Interferon (IFN) activates the signal transducer and activator of transcription (STAT) pathway to regulate immune responses. The protein inhibitor of activated STAT (PIAS) family has been suggested to negatively regulate STAT signaling. To understand the physiological function of PIAS1, we generated Pias1(-/-) mice. Using PIAS1-deficient cells, we show that PIAS1 selectively regulates a subset of IFN-gamma- or IFN-beta-inducible genes by interfering with the recruitment of STAT1 to the gene promoter. The antiviral activity of IFN-gamma or IFN-beta was consistently enhanced by Pias1 disruption. Pias1(-/-) mice showed increased protection against pathogenic infection. Our data indicate that PIAS1 is a physiologically important negative regulator of STAT1 and suggest that PIAS1 is critical for the IFN-gamma- or IFN-beta-mediated innate immune responses.

The NF-B family of transcription factors is activated by a wide variety of signals to regulate a spectrum of cellular processes. The proper regulation of NF-B activity is critical, since abnormal NF-B signaling is associated with a number of human illnesses, such as chronic inflammatory diseases and cancer. We report here that PIAS1 ( and bacterial lipopolysaccharide (LPS), activate the NF-B signaling pathway. NF-B is a family of dimeric transcription factors composed of members of the Rel family of DNA binding proteins, including NF-B1 (p50 and its precursor p105), NF-B2 (p52 and its precursor p100), c-Rel, RelA (p65), and RelB (11, 18). Upon stimulation, NF-B translocates into the nucleus, where it binds to specific DNA sequences and regulates transcription. NF-B is involved in mediating a wide spectrum of cellular responses, including infections, inflammation, and apoptosis (2, 27). Inappropriate regulation of NF-B is involved in a wide range of human diseases, including cancer, neurodegenerative disorders, arthritis, asthma, and chronic inflammation (3,4,10,12). The NF-B signaling pathway is tightly modulated at various levels by distinct regulatory proteins. For example, the binding of the IB family of proteins prevents the nuclear translocation of NF-B (16). However, a protein factor that can regulate the DNA binding activity of NF-B has not been documented.The PIAS (protein inhibitor of activated STAT) family of proteins consists of four members: PIAS1, PIAS3, PIASx, and PIASy (33). Members of the PIAS family have been suggested to regulate STAT-mediated transcription. Upon cytokine stimulation, PIAS binds to STAT and inhibits STAT-mediated gene activation (1,8,21,22). Among the PIAS family, PIAS1 and PIASy have been shown to inhibit STAT1-dependent transcription through distinct mechanisms. PIAS1 inhibits the transcriptional activity of STAT1 by blocking the DNA binding activity of STAT1. In contrast, PIASy does not affect the DNA binding activity of STAT1. It has been suggested that PIASy may act as a transcriptional corepressor of STAT1. The PIAS family of proteins has also been suggested to regulate a number of other transcription factors, including nuclear hormone receptors (13,28,36,37), LEF1 (31), and p53 (15,26,32).To understand the physiological role of PIAS1, we have recently generated Pias1 null mice (23). Detailed gene activation analysis indicates that PIAS1 selectively regulates a subset of interferon (IFN)-inducible genes. The antiviral activity of IFNs is significantly enhanced by Pias1 disruption. In addition, Pias1 null mice show enhanced protection against pathogenic infection. These results support a physiological role of PIAS1 in the negative regulation of IFN-activated STAT1-mediated gene activation and demonstrate an important role of PIAS1 in innate immune responses.Since STAT1 and the Rel family of proteins share structural similarity in their DNA binding domains (6), we explored the possible involvement of PIAS1 in the regulation of NF-B. Here we report that PIAS1 interacts with ...

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c GS-5885 is a novel hepatitis C virus (HCV) NS5A inhibitor. In a 3-day monotherapy study in treatment-naive genotype 1a (GT1a) and GT1b HCV-infected subjects, median viral load reductions ranged from 2.3 to 3.3 log 10 HCV RNA IU/ml across dosing cohorts (1, 3, 10, 30, or 90 mg once daily). Here, we report viral sequencing and phenotypic analysis of clinical isolates from this study. Detection of baseline NS5A amino acid substitutions at positions 28, 30, 31, or 93 in GT1a was associated with a reduced treatment response. In the GT1b cohort, Y93H was detected in 100% of subjects at day 4 or 14. In the Gt1a cohort, population sequencing detected NS5A resistance-associated mutations at day 4 or 14 for 3/10 subjects at the 1-mg dose and for all subjects dosed at >3 mg. A subset of mutants that confer a low level of reduced susceptibility to GS-5885 was not detected by population sequencing at the 30-and 90-mg doses. Subject-derived M28T, Q30R, L31M, and Y93C mutations all conferred >30-fold reductions in GS-5885 and daclatasvir susceptibilities in vitro. Site-directed NS5A mutants also showed reduced susceptibility to GS-5885. However, all NS5A mutants tested remained fully susceptible to other classes of direct-acting antivirals (DAAs), interferon alpha, and ribavirin. Importantly, the nonoverlapping resistance profile and high potency of GS-5885 support its further development with other direct-acting antivirals for the treatment of chronic HCV. (This study has been registered at ClinicalTrials.gov under registration number NCT01193478.) H epatitis C virus (HCV) infection is a global health issue, with approximately 170 million people infected worldwide (1).The standard of care (SOC) has been pegylated alpha interferon and ribavirin until the more recent approval of two NS3 protease inhibitors, telaprevir and boceprevir, for use in conjunction with pegylated alpha interferon and ribavirin (1-3). The last several years have seen a great expansion of new direct-acting antivirals (DAAs) in clinical development to augment or supplant treatment with pegylated alpha interferon and ribavirin. HCV nonstructural protein 5A (NS5A) has emerged as a viable and attractive viral target for small-molecule inhibition. Although there is no known enzymatic activity for NS5A, it is essential for viral replication (4). The first NS5A replication complex inhibitor to show efficacy in the clinic was daclatasvir (BMS-790052) (5). This compound elicited rapid and profound reductions in HCV RNA and validated NS5A as a clinical target. Sequence analysis of clinical isolates following daclatasvir monotherapy identified the main resistanceassociated mutations (RAMs) at NS5A amino acid positions 28, 30, 31, and 93 (6).In addition to daclatasvir, several other NS5A replication complex inhibitors have entered the clinic, including GS-5885, PPI-461, ABT-267, and GSK2336805 (7-10). In addition, preclinical data have recently been described for several other NS5A replication complex inhibitors, including EDP-239, IDX719, MK-4882, , highlighting ...