 2-5A synthetase increases with a much faster rate, reaching maximum activity at 24 h after treatment with 450 IU/ml. This means that the induction of the enzyme precedes the antiproliferative effects of IFN. (3) There is almost no recovery of the IFN antiproliferative effect following treatment for 72 h with high doses of IFN (1,200-1,800 IU/ml). However, at lower doses, recovery is evident. (4) Removal of IFN after treatment for 3 days with 450 IU/ml resulted in a gradual decrease of 2-5A synthetase activity, reaching the basal level at 72 h after removal. However, there is no reduction of enzyme activity following treatment for 72 h with 1,800 IU/ml of IFN.     

Interferon-like activity in an anti-interferon anti-idiotypic hybridoma antibody

We describe here an anti-idiotypic hybridoma antibody directed against affinity-purified rabbit idiotypic antibodies (Rb-Id) to a homogeneous protein, the recombinant human leukocyte A interferon (rIFN-alpha A). The supernatant of the hybridoma, designated 3-1B, was able to inhibit the neutralization of rIFN-alpha A activity by the idiotypic antibodies. An in vivo passage of uncloned 3-1B cells yielded hybridoma cells (presumably a subclone), designated 3B1, the supernatant of which exhibited interferon-like antiviral activity with both bovine kidney (MDBK) cells and human amnion (WISH) cells. This activity could be absorbed by polymer-bound goat anti-mouse immunoglobulin serum and by Rb-Id coupled to Affi-Gel 10, and could be partially eluted from the latter at pH 2.5. The anti-idiotypic hybridoma antibody was able to compete with 125I-rIFN-alpha A for binding to the Rb-Id and also to interferon receptor-bearing MDBK cells. The clinical significance of an interferon-like anti-idiotypic antibody is discussed.     

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.     

Activities of cAMP-dependent protein kinase and enzymes of 2′,5′-oligoadenylate metabolism in NIH 3T3 cells deepening into the resting state

The activity of cAMP-dependent protein kinase was found to increase continuously in the NIH 3T3 cells, deepening into the resting state. The increase correlated with intracellular level of heat-stable protein inhibitor of the protein kinase rather than with the cAMP content. The elevation of 2',5'-oligo(A) synthetase activity and the decrease in 2'-phosphodiesterase activity were also observed in the cells sinking into the resting state. The variations in enzyme activities were similar to those caused by the increase in the intracellular cAMP content described elsewhere. These results agree with the idea that the cAMP-dependent protein kinase is involved in the regulation of the enzymes of 2',5'-oligo(A) metabolism.     

Recombinant interferon-beta 2 (interleukin-6) induces myeloid differentiation

Human IFN-beta 2 cytokine produced in E. coli was purified to homogeneity by immunoaffinity and ion-exchange chromatography. The cytokine inhibits the growth of myeloleukemic M1 cells and induces their morphological and functional differentiation into macrophages. Differentiation was also observed in the histiocytic lymphoma U937 cells. The effect on U937 was synergized by IFN-gamma and under these conditions IFN-beta 2 produced the induction of (2'-5') oligo(A) synthetase typical to IFN action and to differentiation.