Satoru Ikeda

The essential role of C-terminal residues in regulating the activity of hepatitis C virus RNA-dependent RNA polymerase

We have previously determined the crystal structure of a non-structural 5B (NS5B) protein, an RNA-dependent RNA polymerase (RdRp) of hepatitis C virus (HCV). NS5B protein with the hydrophobic C-terminal 21 amino acid residues truncated, designated NS5B(570), shows a typical nucleotide polymerase structure resembling a right-hand shape. In the crystal structure, a C-terminal region between Leu545 and His562 occupies a putative RNA-binding cleft of this polymerase and seems to inhibit the polymerase activity. Varieties of recombinant NS5B proteins (NS5B(552), NS5B(544), NS5B(536) or NS5B(531), with C-terminal 39, 47, 55 or 60 amino acid residues truncated, respectively) were systematically constructed to elucidate effects of the region on the polymerase activity. NS5B(544), NS5B(536) and NS5B(531) showed markedly higher RdRp activities compared to the activities of NS5B(570) or NS5B(552). Furthermore, when the hydrophobic amino acid residues Leu547, Trp550 and Phe551 (LWF) in NS5B(570) and NS5B(552) were changed to alanine, their activities were higher than that of the original NS5B(570). The crystal structures of the various recombinant NS5B proteins were also determined. Structural comparison of the NS5B proteins indicates that the activation was caused by elimination of a unique hydrophobic interaction between the three C-terminal residues and a shallowly concave pocket consisting of thumb and palm domains.

Effects of Mutation in Hepatitis C Virus Nonstructural Protein 5A on Interferon Resistance Mediated by Inhibition of PKR Kinase Activity in Mammalian Cells

The IFN‐induced double‐stranded RNA (dsRNA)‐activated protein kinase PKR is one of the key molecules in the antiviral effects of IFN. To clarify the effects of hepatitis C virus nonstructural protein 5A (NS5A) on antiviral activity of IFN, in particular on PKR kinase activity, in mammalian cells, we established inducible NS5A‐expressing cell lines derived from human osteosarcoma (Saos‐2). The cells expressing NS5A derived from an IFN‐resistant clone (NS5A‐1b) that interacted with endogenous PKR in vitro, showed a suppressive effect on IFN function as determined by interference with vesicular stomatitis virus (VSV) infection, whereas NS5A (NS5A‐2a) from an IFN‐sensitive clone did not block the antiviral effect of IFN. A mutant with deletion of the IFN sensitivity determining region (ISDR) in NS5A‐1b (NS5A‐ΔISDR) also interacted with PKR and suppressed its activity in vitro. However, neither NS5A‐2a nor the C‐terminal truncated mutant of NS5A‐1b (NS5A‐ΔC) blocked PKR activity. These observations confirmed the previous report that the inhibitory effect of NS5A on IFN activity is mediated at least in part by the repression of PKR. In addition, we showed that IFN sensitivity was determined not only by the ISDR but that the involvement of the C‐terminal region of NS5A‐1b is important for the suppression of PKR activity.

MDM2 is a novel E3 ligase for HIV-1 Vif

The human immunodeficiency virus type 1 (HIV-1) Vif plays a crucial role in the viral life cycle by antagonizing a host restriction factor APOBEC3G (A3G). Vif interacts with A3G and induces its polyubiquitination and subsequent degradation via the formation of active ubiquitin ligase (E3) complex with Cullin5-ElonginB/C. Although Vif itself is also ubiquitinated and degraded rapidly in infected cells, precise roles and mechanisms of Vif ubiquitination are largely unknown. Here we report that MDM2, known as an E3 ligase for p53, is a novel E3 ligase for Vif and induces polyubiquitination and degradation of Vif. We also show the mechanisms by which MDM2 only targets Vif, but not A3G that binds to Vif. MDM2 reduces cellular Vif levels and reversely increases A3G levels, because the interaction between MDM2 and Vif precludes A3G from binding to Vif. Furthermore, we demonstrate that MDM2 negatively regulates HIV-1 replication in non-permissive target cells through Vif degradation. These data suggest that MDM2 is a regulator of HIV-1 replication and might be a novel therapeutic target for anti-HIV-1 drug.

Potent and selective inhibition of Tat-dependent HIV-1 replication in chronically infected cells by a novel naphthalene derivative JTK-101

In search for effective human immunodeficiency virus type 1 (HIV-1) transcription inhibitors, we have evaluated more than 100,000 compounds for their inhibitory effects on HIV-1 long terminal repeat (LTR)-driven reporter gene expression, and identified a novel naphthalene derivative, JTK-101. This compound could suppress tumour necrosis factor (TNF)-α-induced HIV-1 production in latently infected OM-10.1 cells at nanomolar concentrations. JTK-101 could also potently inhibit constitutive HIV-1 production in MOTL-4/IIIB. However, the antiviral activity of JTK-101 was found to be much weaker in acutely infected cells and the chronically infected cells U937/IIIB cells than in OM-10.1 and MOLT-4/IIIB cells. JTK-101 selectively suppressed TNF-α-induced HIV-1 mRNA synthesis in OM-10.1 cells in a dose-dependent fashion. JTK-101 modestly inhibited TNF-α-induced HIV-1 LTR-driven reporter gene expression, but potently inhibited Tat-induced gene expression. Immunoblot analysis revealed that low-level expression of the Tat cofactors CDK9 and cyclin T1 might contribute to the diminished antiviral activity in U937/IIIB cells. Furthermore, JTK-101 could not inhibit HIV-1 replication in chronically infected monocytes/macrophages, in which CDK9 and cyclin T1 were undetectable. These results suggest that JTK-101 exerts its anti-HIV-1 activity through the inhibition of known or unknown Tat cofactors, presumably CDK9/cyclin T1.

Isolation and gene analysis of interferon α-resistant cell clones of the hepatitis C virus subgenome

Hepatitis C virus (HCV) proteins appear to play an important role in IFN-resistance, but the molecular mechanism remains unclear. To clarify the mechanism in HCV replicon RNA harboring Huh-7 cells (Huh-9-13), we isolated cellular clones with impaired IFNalpha-sensitivity. Huh-9-13 was cultured for approximately 2 months in the presence of IFNalpha, and 4 IFNalpha-resistant cell clones showing significant resistances were obtained. When total RNA from clones was introduced into Huh-7 cells, the transfected cells also exhibited IFNalpha-resistance. Although no common mutations were present, mutations in NS3 and NS5A regions were accumulated. Transactivation of IFNalpha and IFNalpha-stimulated Stat-1 phosphorylation were reduced, and the elimination of HCV replicon RNA from the clones restored the IFNalpha signaling. These results suggest that the mutations in the HCV replicon RNA, at least in part, cause an inhibition of IFN signaling and are important for acquisition of IFNalpha resistance in Huh-9-13.

Novel benzthiodiazepinones as antiherpetic agents: SAR improvement of therapeutic index by alterations of the seven-membered ring.

A series of novel benzthiodiazepinones was studied as antiherpetic agents. Significant improvements in potency and therapeutic index in a viral replication assay were realized over the starting molecule. The role of stereospecific substitution on the diazepine ring and optimal nitrogen substitution were investigated.