Recruitment of multiple interferon regulatory factors and histone acetyltransferase to the transcriptionally active interferon a promoters.

Type I interferon (IFN) plays a critical role in the innate immunity against viral infection. Expression of IFNA genes in infected cells is cell type-dependent and is regulated at the transcriptional level. The present study is focused on the molecular mechanism underlying the differential expression of human IFNA1 and A2 genes. Two nucleotides, at positions -98 and -81 of IFNA1 and A2 promoter, were pivotal to the differential expression. The DNA pull-down and chromatin precipitation assays have shown that nuclear interferon regulatory factor (IRF)-3 and IRF-7 as well as IRF-1 bind to IFNA1 virus-responsive element (VRE). Interestingly, overexpression of IRF-7 increased the otherwise weak binding of both IRF-3 and IRF-7 to IFNA2 VRE. These data together with the results of two-step chromatin immunoprecipitation strongly suggest that the IRF-3 and IRF-7 bind to IFNA1 promoter as a dimer. Furthermore, binding of IRF-3 and IRF-7 to IFNA VRE is associated with the presence of acetylated histone H3, suggesting that histone acetyltransferase(s) is tethered together with virus-activated IRF-3 and IRF-7 to the IFNA1 promoter. In addition, the constitutively active IRF-3 (5D) and IRF-7 (2D) mutants activate the endogenous IFNA genes in uninfected cells; however, the expression profile of IFNA is not identical to that induced by viral infection.

Downregulation of IRF-3 levels by ribozyme modulates the profile of IFNA subtypes expressed in infected human cells.

As an early response to viral infection, cells express a number of cellular genes that play a role in innate immunity, including alpha/beta interferons (IFN). IFN-alpha/beta are encoded by a single IFNB gene and multiple, closely related IFNA genes. The induction of these IFN genes in infected cells occurs at the transcriptional level, and two transcription factors of the IRF family, IRF-3 and IRF-7, were shown to play a role in their activation. While the expression of IRF-3 alone was shown to be sufficient for induction of the IFNB gene, induction of all the IFNA subtypes in human cells required the presence of IRF-7. Since IRF-3 is expressed constitutively in all cells examined, the role of IRF-3 in the induction of IFNA genes has not been clarified. Using ribozyme targeted to IRF-3 mRNA, we found that the downregulation of IRF-3 levels in the infected cells inhibited not only the induction of IFNB gene but also the expression of IFNA genes. Furthermore, downmodulation of IRF-3 levels altered the expression profile of IFNA subtypes induced by viral infection. These studies suggest that the ratio between the relative levels of IRF-3 and IRF-7 is a critical determinant for the induction of the individual IFNA subtypes in infected cells.

Analysis of functional domains of interferon regulatory factor 7 and its association with IRF-3.

IRF-7 plays an essential role in virus-activated transcription of IFNA genes. To analyze functional domains of IRF-7 we have constructed an amino-terminal deletion mutant of IRF-7 (237-514) which exerted a dominant negative (DN) effect on virus-induced expression of the endogenous Type I IFN genes. Focusing on the molecular mechanism underlying the dominant negative effect of IRF-7 DN, we found that virus-activated transcription of endogenous IFNA genes requires full-length IRF-7 and that Serine 483 and 484 play an essential role. While IRF-7 DN had no effect on virus-stimulated nuclear translocation of IRF-3 and IRF-7, the binding of IRF-7 DN to IRF-3 and IRF-7 was detected by GST pull-down assay as well as by immunoprecipitation in infected cells, indicating that IRF-7 DN targets both IRF-7 and IRF-3. The region by which IRF-7 interacts with IRF-3 was mapped between amino acid 418 and 473. Overexpression of IRF-7 DN in virus-infected 2FTGH cells resulted in an inhibition of IFN synthesis and in a significant reduction of binding of both IRF-3 and IRF-7 to the IFNA1 promoter. Interestingly, the IRF-7 DN-mediated suppression of IFNA gene expression can be negated by overexpression of IRF-3. Altogether these results suggest that the IRF-3/IRF-7 complexes are biologically active and are involved in virus-activated transcription of endogenous IFNA genes.

