Differentiation-dependent activation of interferon-stimulated gene

We have recently shown that the adenovirus EIA gene products block interferon-a-induced signal transduction and transcription factor NF-cB-mediated gene induction. Here we report that the same responses are also blocked in undifferentiated F9 teratocarcinoma cells. The block was removed upon cellular differentiation and regained upon the introduction of viral EIA into the differentiated cells. In undifferentiated cells, interferon-,8 failed to induce the transcription of interferon-responsive genes because of a lack of activation of the cognate trans-acting factors. As a result, in these cells, virus replication was not inhibited by interferon. Similarly, in undifferentiated but not in differentiated F9 cells, tumor necrosis factor a failed to stimulate NF-#c-mediated transcription of a reporter gene because of a failure in the activation of NF-#cB trans-acting factor. These results suggest that a ceUlular ElA-like activity, present in undifferentiated F9 cells, and adenoviral ElA use similar mechanisms for repressing the expression of specifi cellular genes. Interferons (IFNs) are multifunctional cytokines that are components of the host defenses against viral and parasitic infections and certain tumors. IFNs also have immunoregulatory functions and they affect cell proliferation and differentiation (1, 2). Postreceptor signaling by IFN-a/P is mediated by the activation of the IFN-stimulated gene factors (ISGFs). Once activated, these factors bind to the IFNstimulated response elements (ISREs) of the IFN-a/,inducible genes. Although murine ISGFs have not been well-characterized, several such factors have been identified in the human cells. Among these, the activation and functioning of one, ISGF3, closely correlates with the induced transcription of IFN-stimulated genes (ISGs) (3-7). ISGF3 is composed of two functional subunits, ISGF3y and ISGF3a. Inactive ISGF3a, present in the cytoplasm, gets activated by phosphorylation (8-15) within seconds after IFN-a/,f binds to the receptor. Activated ISGF3a (11-13) binds to ISGFy (14) and the complex translocates to the nucleus where it binds to the ISRE-containing genes and stimulates their transcription. We (15) and others (16, 17) have shown that the adenoviral ElA proteins inhibit the IFN responses by blocking the activation of ISGF3. In a more recent study, we have shown (18) that the tumor necrosis factor a (TNF-a)mediated induction of interleukin 6 (IL-6) gene, through an NF-KB element, was also blocked by EIA gene products. Detailed analysis revealed that ElA alters the composition of the NF-KB complex, which binds to its cognate element in the IL-6 gene (18). Induction of IFN biosynthesis is dependent upon the state of cellular differentiation (19-21). In undifferentiated embryonal carcinoma (EC) cells, virus infection or double-stranded RNA fails to induce IFN-,8 synthesis (19-21). This deficiency can be overcome by either inducing cellular differentiation or expressing the trans-acting factor interferon regulatory factor 1 (IRF-1), introduced by transfection, in the undifferentiated cells (21). Several studies have shown that the undifferentiated EC cells also do not respond to IFN-a/,B (22-24), although others claimed that they respond partially (25). The undifferentiated F9 cells have been shown to contain an ElA-like activity that is lost upon retinoic acid-induced differentiation (26-28). However, ectopic expression of adenoviral EIA gene results in the reversal of the fully differentiated state (27). Concomitantly, expression of a large number ofdifferentiation-specific genes was also reversed by ElA. In the present work, we report that the cellular E1Alike activity of F9 cells, like the viral ElA, inhibits the ISGF3-dependent and NF-KB-dependent gene expression. MATERIALS AND METHODS Cells, Viruses, and Plasmids. For the present investigation, we employed F9 EC cells in three states of differentiation: undifferentiated (F9), terminally differentiated by treatment of cells with retinoic acid (dF9), and terminally differentiated cells stably transfected with the adenovirus EIA12S gene (dF9-ElA). Cells were cultured in Dulbecco's modified Eagle's medium (27). dF9 cells were obtained by growing the F9 cells in medium containing retinoic acid (0.1 uM). mIFN-fl and mIFN-y were from Lee Biomolecular Laboratories (San Diego) and Genentech, respectively. Phorbol 12-myristate 13-acetate (PMA) and TNF-a were purchased from Sigma and Genentech, respectively. pRM-CAT, vesicular stomatitis virus (VSV), and encephalomyocarditis virus were described (15). IP10-CAT gene was provided by T. Hamilton (The Cleveland Clinic Foundation). IRF-1 and (2'5')oligoadenylate synthetase cDNAs were as described (15, 21). Antiviral Assays. Antiviral assays were carried out using VSV and encephalomyocarditis virus as described (15). After 30 h ofinfection, the plates were stained with methylene blue. The dye from the plates was extracted and the absorbance at 595 nm was measured. Average data from triplicate samples are presented. Transcriptional Analysis. Procedures for Northern blot analyses, nuclear run-off transcription, DNA transfections, chloramphenicol acetyltransferase (CAT) assays, and electrophoretic-mobility-shift assays were described in our publications (3, 15). Abbreviations: IFN, interferon; ISGF, IFN-stimulated gene factor; ISRE, IFN-stimulated response element; ISG, IFN-stimulated gene; TNF-a, tumor necrosis factor a; IL-6, interleukin 6; EC, embryoneal carcinoma; IRF-1, interferon regulatory factor 1; PMA, phorbol 12-myristate 13-acetate; VSV, vesicular stomatitis virus; CAT, chloramphenicol acetyltransferase. *To whom reprint requests should be addressed. 3167 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 3168 Cell Biology: Kalvakolanu and Sen