Double-stranded RNAs as gene activators.
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Animals↗
Biomedical subjects
G Gribaudo
Publications and source records attributed to G Gribaudo.
Trans-activation of the mouse cytomegalovirus immediate early gene enhancer by ras oncogenes.
The ras gene family encodes 21K proteins that reside on the inner face of the plasma membrane and bind GTP and GDP with an equally high affinity. Cotransfection of NIH 3T3 cells with a mammalian expression vector containing a viral Harvey-ras (v-Ha-ras) cDNA, together with a plasmid (pCMVCAT) carrying the immediate early (IE) enhancer of the murine cytomegalovirus (MCMV) linked to the chloramphenicol acetyltransferase (CAT) reporter gene strongly stimulated CAT activity. Basal levels of pCMVCAT expression as well as trans-activation by v-ras plasmid were both inhibited by cotransfection of an expression vector containing the dominant inhibitory mutant gene Ha-ras Asn-17. This indicates that the p21ras protein is responsible for these activities. High pCMVCAT activation was also observed in cell lines carrying stably transfected ras oncogenes, activated by point mutation or amplification. To define the cis-acting DNA elements in the MCMV IE enhancer responsible for this trans-activation by p21ras protein, we constructed several plasmids containing the CAT gene under control of MCMV IE enhancers that were deleted in different regions. The CAT assays demonstrated that several sequences were responsive to p21ras protein. These sequences are scattered throughout the IE enhancer, upstream of the transcription start site, and contain responsive elements that are homologous to the binding sites for cellular transcription factors such as NF kappa B, AP1, ATF and SP1. Activation of the p21ras protein may thus be one of the signals that regulate IE genes transcription during MCMV infection.
3T3 Cells↗
Presence of murine retroviral sequences in human cell line transplanted in immunosuppressed SCID-mice.
The SCID-hu mouse, a severe combined immunodeficient mouse transplanted with human hematopoietic cells, has been proposed as an experimental model for HIV-1 infection. However, like other animal models for infectious diseases, the SCID-hu mouse should provide the replication of the experimental agent without conferring any alteration during growth in the host. In this study using Southern blot hybridization, in situ hybridization and PCR analysis we demonstrate the presence of specific murine retroviral sequences in human cells recovered from the in vivo passages in SCID mice. Since the acquisition of endogenous retrovirus by human cells was demonstrated after their growth in nude mice, the data reported here extend this observation to the SCID model.
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Mouse soluble IFN gamma receptor as IFN gamma inhibitor. Distribution, antigenicity, and activity after injection in mice.
With the purpose to use a soluble receptor as an IFN gamma antagonist in vivo, we assessed the immunogenicity, the half-life, the bioavailability, and the activity of the soluble receptor after injection into mice. No significant immunogenicity was detected after multiple chronic injections of mouse (Mo)IFN gamma-R or injections of MoIFN gamma-R in emulsion with CFA. Pharmacokinetic experiments using radiolabeled MoIFN gamma-R revealed that the half-life of the soluble MoIFN gamma-R is 3 h in the blood and 6 h in the lymphoid organs. When the MoIFN gamma-R protein was traced by the capacity of the animal sera to displace the binding of radiolabeled MoIFN gamma-R from affinity purified rabbit anti-MoIFN gamma-R antibodies, a blood half-life of 1 h was determined. Finally, the capacity of the injected receptor to bind and neutralize the IFN gamma-mediated antiviral activity was assessed in vivo. Treatment with IFN-gamma or IFN-alpha A/D protected the mice from the lethal infection with encephalomyocarditis virus. Mice treated with MoIFN-gamma-R neutralized the protective effect of IFN-gamma.
Animals↗
A monoclonal antibody to the NH2-terminal segment of human IFN-gamma selectively interferes with the antiproliferative activity of the lymphokine.
