Amino acid (aa) mutations in the interferon-sensitivity determining region (ISDR) (aa position 237-276 of the nonstructural region 5A [NS5A] protein consisting of 447 amino acids) of hepatitis C virus (HCV) are related to increased interferon sensitivity and low viral load, but its mechanism has not been clarified. Recently, the NS5A protein has been reported to have a transcriptional activation function, like other viral transactivator proteins known to repress interferon-induced gene expression, and the ISDR overlaps one of the acidic amino acid regions, putative domains conferring this activity. In the present study, we investigated the transcriptional activation function of the ISDR itself and the effect of amino acid mutations in the ISDR on this activity. The full-length or truncated NS5A cDNA with different ISDR sequences was cloned into a yeast or mammalian expression vector to form a fusion protein consisting of the GAL4 DNA-binding domain (GAL4-DBD) and NS5A protein. Following transfection, the transcriptional activities of these constructs were determined using beta-galactosidase (yeast) or chloramphenicol acetyltransferase (CAT) (mammalian cell) reporter gene expression under the control of GAL4 binding sites. In yeast, both the full-length sequence of NS5A-R (a clone with one aa mutation in the ISDR) and NS5A-S (a derivative of NS5A-R with six aa mutations in the ISDR) had no distinguishable transcriptional activity, whereas an amino-terminal deletion construct of NS5A-R (aa position 228-447) lacking 227 aa, showed remarkable activity with the relative value of 117.0 over that of the backbone vector. The same deletion mutant of NS5A-S produced five times higher activity with the relative value of 575.0, indicating that aa mutations in the ISDR profoundly affect this transcriptional activity. In a hepatoma cell line, HuH-7, the transcriptional activity was more prominent with a construct consisting of only the ISDR and short flanking sequences (aa 228-284) than larger deletion constructs of NS5A-R. Analysis using six different ISDR clones revealed that different mutations enhanced this activity to various extent compared with the wild-type ISDR. In particular, site-directed mutagenesis targeted to the aa position 252 showed that this aa residue had profound influence on the activity. These results suggest that the ISDR has a transcriptional activity, and it is enhanced by aa mutations that are also related to decreased viral load and increased interferon sensitivity. The possible association between transcriptional activation and interferon sensitivity or viral replication should be studied further.