Faculty Bibliography

Interferon-alpha (IFN alpha) and interferon-gamma (IFN gamma) each induce in susceptible target cells a state of resistance to viral replication and reduced cellular proliferation, presumably through different mechanisms: these two polypeptides are unrelated by primary sequence and act through distinct cell-surface receptors to induce expression of largely non-overlapping sets of genes. However, acting in concert, they can produce synergistic interactions leading to mutual reinforcement of the physiological response. In HeLa cells, this synergistic response was initiated by cooperative induction of IFN alpha stimulated genes (ISGs). These normally quiescent genes were rapidly induced to high rates of transcription following exposure of cells to IFN alpha. Although they were only negligibly responsive to IFN gamma, combined treatment of cells with IFN gamma followed by IFN alpha resulted in an approximately 10-fold increase in ISG transcription. ISG transcription is dependent upon ISGF3, a positive transcription factor specific for a cis-acting regulatory element in ISG promoters. IFN gamma treatment induced increased synthesis of latent ISGF3, which was subsequently activated in response to IFN alpha to form approximately 10-fold higher levels than detected in cells treated with IFN alpha alone. ISGF3 is composed of two distinct polypeptide components, synthesis of one of which was induced by IFN gamma, increasing its cellular abundance from limiting concentrations to a level which allowed formation of at least 10 times as much active ISGF3. Cell lines vary in their constitutive levels of the inducible component of ISGF3 and in the ability of IFNs to increase its synthesis.(ABSTRACT TRUNCATED AT 250 WORDS)

Virus inducible elements (IE) in promoters of mouse alpha-interferon and human beta 1-interferon genes contain multiple copies of the hexanucleotide sequence AGT-GAA or its variants which are also found in the interferon-stimulated response element of genes transcriptionally induced by interferon. We have examined the similarities between virus and interferon induction of gene expression and the role of AGTGAA and AAT-GAA hexamers in these responses. Hybrid plasmids were constructed by inserting the IE region, the alpha 4 promoter, or the multiple copies of AGTGAA or AAT-GAA 5' to the inactive-45 human immunodeficiency-chloramphenicol acetyltransferase hybrid gene, and their inducible expression was studied in a transient expression assay. In L-cells, multiple hexamers were efficiently induced both by infection with Newcastle disease virus and by interferon treatment; while the alpha 4 promoter and the IE inducible region were induced predominantly by virus rather than by interferon. In order to dissociate the effect of virus and endogenous interferon on the induction process, we examined the gene expression in Vero cells, which have undergone homozygous deletion of type 1 interferon genes, and in VNPT-159 cells, which were derived from Vero cells by insertion of an inducible human interferon beta 1 gene. The results show that while the alpha 4 promoter was efficiently induced only by virus in both cell types, the constructs containing shorter segments of the IE were induced by both virus and interferon in Vero cells. However, the inducibility by interferon was not detected in VNPT-159 cells, suggesting that the presence of endogenous interferon suppresses interferon-induced expression of hexanucleotide repeats and the short inducible region. In contrast, virus inducibility of endogenous interferon-stimulated genes, ISG-15 and ISG-54, was about 100-fold more efficient in VNPT-159 cells than in Vero cells, suggesting that this induction is largely mediated through synthesis of endogenous interferon. Hence, endogenous interferon may play a role in the autoregulation of both interferon genes and interferon-stimulated genes

The signal transduction pathway through which interferon-alpha (IFN alpha) stimulates transcription of a defined set of genes involves activation of DNA-binding factors specific for the IFN alpha-stimulated response element (ISRE). IFN-stimulated gene factor-3 (ISGF3), the positive regulator of transcription, was derived in response to IFN alpha treatment from preexisting protein components that were activated first in the cell cytoplasm prior to appearance in the nucleus. Nuclear translocation of ISGF3 required several minutes and could be inhibited by NaF. Formation of active ISGF3 was mimicked in vitro by mixing cytoplasmic extracts from IFN alpha-stimulated cells with extracts of cells treated to contain high amounts of the unactivated factor. Active ISGF3 was found to be formed from association of two latent polypeptide precursors that were distinguished biochemically by differential sensitivity to N-ethyl maleimide. One precursor was modified in response to IFN alpha occupation of its cell-surface receptor, thus enabling association with the second subunit. The resulting complex then was competent for nuclear translocation and binding to ISRE. Cytoplasmically localized transcription factor precursors thus serve as second messengers to translate directly an extracellular signal into specific transcriptional activity in the nucleus