To gain more information about the relationship between the structure of IFN-gamma and its activity, a peptide corresponding to a hydrophilic peak between amino acids 4 and 16 was used to immunize mice and generate mAb. mAb IGMB-15 reacts to both native and rIFN-gamma and neutralizes the antiproliferative activity of IFN-gamma without affecting its antiviral activity or its ability to up-regulate HLA-DR Ag expression. Moreover, we observed that mAb IGMB-15 was unable to inhibit the binding of radiolabeled IFN-gamma to its cellular receptor. These findings show that the NH2-terminal region may somehow be involved in the biologic activity of IFN-gamma. Besides, the capability of mAb IGMB-15 to inhibit the antiproliferative but not the antiviral activity of IFN-gamma in the same cell (HEp-2) suggests the presence of different elements involved in signal transduction, which may account for the multiple activities of the lymphokine.
Amino Acid Sequence↗
Interferons inhibit onset of murine cytomegalovirus immediate-early gene transcription.
Treatment of NIH 3T3 fibroblasts with interferon-alpha or interferon-gamma (IFN-alpha or IFN-gamma) significantly reduced murine cytomegalovirus (MCMV) replication. Determination of viral DNA in the nuclei of the infected cells before onset of DNA replication demonstrated that virus uptake, transport to the nucleus, and DNA stability were not decreased. Analysis of the virus specified mRNAs soon after infection revealed that in the cells exposed to IFNs expression of the immediate early (IE) genes was strongly reduced. Nuclear run-off transcription analysis showed that this inhibition is due to significant reduction of IE gene transcription rates following IFN treatment. Since transcription of the MCMV IE region is regulated by a strong enhancer element, a construct containing the chloramphenicol acetyltransferase (CAT) reporter gene, driven by an 1.2 kb segment spanning the enhancer and IE1/3 promoter region of the IE transcription unit, was transfected into NIH 3T3 cells. Treatment with IFN-alpha or IFN-gamma after transfection strongly reduced CAT activity compared to untreated controls. In an attempt to define a negative IFN-responsive element in the IE enhancer, a series of deletion mutants driving the CAT reporter gene were transfected into NIH 3T3 cells that were then treated with IFN-alpha. With the sole exception of the construct containing the minimal MCMV IE1/3 promoter (-102 to the cap site), all other deletion mutants were strongly down-regulated by IFN-alpha-treatment. Taken as a whole, these results suggest that IFNs inhibit MCMV replication by impairing the transcription of the IE transcription units, and that this negative regulation is carried out by sequences scattered throughout the IE enhancer region.
3T3 Cells↗
Effect of interferon-alpha on immediate early gene expression of murine cytomegalovirus.
Interferon-alpha (IFN-alpha) significantly reduced the replication of murine cytomegalovirus (MCMV) in mouse embryo fibroblasts derived from the susceptible mouse strain C3H/HeJ. When infectious virus production was measured, a strong decrease in virus titer was observed in IFN-treated cells at a multiplicity of infection (moi) of 1 and 0.5 pfu/cell. Analysis of virus-specified mRNAs by Northern blot assay revealed that IFN-alpha had a significant effect on the expression of viral mRNAs at 48h. In particular, the mRNAs of the major immediate early (IE) transcription units, IE1, IE2, and IE3, were impaired by IFN-alpha. In addition, decrease of IE1 mRNA synthesis was accompanied by a reduction of the major IE product (pp89), as revealed by Western blot assay. These results suggest that IFN-alpha may inhibit MCMV replication by directly impairing IE gene transcription.
Animals↗
Effects of interferon alpha on murine cytomegalovirus replication.
The present study examined the protective effect of IFN-alpha against mouse cytomegalovirus (MCMV) infection in embryo fibroblasts (MEF) of genetically resistant (C3H/HeJ) and susceptible (C57BL/6) mouse strains. At a M.O.I. of 1 IFN-alpha was protective in C3H/HeJ-MEF but not in C57BL/6-MEF. Dot-blot analysis during MCMV replication in C3H/HeJ-MEF showed that IFN-alpha pretreatment reduced the steady state level of immediate early and late mRNAs but partially reduced early gene expression.
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Interferon action: binding of viral RNA to the 40-kilodalton 2'-5'-oligoadenylate synthetase in interferon-treated HeLa cells infected with encephalomyocarditis virus.