Regulation of the promoter activity of interferon regulatory factor-7 gene. Activation by interferon snd silencing by hypermethylation.

The molecular mechanism by which virus induces expression of the early inflammatory genes has not yet been completely elucidated. Previous studies indicated that the virus-mediated transcription of type I interferon (IFN) genes required activation of two members of IFN regulatory factor (IRF) family, IRF-3 and IRF-7, where the expression of IRF-7 was found to be indispensable for the induction of IFNA genes. To determine the factors that regulate expression of IRF-7 gene, as well as its inducibility by type I IFNs, we have isolated and characterized the promoter and first intron of the human IRF-7 gene. This region shows a presence of two potential interferon-sensitive response elements (ISRE/IRF-E). However, only the ISRE present in the first intron was functional and conferred interferon inducibility in a transient transfection assay. Using a pull-down assay with an oligodeoxynucleotide corresponding to this ISRE immobilized to magnetic beads, we have demonstrated that this ISRE binds ISGF3 complex and IRF-1 from the extract of IFN-treated cells but not from the untreated cells. We have further shown that the previously observed lack of expression of IRF-7 in 2fTGH fibrosarcoma cell line, correlated with hypermethylation of the CpG island in the human IRF-7 promoter. The repression of the promoter activity was relieved by treatment with DNA methyltransferase inhibitor 5-aza-deoxycytidine. In vitro methylation of IRF-7 promoter silenced IRF-7 directed expression of luciferase gene in HeLa cells that express endogenous IRF-7 gene. Whether silencing of IRF-7 by methylation is instrumental for the process of tumorigenesis remains to be determined.

Reconstitution of virus-mediated expression of interferon alpha genes in human fibroblast cells by ectopic interferon regulatory factor-7.

Type I interferons constitute an important part of the innate immune response against viral infection. Unlike the expression of interferon (IFN) B gene, the expression of IFNA genes is restricted to the lymphoid cells. Both IFN regulatory factor 3 and 7 (IRF-3 and IRF-7) were suggested to play positive roles in these genes expression. However, their role in the differential expression of individual subtypes of human IFNA genes is unknown. Using various IFNA reporter constructs in transient transfection assay we found that overexpression of IRF-3 in virus infected 2FTGH cells selectively activated IFNA1 VRE, whereas IRF-7 was able to activate IFNA1, A2, and A4. The binding of recombinant IRF-7 and IRF-3 to these VREs correlated with their transcriptional activation. Nuclear proteins from infected and uninfected IRF-7 expressing 2FTGH cells formed multiple DNA-protein complexes with IFNA1 VRE, in which two unique DNA-protein complexes containing IRF-7 were detected. In 2FTGH cells, virus stimulated expression of IFNB gene but none of the IFNA genes. Reconstitution of IRF-7 synthesis in these cells resulted, upon virus infection, in the activation of seven endogenous IFNA genes in which IFNA1 predominated. These studies suggest that IRF-7 is a critical determinant for the induction of IFNA genes in infected cells.

Lipopolysaccharide inhibits virus-mediated induction of interferon genes by disruption of nuclear transport of interferon regulatory factors 3 and 7.

We have studied the effects of lipopolysaccharide (LPS) on the Newcastle disease virus (NDV)-mediated induction of cytokine genes expression. Raw cells treated with LPS before or after virus infection showed down-regulation in the expression of interferon A and, to a lesser extent, interferon B genes. In contrast, induction of the interleukin (IL)-6 gene was enhanced. The effects of LPS were not a result of the suppression of virus replication, because the transcription of viral nucleocapsid gene was not affected. Consistent with these findings, LPS also suppressed the NDV-mediated induction of chloramphenicol acetyltransferase reporter gene driven by murine interferon A4 promoter in a transient transfection assay. Furthermore, LPS inhibited virus-mediated phosphorylation of interferon regulatory factor (IRF)-3 and the consequent translocation of IRF-3 from cytoplasm to nucleus. The LPS-mediated inhibition of IFNA gene expression was much weaker in infected Raw cells that constitutively overexpressed IRF-3. The nuclear translocation of IRF-7 in infected cells was also inhibited by LPS. These data suggest that LPS down-regulates the virus-mediated induction of IFNA genes by post-translationally targeting the IRF-3 and IRF-7 proteins.

