MUTATIONS IN THE INTERFERON-SENSITIVITY DETERMINING REGION OF HEPATITIS-C VIRUS AND TRANSCRIPTIONAL ACTIVITY OF THE NONSTRUCTURAL REGION 5APROTEIN

Amino acid (aa) mutations in the interferon-sensitivity determining re gion (ISDR) (aa position 237-276 of the nonstructural. region 5A [NS5A ] protein consisting of 447 amino acids) of hepatitis C virus (HCV) ar e related to increased interferon sensitivity and low viral load, but its mechanism has not been clarified. Recently, the NS5A protein has b een reported to have a transcriptional activation function, like other viral transactivator proteins known to repress interferon-induced gen e expression, and the ISDR overlaps one of the acidic amino acid regio ns, putative domains conferring this activity. In the present study, w e investigated the transcriptional activation function of the ISDR its elf and the effect of amino acid mutations in the ISDR on this activit y. The full-length or truncated NS5A cDNA with different ISDR sequence s was cloned into a yeast or mammalian expression vector to form a fus ion protein consisting of the GAL4 DNA-binding domain (GAL4-DBD) and N S5A 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 th e 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 amin o-terminal deletion construct of NS5A-R (aa position 228-447) lacking 227 aa, showed remarkable activity with the relative value of 117.0 ov er that of the backbone vector. The same deletion mutant of NS5A-S pro duced five times higher activity with the relative value of 575.0, ind icating that aa mutations in the ISDR profoundly affect this transcrip tional activity. In a hepatoma cell line, HuH-7, the transcriptional a ctivity was more prominent with a construct consisting of only the ISD R and short flanking sequences (aa 228-284) than larger deletion const ructs of NS5A-R, Analysis using six different ISDR clones revealed tha t different mutations enhanced this activity to various extent compare d with the wild-type ISDR. In particular, site-directed mutagenesis ta rgeted 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 sensitiv ity. The possible association between transcriptional activation and i nterferon sensitivity or viral replication should be studied further.