A Regulatory Domain within the Virus‐Response Element of the Interferon α1 Gene Acts as a Transcriptional Repressor Sequence and Determinant of Cell‐Specific Gene Expression

Abstract

Type-I interferons are encoded by a multigene family, the major members of which are at least 13 IFN A subtypes and a single IFN B gene. IFNs A and B are induced in response to similar stimuli, such as virus infection and double-stranded RNA, but in different cell types: the induction of IFN A is almost exclusively restricted to cells of lymphoid origin, while IFN B has been found to be induced in a variety of cell types including fibroblasts. The virus-responsive enhancer element in the promoter region of IFN A family members is largely responsible for the differential expression of individual subtypes in responsive cells. In this paper we describe experiments which address the issue of the differential expression of IFN A and IFN B in different cell types. We show that IFN-P is induced in a variety of cells of different origin, while not all of these are able to secrete IFN-a. By transfection of reporter gene constructs comprising the virus-responsive enhancer from the IFN A1 and IFN B genes, we show that this differential response is mediated at the level of transcription via these control elements. More detailed analysis of the function of these regions identifies specific sequences within the IFN A1 virus response element that has an inhibitory effect on expression in cells that are normally inducible, and is also implicated in the overall suppression of IFN A induction in non-inducible cells.Keywords: interferon ; transcription ; cell specific ; Sendai virus ; repression.The interferons (IFNs) are a family of secreted cytokines induced in mammalian cells in response to a number of stimuli including virus infection (reviewed in [3 -51). The interferon proteins function via a cell surface receptor, and confer antiviral and antiproliferative effects on cells. The interferons are subdivided into two major classes, type I and type 11, on the basis of their immunological and biological properties. These two types are largely unrelated, although they act through an overlapping network of responsive genes. The work in this paper addresses the induction of type-I gene expression. The type-I IFNs are encoded by a multigene family located on chromosome 9 in human cells [6][7][8] (chromosome 4 in mouse) and may be further subdivided into three groups. IFN-a is expressed almost exclusively in cells of lymphoid origin, and consists of several subtypes derived from around 13 functional genes. IFN-P was initially identified as being produced by fibroblast cells [9] and is derived from a single gene. Expression of IFN-/I is more widespread than TFN-a, with most cell types being capable of its secretion. The third identified member of the type I IFN family is trophoblast IFN (IFN-w) Virus induction of the expression of interferons has been demonstrated to occur at the level of gene transcription (reviewed in [12-141), although regulation may also occur at other levels, including that of mRNA instability [I 5, 161. Transcriptional enhancer sequences through which virus induction is mediated have been identified for both hu...

The Interferon Regulatory Factor, IRF5, Is a Central Mediator of Toll-like Receptor 7 Signaling

“…Gal4-IRF5 (19), Gal4-IRF7 (49), FLAG-IRF5 (19), IRF7-GFP, and IRF5-GFP (19) were described previously. The IFNA1 and IFNB-secreted alkaline phosphatase reporter plasmids were as described (50). TRAF2 and TRAF6 mutant constructs were from H. Wesche (Tularik, CA) (51).…”

“…Additionally, cells were transfected with 50 ng of the ␤-galactosidase expression plasmid to allow for the normalization of the data. 20 h after transfection cells were divided and seeded into 24-well plates and stimulated with 10 M R-848 or infected with NDV (240 hemagglutination units; 1 hemagglutinin unit is defined as the amount of virus needed to agglutinate an equal volume of standardized red blood cell suspension) or VSV (multiplicity of infection 2) for an additional 16 h. The secreted alkaline phosphatase activity was determined as described (50). All data are expressed as the mean relative stimulation Ϯ S.D.…”

Virus-induced Heterodimer Formation betweenIRF-5 and IRF-7 Modulates Assembly of theIFNA Enhanceosome in Vivo and Transcriptional Activity of IFNA Genes

“…For the SAP assays, equal amounts (2.5 g) of reporter plasmid and IRF-expressing plasmid were co-transfected with the ␤-galactosidase expression plasmid (50 -100 ng). The transfected cells were divided 14 h later and infected with NDV for 16 h. The alkaline phosphatase (SAP) assays were performed as described previously (44,46). Each experiment was repeated 3 times.…”

“…Similarly, IRF-1, IRF-3, IRF-5, and IRF-7 expression plasmids (20,21,27,43); HuIFNA1 and -A14 soluble alkaline phosphatase (SAP) reporter plasmids (44,45); and polyclonal IRF-1, IRF-3, and IRF-7 antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). M2 anti-FLAG monoclonal antibody was obtained from Sigma.…”

Downregulation of IRF-3 Levels by Ribozyme Modulates the Profile of IFNA Subtypes Expressed in Infected Human Cells

“…Virusresponsive elements (VRE) were identified in promoters of IFNA and IFNB genes that alone can confer virus-mediated activation. The VRE show sequence motifs that are highly conserved in both IFNA and IFNB promoters (2,28). The molecular mechanisms of activation of IFNB gene expression by virus infection or double-stranded (ds) RNA have been studied extensively (4,6,8,9,17,25,26,27,28).…”