Role of the interferon regulatory factors (IRFs) in virus-mediated signaling and regulation of cell growth.

As a response to viral infection, cells express the early inflammatory genes that encode small proteins generally called cytokines or chemokines. These protein can activate immune responses to viral infection as well as to modulate directly the outcome of viral infection. The group of proteins with the direct antiviral effects have been called interferons. The stimulation of interferon synthesis in infected cells is regulated on a transcriptional level and two families of cellular transcriptional factors seem to play a critical role in the transcriptional activation of interferon genes. The first one are the proteins of NF-kappaB family and the second is the family of the interferon responsive factors. While both of the types of the transcriptional factors are important for the induction of interferon beta gene, the NF-kappaB factor do not seems to participate in the induction of interferon alpha genes. The present review is focused on the recently identified new members of cellular IRF family and their role in virus mediated response, responses and cell growth. In addition the HHV-8 encoded vIRFs are described.

Characterization of the interferon regulatory factor-7 and its potential role in the transcription activation of interferon A genes.

The family of interferon regulatory factors (IRFs) plays an important role in modulating cellular responses to viral infection and cytokines, including IFNs. The transcription factors that are involved in the transcriptional activation of the IFNB gene have been extensively studied. However, the molecular mechanism by which virus activates the expression of the IFNA gene remains to be defined. Recently, we have identified a new IRF-7 isoform, denoted as IRF-7H, which encodes a protein of 514 amino acids and is most closely related to the IRF-3. The expression of IRF-7 is restricted to the lymphoid cell types and is inducible by virus, lipopolysaccharide, and IFNA. The functional characterization of IRF-7H reveals a presence of transactivation domain located carboxyl-terminal to its DNA binding domain. Overexpression of IRF-7H results in an activation of IFNA promoter in transient transfection assay and a strong enhancement of virus-mediated activation of this promoter. Whereas in uninfected cells, overexpressed IRF-7H is present mainly in the cytoplasm, viral infection facilitates the transfer of IRF-7H to the nucleus; overexpression of IRF-3 interferes with the virus-induced translocation of IRF-7H. Thus, IRF-7 exhibits functional similarity to IRF-3; however, the preferential expression of IRF-7 in lymphoid cells (the cell type that expresses IFNA) suggests that IRF-7 may play a critical role in regulating the IFNA gene expression.

Primary activation of interferon A and interferon B gene transcription by interferon regulatory factor 3.

The family of interferon (IFN) regulatory factors (IRFs) encodes DNA-binding transcription factors, some of which function as modulators of virus-induced signaling. The IRF-3 gene is constitutively expressed in many tissues and cell types, and neither virus infection nor IFN treatment enhances its transcription. In infected cells, however, IRF-3 protein is phosphorylated at the carboxyl terminus, which facilitates its binding to the CBP/p300 coactivator. In the present study, we demonstrate that overexpression of IRF-3 significantly enhances virus-mediated transcription of the IFNA and IFNB genes in infected cells as well as IFN synthesis. IRF-3-mediated activation of IFN genes depends in part on carboxyl-terminal phosphorylation of a cluster of Ser/Thr residues, because a mutant with Ser/Thr to Ala substitutions activates the IFN promoter less efficiently. However, overexpression of IRF-3 in human 2FTGH cells alone results in the induction of an antiviral state, which depends on functional IFN signaling, because IRF-3 does not induce an antiviral state in mutant 2FTGH cells defective in either JAK-1 or p48 functions; also no antiviral effect of IRF-3 could be demonstrated in Vero cells that lack the IFNA and IFNB genes. This finding indicates that the observed antiviral activity of IRF-3 in 2FTGH cells results mainly from the induction of IFNs. Furthermore, E1A protein inhibited IRF-3-mediated stimulation of the IFNA4 promoter in transient expression assays; this inhibition could be reversed partially by overexpression of CBP/p300 and was not demonstrated with the mutant of E1A that does not bind p300. These results identify IRF-3 and CBP/p300 as integral components of the virus-induced complex that stimulates type 1 IFN gene transcription. The observation that adenovirus E1A antagonizes IRF-3 mediated activation suggests that E1A and IRF-3 may compete for binding to CBP/p300 and implicates a novel mechanism by which adenovirus may overcome the antiviral effects of the IFN pathway.

