In vitro characterization of baloxavir acid, a first-in-class cap-dependent endonuclease inhibitor of the influenza virus polymerase PA subunit.

Cap-dependent endonuclease (CEN) resides in the PA subunit of the influenza virus and mediates the critical "cap-snatching" step of viral RNA transcription, which is considered to be a promising anti-influenza target. Here, we describe in vitro characterization of a novel CEN inhibitor, baloxavir acid (BXA), the active form of baloxavir marboxil (BXM). BXA inhibits viral RNA transcription via selective inhibition of CEN activity in enzymatic assays, and inhibits viral replication in infected cells without cytotoxicity in cytopathic effect assays. The antiviral activity of BXA is also confirmed in yield reduction assays with seasonal type A and B viruses, including neuraminidase inhibitor-resistant strains. Furthermore, BXA shows broad potency against various subtypes of influenza A viruses (H1N2, H5N1, H5N2, H5N6, H7N9 and H9N2). Additionally, serial passages of the viruses in the presence of BXA result in isolation of PA/I38T variants with reduced BXA susceptibility. Phenotypic and genotypic analyses with reverse genetics demonstrate the mechanism of BXA action via CEN inhibition in infected cells. These results reveal the in vitro characteristics of BXA and support clinical use of BXM to treat influenza.

Hepatitis C virus hijacks P-body and stress granule components around lipid droplets.

The microRNA miR-122 and DDX6/Rck/p54, a microRNA effector, have been implicated in hepatitis C virus (HCV) replication. In this study, we demonstrated for the first time that HCV-JFH1 infection disrupted processing (P)-body formation of the microRNA effectors DDX6, Lsm1, Xrn1, PATL1, and Ago2, but not the decapping enzyme DCP2, and dynamically redistributed these microRNA effectors to the HCV production factory around lipid droplets in HuH-7-derived RSc cells. Notably, HCV-JFH1 infection also redistributed the stress granule components GTPase-activating protein (SH3 domain)-binding protein 1 (G3BP1), ataxin-2 (ATX2), and poly(A)-binding protein 1 (PABP1) to the HCV production factory. In this regard, we found that the P-body formation of DDX6 began to be disrupted at 36 h postinfection. Consistently, G3BP1 transiently formed stress granules at 36 h postinfection. We then observed the ringlike formation of DDX6 or G3BP1 and colocalization with HCV core after 48 h postinfection, suggesting that the disruption of P-body formation and the hijacking of P-body and stress granule components occur at a late step of HCV infection. Furthermore, HCV infection could suppress stress granule formation in response to heat shock or treatment with arsenite. Importantly, we demonstrate that the accumulation of HCV RNA was significantly suppressed in DDX6, Lsm1, ATX2, and PABP1 knockdown cells after the inoculation of HCV-JFH1, suggesting that the P-body and the stress granule components are required for the HCV life cycle. Altogether, HCV seems to hijack the P-body and the stress granule components for HCV replication.

A disulfide-bonded dimer of the core protein of hepatitis C virus is important for virus-like particle production.

Hepatitis C virus (HCV) core protein forms the nucleocapsid of the HCV particle. Although many functions of core protein have been reported, how the HCV particle is assembled is not well understood. Here we show that the nucleocapsid-like particle of HCV is composed of a disulfide-bonded core protein complex (dbc-complex). We also found that the disulfide-bonded dimer of the core protein (dbd-core) is formed at the endoplasmic reticulum (ER), where the core protein is initially produced and processed. Mutational analysis revealed that the cysteine residue at amino acid position 128 (Cys128) of the core protein, a highly conserved residue among almost all reported isolates, is responsible for dbd-core formation and virus-like particle production but has no effect on the replication of the HCV RNA genome or the several known functions of the core protein, including RNA binding ability and localization to the lipid droplet. The Cys128 mutant core protein showed a dominant negative effect in terms of HCV-like particle production. These results suggest that this disulfide bond is critical for the HCV virion. We also obtained the results that the dbc-complex in the nucleocapsid-like structure was sensitive to proteinase K but not trypsin digestion, suggesting that the capsid is built up of a tightly packed structure of the core protein, with its amino (N)-terminal arginine-rich region being concealed inside.

Inhibitory effect of (-)-epigallocatechin and (-)-epigallocatechin gallate against heregulin beta1-induced migration/invasion of the MCF-7 breast carcinoma cell line.

Migration/invasion is involved in the multiple steps of metastasis, resulting in a poor prognosis of breast cancer. (-)-Epigallocatechin-3-gallate (EGCG) in green tea inhibits the metastasis of some cancer cell lines. Cell migration/invasion assays using Boyden chambers demonstrated that (-)-epigallocatechin (EGC), another green tea catechin, inhibited heregulin-beta1 (HRG)-induced migration/invasion of MCF-7 human breast carcinoma cells to approximately the same extent as EGCG. Assays of cytoskeletal reorganization, Western blotting and immunoprecipitation suggested that EGCG inhibited this migration/invasion by suppressing the HRG-stimulated activation of epidermal growth factor receptor-related protein B2 (ErbB2)/ErbB3/protein kinase B (Akt), whereas EGC did so through pathways including the disruption of the HRG-stimulated activation of ErbB2/ErbB3 but not Akt. EGC, as well as EGCG, could play an important role against the promotion of metastasis of breast cancer cells.

Comparative examination of anti-proliferative activities of (-)-epigallocatechin gallate and (--)-epigallocatechin against HCT116 colorectal carcinoma cells.

We compared anti-proliferative activities of (-)-epigallocatechin gallate (EGCG) and (-)-epigallocatechin (EGC) against HCT116 colorectal carcinoma cells. These catechins inhibited cell growth to nearly the same extent at low cell confluency in plates. However, their inhibitory effect grew weaker as cell confluence increased, and this tendency was more conspicuous for EGC than for EGCG. Both EGCG and EGC activated the phosphorylation of the major MAPKs, ERK, JNK, and p38, in the HCT116 cells as in many other established human cancer cells though to different extents. Cell cycle analyses, DNA fragmentation assays, and TUNEL assays as well as Western blot assays suggested that these catechins inhibited cell growth through mitogen-activated protein kinase (MAPK)-mediated apoptosis rather than cell cycle regulation.