The interferon‐stimulated gene 54 K promoter contains two adjacent functional interferon‐stimulated response elements of different strength, which act synergistically for maximal interferon‐α inducibility

Abstract

The interferon-a(1FN-a)-regulated hamster ISG-54 K gene, which is activated in hamster CHO-12 cells at least 40-fold, was isolated and the promoter region was characterized in detail. Sequence analysis revealed the presence of two elements, closely related to the interferon-stimulated-responseelement (ISRE) consensus sequence [AGTTTCNNTTTC(,C)]. The putative ISRE-I sequence (GGTTTCAATTTCT) is located at position -97 to -85 ; ISRE-I1 (AGTTTTACTTTCT), which differs at three positions from ISRE-I, is found directly upstream of ISRE-I at position -110 to -98. In a transient transfection assay in CHO-12 cells the wild-type hamster ISG-54K-promoterchloramphenicol-acetyl-transferase (CAT) reporter construct showed a 40 -80-fold induction, offering an excellent model to study the functional properties of the two ISRE. To find out whether both elements were functional in interferon regulation of the promoter, selected point mutations were introduced in the -110 to -85 region and in flanking sequences. The (mutated) ISG-54 K promoter was linked to the CAT reporter gene and transiently expressed in CHO cells in the absence and presence of murine (MujIFN-a6. Transfections showed that both the -97 to -85 (ISRE-I) and the -110 to -98 (ISRE-11) segment were needed for optimal interferon induction of the ISG-54 K promoter. However, ISRE-I has an approximately sevenfold stronger activity compared to ISRE-11. Sequential substitution of the three ISRE-I bases, which differ in ISRE-I1 showed that the T at position -105 causes the lower activity of ISRE-11. Transfection of ISG-54 K promoter constructs, in which ISRE-I was replaced by ISRE-11, which generates a promoter with two ISRE-I1 segments, and vice versa (two ISRE-I), provided further evidence for a role of both elements in IFN-a induction. Importantly, all data obtained in transfection studies show that the two ISRE cooperate synergistically. The mechanism of synergism is most probably an indirect interaction between transcription factors binding to the ISRE, because an increase in the spacial arrangement of the two ISRE with a complete helical turn or half a turn did not result in a substantial decrease in promoter activity.Type 1 interferons (IFN-a/IFN-P) are a family of cytokines, which induce multiple cellular changes. The major biological responses of cells treated with interferons are the inhibition of viral replication in these cells and a decrease in cell-growth rate. During the last few years several steps of the molecular mechanism of the interferon-induced signaling pathway have been elucidated, but many aspects remain unclear (for recent reviews [l, 21).Interferons interact with cells through specific cell surface receptors. The interaction of IFN-a/IFN-P with its proper receptor ultimately results in the induction of the expression of a group of IFN-a/IFN-P stimulated genes. I m - a/IFN-P-induced gene expression is directly regulated at the level of transcription [3, 41. Transcriptional stimulation in response to interferon treatment is at least partially mediate...

Combinatorial association and abundance of components of interferon-stimulated gene factor 3 dictate the selectivity of interferon responses.

“…Chloramphenicol acetyltransferase (CAT) plasmids containing the hamster ISG54 promoter with wild-type or modified ISRE sequences have been described (20). Full-length cDNA expression constructs for ISGF3,y (p48) (5), STAT113 (3), STAT91 (2), and Tpr-Met (21) (details available on request) were driven by the cytomegalovirus promoter (22).…”

Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

“…Plasmids encoding human FLAG-tagged TBK-1 and IKKε were kindly provided by John Hiscott (McGill University) and have been described previously (54). The reporter vectors pHISG-54-CAT (CAT, chloramphenicol acetyltransferase) and pIFN-␤-CAT have been described previously (8,61). Firefly luciferase cloned into pCAGGS was used as a transfection control.…”

A Recombinant Influenza A Virus Expressing anRNA-Binding-Defective NS1 Protein Induces High Levels of BetaInterferon and Is Attenuated inMice

“…For the experiments investigating IFN-␤ and ISRE promoter activation, 293T cells were transfected with a mouse IFN-␤ promoter-driven chloramphenicol acetyltransferase (CAT) reporter plasmid, pIFN-CAT (40), or an ISRE-driven CAT reporter plasmid, pHISG-54-CAT (2,5). In addition, an internal control plasmid, pGL2-control (Promega), encoding the firefly luciferase protein, and the appropriate pCAGGS-NS1 expression plasmid were transfected into the cells.…”