Detection of the Human Cytomegalovirus 2.0-kb Immediate Early Gene 1 Transcripts in Permissive and Nonpermissive Infections by RNA in situ Hybridization.

The immediate early gene 1 (IE1) is the first gene to be expressed following the entry of the human cytomegalovirus (HCMV) into the cell and it does not require prior protein synthesis for its expression. Therefore, the IE1 gene is a potential candidate for the development of probes to detect HCMV in various states of infection. Using strand-specific (32)P- or digoxigenin-labeled riboprobes derived from an exon-specific subgenomic fragment of the HCMV Towne IE1 gene, we performed Northern blot analysis and RNA in situ hybridization on HCMV-infected human (permissive cells) and mouse (nonpermissive cells) fibroblasts and on 10 formalin-fixed paraffin-embedded sections of human tissue. By Northern blot analysis and by in situ hybridization, expression of the 2.0-kb IE1 gene was found in permissive as well as in nonpermissive infections. Specific nuclear and cytoplasmic hybridization was found at 5, 10, 24 and 72 h after infection in human fibroblasts. In comparison, hybridization was first detected at 10 h after infection in mouse fibroblasts. Hybridization with the IE1 probe was detected in cells with and without cytopathic changes in the formalin-fixed paraffin-embedded HCMV-infected human tissues. Hybridization patterns of the IE1 riboprobe were compared to those of the HCMV 2.7-kb major early beta-riboprobe which we have previously described [Am J Pathol 141:1247-1254;1992]. Although both riboprobes hybridize to their respective target sequences in the consecutive tissue sections, the patterns of hybridization are different. On occasion, sections of HCMV-infected human tissue showing no specific hybridization for the 2.7-kb riboprobe will show specific in situ hybridization when using the IE1 riboprobe. Our results suggest that RNA in situ hybridization with a probe directed at the IE1 transcripts is an effective method of detecting early and late stages of both permissive and nonpermissive HCMV infections. Copyright 1997 S. Karger AG, Basel

Identification of a member of the interferon regulatory factor family that binds to the interferon-stimulated response element and activates expression of interferon-induced genes.

A family of interferon (IFN) regulatory factors (IRFs) have been shown to play a role in transcription of IFN genes as well as IFN-stimulated genes. We report the identification of a member of the IRF family which we have named IRF-3. The IRF-3 gene is present in a single copy in human genomic DNA. It is expressed constitutively in a variety of tissues and no increase in the relative steady-state levels of IRF-3 mRNA was observed in virus-infected or IFN-treated cells. The IRF-3 gene encodes a 50-kDa protein that binds specifically to the IFN-stimulated response element (ISRE) but not to the IRF-1 binding site PRD-I. Overexpression of IRF-3 stimulates expression of the IFN-stimulated gene 15 (ISG15) promoter, an ISRE-containing promoter. The murine IFNA4 promoter, which can be induced by IRF-1 or viral infection, is not induced by IRF-3. Expression of IRF-3 as a Gal4 fusion protein does not activate expression of a chloramphenicol acetyltransferase reporter gene containing repeats of the Gal4 binding sites, indicating that this protein does not contain the transcription transactivation domain. The high amino acid homology between IRF-3 and ISG factor 3 gamma polypeptide (ISGF3 gamma) and their similar binding properties indicate that, like ISGF3 gamma, IRF-3 may activate transcription by complex formation with other transcriptional factors, possibly members of the Stat family. Identification of this ISRE-binding protein may help us to understand the specificity in the various Stat pathways.

Stimulation of interferon and cytokine gene expression by imiquimod and stimulation by Sendai virus utilize similar signal transduction pathways.