The 40-kDa 2'-5'-oligoadenylate [(2'-5') (A)n] synthetase isoenzyme was proven to be a mediator of the inhibition of encephalomyocarditis virus (EMCV) replication by interferon (IFN). When activated by double-stranded RNA, this enzyme converts ATP into 2'-5'-oligoadenylate [(2'-5') (A)n], and (2'-5') (A)n was found to accumulate in IFN-treated, EMCV-infected cells. The only known function of (2'-5') (A)n is the activation of RNase L, a latent RNase, and this was also implicated in the inhibition of EMCV replication. Intermediates or side products in EMCV RNA replication, presumed to be partially double stranded, were shown to activate (2'-5') (A)n synthetase in vitro. These findings served as the basis of the long-standing hypothesis that the activator of (2'-5') (A)n synthetase in IFN-treated, EMCV-infected cells is the viral RNA. To test this hypothesis, we have generated a polyclonal rabbit antiserum to the human 40-kDa (2'-5') (A)n synthetase. The antiserum immunoprecipitated, from IFN-treated HeLa cells that had been infected with EMCV, the 40-kDa (2'-5') (A)n synthetase protein in complex with both strands of EMCV RNA. The immunoprecipitate was active in (2'-5') (A)n synthesis even without addition of double-stranded RNA, whereas the immunoprecipitate from IFN-treated, uninfected cells was not. These and other results demonstrate that in IFN-treated, EMCV-infected cells, viral RNA is bound to the (2'-5') (A)n synthetase and suggest that the agent activating the (2'-5') (A)n synthetase is the bound viral RNA.
2',5'-Oligoadenylate Synthetase↗
Activation of interferon-inducible genes in vivo by synthetic double-stranded RNA, poly rI:rC.
We analyzed the effects of synthetic dsRNA (poly rI:rC) treatment on the expression in vivo of two interferon (IFN)-inducible genes. DBA/2 mice were injected i.p. with poly rI:rC and its effect on the levels of the following mRNAs was determined; 202, 2'-5' oligoadenylate synthetase (2-5A synthetase) and beta-actin. After poly rI:rC treatment the levels of the 202 and 2-5A synthetase mRNAs in the spleen and in bone marrow peaked between 12 and 24 h and decreased thereafter, whereas beta-actin levels remained unchained unchanged. Pretreatment of DBA/2 mice with sheep anti-murine IFN-alpha/beta antibodies before rI:rC injection strongly diminished the induction of 202 mRNA indicating that IFN-alpha/beta mediated this induction.
Animals↗
Natural antibodies to IFN-gamma in man and their increase during viral infection.
Natural antibodies to IFN-gamma were found in healthy individuals ranging from newborn babies to adults and, at higher levels, in patients suffering from different viral infections. During a viral infection, the titer of anti-IFN-gamma antibodies was observed to be correlated with the stage of the disease. Antibodies specific to IFN-gamma were affinity purified both from sera taken from healthy individuals and sera from viral-infected patients, by using a rIFN-gamma-coupled CNBr-activated Sepharose 4B column. The antibodies were found to be of the IgG class, and maintained their ability to bind rIFN-gamma. They were then tested for neutralizing activity and none of the IgG preparations we analyzed impaired the antiviral activity of rIFN-gamma. This finding suggests that the antigenic determinants recognized by these antibodies on the IFN-gamma molecule are located outside the site, on the IFN-gamma molecule, responsible for its antiviral activity.
Age Factors↗
Cell and type specificity of interferon action. Unusual characteristics of the transcriptional control of gene expression by interferon-gamma in T cells.