Transcriptional regulation of the gene encoding a guanylate-binding protein (GBP) by the two interferon (IFN) types was studied. GBP gene transcription was regulated by alpha IFN in a manner identical to that of previously described IFN-stimulated genes (ISGs): rapid induction, without a need for protein synthesis, followed by a protein synthesis-dependent suppression of transcription to basal levels within 6 h. Transcriptional induction by gamma IFN was equally rapid and independent of ongoing protein synthesis but remained at elevated levels for greater than 24 h. Experiments employing combined treatments with IFNs of both types revealed that induction of the GBP gene by gamma IFN overrides the alpha IFN-induced active repression and reverses the alpha IFN-induced repressed state. Moreover, the alpha IFN-mediated repression of ISG54, a gene normally responsive to only alpha IFN, is also reversed by gamma IFN. Induction of GBP by gamma IFN is presumably mediated by a factor different from the recently described activator Interferon Stimulated Gene Factor 3 (ISGF3) because induction of this factor was not observed upon treatment of cells with gamma IFN. Finally, a complex set of reinforcing or synergistic effects were observed when induction of the GBP gene was evoked by a combined treatment with the two IFN types

Human cultured cell lines deficient in their ability to respond to type I interferon (IFN) fail to interrupt cellular proliferation or to induce an antiviral state following exposure to IFN alpha. Comparison of non-responsive Daudi and HeLa cell lines with IFN-responsive partner cell lines and examination of non-responsive Raji cells showed that the defective cell lines expressed type I IFN receptors of typical number and affinity and bound IFN equivalently compared to the normal cells. However, transcriptional induction of interferon-stimulated genes (ISGs) was greatly reduced and delayed in these cell lines, leading to reduced accumulation of ISG mRNA. Furthermore, the rapid activation of IFN-stimulated promoter binding factors whose appearance correlates with ISG transcriptional induction, did not occur in non-responsive cells. Thus, the primary defect of these cells leading to an impaired physiological response to IFN appears to be an inability to activate promoter-binding factors necessary to trigger ISG transcription, an obligate early step in antiviral and antiproliferative physiology

Nuclear proteins induced by interferon (IFN) treatment of human cells are capable of forming two specific complexes with DNA fragments containing the IFN-stimulated response element (ISRE). These two complexes, designated B2 and B3, are distinguished by differential migration in gel retardation assays. The factor that forms the B3 complex, termed IFN-stimulated gene factor 3 (ISGF-3), preexists in cells, is activated upon IFN treatment, and appears with kinetics paralleling those for transcriptional activation of IFN-stimulated genes. The factor that forms the B2 complex (ISGF-2) appears following a time lag after IFN treatment during which protein synthesis must occur. By extensive point mutagenesis of the ISREs from two IFN-stimulated promoters (ISG54 and ISG15), we demonstrate that the B2 and B3 complexes are formed by factors binding to the same DNA sequence. Mutations at this site decrease or eliminate transcriptional activation and impair binding of both ISGF-2 and ISGF-3. This analysis has shown that the ISGF-3 binding site is slightly broader than the ISGF-2 binding site, which is completely contained within the sequence necessary both for ISGF-3 binding and for transcriptional activation. The evidence strongly implicates ISGF-3 as the positive transcriptional regulator of IFN-stimulated genes

Human alpha- and beta-interferons (IFNs) stimulate rapid but transient increases in transcription from a set of previously quiescent genes. Protein synthesis is not required for initial stimulation, but duration of the response is limited to a few hours by a process requiring synthesis of new proteins. An IFN-stimulated response element (ISRE) was identified 5' to an inducible gene by deletion analysis and point mutagenesis, and sequence comparisons with other promoters defined the consensus element YAGTTTC(A/T)YTTTYCC. Two classes of IFN-inducible nuclear factors were found that bind to the ISRE. The most rapidly induced factor appeared without new protein synthesis, whereas a second factor required active protein synthesis for its appearance and maintenance. The kinetics of appearance and loss of these binding activities correlate with the activation and repression of IFN-stimulated genes. These different IFN-activated or induced factors may bind sequentially to the same essential promoter element to first increase and then repress transcription

Genes in human chromosomes that normally require induction by alpha-interferon are activated after calcium phosphate (CaPO4) transfection, but not after DEAE-dextran transfection. The c-fos gene and genes stimulated by gamma-interferon also are affected by CaPO4-DNA precipitates, but the calcium ionophore A23187 stimulates only c-fos among this group. These results suggest caution not only in choosing gene transfer methods, but also in interpreting experiments aimed at understanding the role of second messengers in gene activation

Interferon treatment of cell cultures results in the rapid transcriptional induction of a specific set of genes. In this paper we explore the effect of cellular infection by several adenoviruses, both wild type and mutant, on the expression of these genes. Infection with adenovirus induces the transcription of the interferon-stimulated genes in the absence of any protein synthesis. In fact, the inhibition of protein synthesis during a wild-type infection produces enhanced stimulation of transcription of these genes. Experiments with viral mutants indicate the ability to specifically suppress this transcription maps to the E1A gene. In addition, the E1A gene products are capable of suppressing the specific transcriptional induction of interferon-stimulated promoters during cotransfection experiments and therefore presumably during viral infection. The dual effect of adenovirus on the expression of interferon-stimulated genes may represent an example of action and evolutionary reaction between virus and host