The imidazoquinolineamine derivative 1-(2-methyl propyl)-1H-imidazole [4,5-c]quinoline-4-amine (imiquimod) has been shown to induce alpha interferon (IFN-alpha) synthesis both in vivo and in peripheral blood mononuclear cells in vitro. In this study, we show that, in these cells, imiquimod induces expression of several IFNA genes (IFNA1, IFNA2, IFNA5, IFNA6, and IFNA8) as well as the IFNB gene. Imiquimod also induced the expression of interleukin (IL)-6, IL-8, and tumor necrosis factor alpha genes. Expression of all these genes was transient, independent of cellular protein synthesis, and inhibited in the presence of tyrosine kinase and protein kinase C inhibitors. Infection with Sendai virus led to expression of a similar set of cytokine genes and several of the IFNA genes. Imiquimod stimulates binding of several induction-specific nuclear complexes: (i) the NF-kappa B-specific complexes binding to the kappa B enhancer present in the promoters of all cytokine genes, but not in IFNA genes, and (ii) the complex(es) binding to the A4F1 site, 5'-GTAAAGAAAGT-3', conserved in the inducible element of IFNA genes. These results indicate that imiquimod, similar to viral infection, stimulates expression of a large number of cytokine genes, including IFN-alpha/beta, and that the signal transduction pathway induced by both of these stimuli requires tyrosine kinase and protein kinase activity.

Virus-mediated induction of interferon A gene requires cooperation between multiple binding factors in the interferon alpha promoter region.

Transcriptional activation of interferon A (IFNA) gene in virus-infected cells is controlled by a 35-nucleotide inducible element that is cell type specific. Within this region, two elements, alpha F1 and IRF-1 binding sites, were shown by mutation analysis to play a crucial role in the expression of inducible element. In this study, we have analyzed the binding of nuclear proteins to the alpha F1 sequence and have shown that the induction is associated with the formation of a novel complex alpha F1/B, which contains at least two DNA binding proteins of 68 and 96 kDa. In contrast, no binding of the purified interferon regulatory factor 1 (IRF-1) either to the alpha F1 or IRF-1 binding sites could be detected in vitro. However, the oligonucleotides corresponding to alpha F1 or IRF-1 binding sites competed efficiently for the induction of IFNA4 promoter region in a transient transfection assay. We suggest that the induction of IFNA promoter region requires cooperation between alpha F1 binding proteins and IRF-1. Interestingly, our data also show that the inability of IFNA6 promoter to be expressed in infected L-cells may be a result of a viral-induced repressor, which could act by binding and inactivating alpha F1 or by competing for the IRF-1 binding site. These results suggest that cell-specific expression of IFNA genes results from core-cruitment of trans-acting factors that bind to alpha F1 and the IRF-1 binding site with the cell-specific virus-induced activator or repressor.

Localization of the human cytomegalovirus 2.7-kb major early beta-gene transcripts by RNA in situ hybridization in permissive and nonpermissive infections.

During the early phase of a human cytomegalovirus (HCMV) infection, the 2.7-kb early gene is by far the most abundantly transcribed RNA. Using strand-specific 32P or digoxigenin-labeled riboprobes derived from a subgenomic fragment of the HCMV Towne 2.7-kb early gene, we have performed Northern blot analysis and RNA in situ hybridization on human and mouse fibroblasts infected with HCMV and on 23 formalin-fixed paraffin-embedded sections of tissue obtained at autopsy. By Northern blot analysis, expression of the 2.7 kb early gene was found only in permissive infections. In contrast, specific hybridization was detected in both permissive and nonpermissive cells by RNA in situ hybridization. In nonpermissive cells, hybridization was weak and predominantly nuclear. In permissive cells, strong nuclear and cytoplasmic hybridization was noted. Specific hybridization to cells with and without cytopathic changes was detected with the anti-sense probe in CMV infected tissue obtained at autopsy. When the sense riboprobe was employed, no specific hybridization was found under nondenaturing conditions. These results suggest that in situ hybridization with a probe directed at the 2.7-kb early gene is an effective method of detecting both permissive and nonpermissive HCMV infections in different stages of infection and in localizing the expression of the major early gene in various cell types.