We have examined the mechanisms by which interferon (IFN)-gamma and IFN-alpha regulate the expression of 2'-5'-oligoadenylate synthetase (2-5A synthetase) and class I major histocompatibility complex antigens in murine T cells and in cell types of other histological origin. When treated with IFN-alpha both fibroblasts and T cell lines displayed a marked increase of the 2-5A synthetase activity and of the corresponding mRNA. The augmentation of the enzyme activity in T cells was induced by IFN-alpha at the transcriptional level, as determined by nuclear run-on analysis. In contrast IFN-gamma was capable of increasing 2-5A synthetase activity only in fibroblasts, but not in T cells. Nuclear run-on assays revealed that the 2-5A synthetase gene in T cells is not transcriptionally activated by IFN-gamma. After IFN-alpha and -gamma treatment we also observed a significant increase in class I gene expression in fibroblasts and T cell lines as measured both on the cell surface and by cytoplasmic RNA accumulation. In the case of the T cell line, DO1110, the observed increase in the steady-state levels of class I transcripts was a consequence of a high rate of H-2 gene transcription as demonstrated by run-on analysis. However, the molecular mechanisms involved in this IFN-dependent H-2 gene transcriptional activation are different between IFN-alpha and IFN-gamma. When the T cell lines DO1110, L12-R4 and EL4 were transfected with a plasmid containing a reporter gene (chloramphenicol acetyltransferase) under the control of a regulatory IFN-responsive DNA element of 237 bp or 1.4 kb, IFN-alpha was able to activate the transcription of these constructs. In contrast, IFN-gamma did not recognize the IFN-responsive element which, by itself, activated transcription of the reporter gene in response to IFN-gamma in other cellular types of non-T cell origin. Therefore, in the T cell lines examined, IFN-gamma increases the H-2 gene expression by acting on DNA elements located upstream of the regulatory segment used in this study or downstream of the cap site. This suggests a possible cell specificity in the activation of an IFN-responsive element, that in turn may regulate the IFN-inducible gene expression in a cell-specific fashion. Thus, the differential biological activities of IFN-gamma on T cells could be generated by a differential gene activation at the transcriptional level.
2',5'-Oligoadenylate Synthetase↗
Inhibition of interferon-gamma antiviral and antiproliferative activities by ras oncogene expression.
In this study, we evaluated the effect of ras oncogene activation on cell response to interferons (IFNs). For this purpose, we treated NIH 3T3 murine fibroblasts transformed by transfection with K-, Ha-, or N-ras oncogenes, either mutated or amplified, for 24 hours with IFN-gamma or IFN-alpha. We evaluated cell response by measuring virus replication, [3H]thymidine incorporation, 2',5'-oligoadenylate synthetase activation, and class I antigen induction. Transformed cells were much less responsive to IFN-gamma antiviral and antiproliferative activities than normal NIH 3T3 cells. Similarly, the induction of 2',5'-oligoadenylate synthetase following IFN-gamma treatment was completely depressed in transformed cells. Only class I antigens, measured at the cell surface and mRNA levels, appeared partially inducible by IFN-gamma in ras-transformed cells. When the same cell lines were treated with IFN-alpha, we observed full response. Because both normal and ras-transformed NIH 3T3 cells were able to bind [125I]IFN-gamma with comparable Kd values (8.3 X 10(-11) M vs. 3 X 10(-11) M, respectively), these findings suggest that ras oncogenes may differentially impair IFN-gamma activities by affecting activation of IFN-inducible genes downstream from the receptor binding event.
2',5'-Oligoadenylate Synthetase↗
Characterization of cytoplasmic and nuclear polypeptides induced by interferon-gamma in a murine pre-B cell leukemia.
The effects of interferon-gamma (IFN-gamma) on protein synthesis of a pre-B cell leukemia, L1210, have been studied by two-dimensional gel electrophoresis. In total cell extracts, at least ten proteins were induced de novo, or increased in their expression, after an 18-h IFN-gamma treatment, whereas in the nuclear extracts eight proteins were specifically induced. Of these, increased synthesis of a 40-kDa/pI 5.9 cytoplasmic protein was the most prominent and reproducible. Most of these proteins appear to be specific for a defined step of differentiation, since they are not found in other B cell leukemias upon IFN-gamma treatment. Others appear to be tissue specific, since they are not induced in fibroblasts nor in T cells. In addition, synthesis of some of the induced proteins appeared to require rapid transcription of new mRNA, because actinomycin D markedly inhibited their formation when added immediately before IFN-gamma. In keeping with this finding, in vitro translation of mRNA from IFN-gamma-treated L1210 cells into a rabbit reticulocyte lysate system, followed by analysis of the labeled proteins by two-dimensional gel electrophoresis, revealed the appearance of at least seven proteins. Taken as a whole, these results demonstrate that in leukemic pre-B cells IFN-gamma induces the transcriptional activation of genes coding for cytoplasmic and nuclear proteins, some of which could be employed as specific cell activation markers.
