An indoleamine 2,3-dioxygenase-negative mutant is defective in Stat1 DNA binding: Differential response to IFN-gamma and IFN-alpha

We have previously reported the isolation of mutant cell lines from the hum an carcinoma line ME180 that are resistant to the antiproliferative effect of interferon-gamma (IFN-gamma). These cell lines were defective in the ind uction of indoleamine 2,3-dioxygenase (IDO), a key enzyme of tryptophan cat abolism. One of these cell lines, 3B6A, was chosen for further study. This cell line was also defective in the ability of IFN-gamma to protect against vesicular stomatitis virus (VSV) infection. However it maintained a normal antiviral response to IFN-alpha. A promoter-chloramphenicol acetyltransfer ase (CAT) construct containing the promoter region of IDO, which includes I FN-gamma activation site (GAS), IFN-stimulated response element-1 (ISRE-1), and ISRE-2 regions, was not expressed in 3B6A in the presence of IFN-gamma , indicating that the defect was likely to be in either Stat1 or IFN regula tory factor-1 (IRF-1), transcription factors known to bind to these cis-act ing sequences. The induction of other IFN-gamma-inducible genes, such as tr yptophanyl-tRNA synthetase (hWRS), was also affected. Electrophoretic mobil ity shift assays (EMSA) comparing nuclear extracts from parental and mutant cells indicated that Stat1 from the mutant did not bind to GAS sequences. However, Western blot analysis indicated that Stat1 protein was present. Th is IDO-negative phenotype can be reversed by transfection with a Stat1 expr ession vector. DNA sequencing of the Stat1 cDNA from wild-type and 3B6A cel ls indicated that an amino acid change occurred in the Stat1 protein of the mutant at W573, a tryptophan conserved in all known Stat proteins. We hypo thesize that a change in this region of the Stat protein affects the respon se to IFN-gamma but not to IFN-alpha.