Distinct activation of murine interferon-alpha promoter region by IRF-1/ISFG-2 and virus infection.

Virus infection in mouse L929 cells activates expression of interferon-alpha 4 (IFN-alpha 4), but not IFN-alpha 6. The integrity of a symmetrical sequence, GTAAAGAAAGT (alpha F1 site); (-103 to -93), present in the 35 nucleotide (nt) long inducible element (IE) (-109 to -75) of the alpha 4 promoter region is essential for the virus-induced expression. In the present study, we have shown that the interferon regulatory factor 1 (IRF-1) can induce expression of both IFN-alpha 4 and -alpha 6 in a transient expression assay. Virus infection cooperates with IRF-1 and further enhances transcription from the alpha 4 promoter, but inhibits the IRF-1-mediated expression from the alpha 6 promoter. The virus-mediated induction is determined by both IRF-1 and alpha F1 sites, while activation by IRF-1 in a cotransfection assay is not greatly influenced by the alpha F1 sequence. The activation of IFN-alpha gene promoters by IRF-1 was limited to the transient expression assay. The integrated alpha 4 promoter or the endogenous IFN-alpha genes could not be induced by transfection with IRF-1 expressing plasmid and IRF-1 did not up-regulate expression of the endogenous IRF-1 gene. However, expression of IRF-1 alone was sufficient to up-regulate the expression of two IFN stimulated genes, 2',5' oligoadenylate synthetase (OAS) and interferon stimulated (ISG)-15 gene. These results suggest that induction of IFN-alpha gene expression by virus infection requires cooperation between IRF-1 and another factor(s) that binds to the alpha F1 sequence.

Virus-associated RNAs (VA-I and VA-II). An efficient target for the detection of adenovirus infections by in situ hybridization.

The identification of adenovirus in tissue can be difficult. In situ hybridization for adenovirus nucleic acids may aid in the demonstration of adenovirus infections. To develop a probe against adenovirus, a 978 bp fragment of DNA containing the VA-I, VA-II, and a portion of the L-1 regions of the adenovirus type 2 genome was cloned into the SK+ vector. These regions were selected because they are generally conserved among adenoviruses and are abundantly transcribed during the lytic cycle. Sense and antisense tritium or Digoxigenin-labeled riboprobes were generated using in vitro transcription and applied to formalin-fixed paraffin-embedded sections of HeLa cells infected with adenovirus type 2. Extensive in situ hybridization of the antisense riboprobe to HeLa cells with cytopathic changes was found. The number of cells to which the probe hybridized decreased proportionately with dilution of infected with noninfected cells. The control sense riboprobe showed only scattered breakthrough hybridization and in these cells hybridization was mainly located in the nucleus. Northern blot analysis of RNA from infected HeLa cells confirmed the in situ hybridization results. No hybridization was detected when cultured cells infected with herpes simplex virus, Epstein-Barr virus, cytomegalovirus, or human immunodeficiency virus were examined. Specific hybridization was detected in tissues obtained at autopsy from four patients with culture proven adenovirus infection. These observations suggest that this probe is useful in the diagnosis of adenovirus in formalin-fixed paraffin-embedded material.

Identification of a novel virus-responsive sequence in the promoter of murine interferon-alpha genes.

We have previously shown that the infection of mouse L-cells with Newcastle disease virus activates transcription of the alpha 4 but not the alpha 6 interferon gene and that the induction is mediated by a 35-base pair inducible element (IE) found in the alpha 4 promoter (-109 to -75). In the present study, we show that the inactivity of the alpha 6 promoter can be mapped to 2 out of 6 nucleotides in which the alpha 6 differs from alpha 4 IE. The symmetrical sequence, GTAAAGAAAGT (-103 to -93), present in the alpha 4 IE is essential for its inducibility and binding of nuclear protein(s) to the alpha 4 IE.

Virus infection and interferon can activate gene expression through a single synthetic element, but endogenous genes show distinct regulation.

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.