Animals↗
Differential control of T cell functions by IFN-alpha/beta and IFN-gamma.
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3T3 Cells↗
Interferon-gamma is not an antiviral, but a growth-promoting factor for T lymphocytes.
The effects of interferon (IFN)-gamma or IFN-alpha/beta on virus yield, (2'-5')oligo(A) synthetase activation, H-2 antigen expression and proliferation of T lymphocytes have been investigated. Under the culture conditions used, vesicular stomatitis virus or Semliki Forest virus replication in T cells was not impaired by the addition of IFN-gamma, whereas it was completely inhibited by the addition of IFN-alpha/beta. In contrast, B cell lines, macrophage-transformed cell lines and fibroblasts were fully protected by both IFN-gamma as well as IFN-alpha/beta following virus infection. The lack of sensitivity of T lymphocytes to the antiviral effects of IFN-gamma was not due to absence of specific membrane receptors, since in saturation binding experiments with 125I-labeled murine IFN-gamma most T cell lines displayed a number of binding sites and a degree of affinity comparable to those found on B cells, which are fully sensitive to IFN-gamma antiviral activity. Analysis of IFN-induced dsRNA-dependent (2'-5')oligo(A) synthetase activity, one of the biochemical markers for cellular responses to IFN, showed that it was not induced in T lymphocytes after IFN-gamma treatment, whereas IFN-alpha/beta induced high levels. Both IFN-gamma and IFN-alpha/beta enhanced H-2 antigen expression on T cells as well as on cells of different histological type. Moreover, when IFN-gamma was tested for its antiproliferative activity on T cells, it was found to consistently potentiate the response of these cells to mitogens or growth factors, rather than inhibit their proliferation. Taken as a whole these results suggest that on T lymphocytes IFN-gamma should not be regarded as an antiviral agent, but rather as a modulator of T cell growth and functional differentiation, transducing intracellular signals dissimilar to those observed with target cells of different origin.
2',5'-Oligoadenylate Synthetase↗
Interferons as gene activators. Characteristics of an interferon-activatable enhancer.
Previously we linked a 0.8-kilobase segment (including the 5'-flanking region and the 5'-terminal exon) of an interferon-activatable mouse gene (202 gene) to the chloramphenicol acetyltransferase gene and transfected the construct into mouse Ltk- cells (Samanta, H., Engel, D. A., Chao, H. M., Thakur, A., Garcia-Blanco, M. A., and Lengyel, P. (1986) J. Biol. Chem. 261, 11849-11858). Treatment of these cells with mouse beta-interferon increased the expression of the chloramphenicol acetyltransferase gene 5-10-fold. Here we demonstrate that this segment from the 202 gene has characteristics of an interferon-activatable enhancer: (a) it can activate a heterologous promoter (SV40 early promoter), (b) it is active in both the appropriate and the inverted orientation and in either upstream or downstream locations from the promoter activated, and (c) treatment of cells with interferon increases its activity severalfold.
Animals↗
Time-dependent differential effects of natural and recombinant murine interferon-gamma on ornithine decarboxylase activity of tumor cells.
Incubation of quiescent tumor cells with fetal calf serum induced ornithine decarboxylase (ODCase) activity concomitantly with mitogenic stimulation. Pretreatment of cells with highly purified natural or recombinant murine interferon-gamma (MuIFN-gamma) for 5 h caused a dose-dependent increase of ODCase activity induced by fetal calf serum (FCS). Pretreatment of target cells with IFN-gamma for 5 h in absence of FCS stimulation did not induce ODCase activity. When pretreatment of cells with natural or recombinant MuIFN-gamma was prolonged for 18 h both ODCase activity and DNA synthesis induced by FCS were suppressed. By contrast when a mixture of MuIFN-alpha and -beta was used, ODCase activity was significantly suppressed after 5 h pretreatment compared to untreated controls. These results suggest that IFN-gamma exerts a differential effect on mitogen-stimulated events depending on the dose and the time of addition.
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