Double-Stranded Structure of the Polyinosinic-Polycytidylic Acid Molecule to Elicit TLR3 Signaling and Adjuvant Activity in Murine Intranasal A(H1N1)pdm09 Influenza Vaccination.

Polyinosinic-polycytidylic acid (PIC) is a potent double-stranded RNA (dsRNA) adjuvant useful in intranasal influenza vaccination. In mice, the intensity and duration of immune responses to PIC correlated with the double-stranded chain length. A rational method to avoid PIC chain extension in PIC production is to use multiple short poly(I) molecules and one long poly(C) molecule for PIC assembly. In this study, we elucidate that a newly developed uPIC100-400 molecule comprising multiple 0.1 kb poly(I) molecules and one 0.4 kb poly(C) molecule effectively enhanced the immune responses in mice, by preventing the challenged viral propagation and inducing hemagglutinin-specific IgA, after intranasal A(H1N1)pdm09 influenza vaccination. Reduced intraperitoneal toxicity of PIC prepared with multiple short poly(I) molecules in mice indicates the widened effective range of uPIC100-400 as an adjuvant. In contrast to uPIC100-400, the PIC molecule comprising multiple 0.05 kb poly(I) molecules failed to elicit mouse mucosal immunity. These results were consistent with TLR3 response but not retinoic acid inducible gene I (RIG-I)-like receptor response in the cell assays, which suggests that the adjuvant effect of PIC in mouse intranasal immunization depends on TLR3 signaling. In conclusion, the double-stranded PIC with reduced toxicity developed in this study would contribute to the development of PIC-adjuvanted vaccines.

Mechanism of Action of the Anti-Influenza Virus Active Kampo (Traditional Japanese Herbal) Medicine, Hochuekkito.

When the Kampo medicine, Hochuekkito (Hochu), was administered to normal mice for 2 weeks, influenza virus titer was reduced. The mechanism of action of Hochu was examined using the plaque assay method. It was suggested that Hochu may either obstruct the first stage of the infection process (adsorption and entry) or may directly target viral particles. Using the plaque assay method, these 2 modes of action could not be differentiated. Virus RNA in the infected cell was verified by quantitative real-time polymerase chain reaction. An equal inhibition effect was obtained when Hochu was preprocessed for normal cells and when they were made to act simultaneously with virus adsorption. The viral load at the cell surface following UV irradiation was higher in the Hochu-administered group as compared with that of the control. Moreover, the affinity of Hochu for the influenza virus was hundred times higher than its affinity for the host cell. The effect of entry obstruction by Hochu was observed via image analysis, where the amount of virus nucleocapsid protein (NP) invading the cell was visualized with FITC-labeled NP antibody. Hochu does not seem to have an effect on nucleic acid synthesis, viral release from infected cells, and on the subsequent second round of infection. In conclusion, Hochu binds to viral particles and forms complexes that can obstruct the entry of influenza virus into cells.

Hochuekkito, a Japanese Herbal Medicine, Restores Metabolic Homeostasis between Mitochondrial and Glycolytic Pathways Impaired by Influenza A Virus Infection.

BACKGROUND: Hochuekkito (HKT), a traditional Japanese herbal medicine (Kampo), has been used to treat symptoms of several diseases. In a recent clinical study, HKT was shown to be protective against the influenza virus infection. However, the underlying mechanism of the prophylactic effect is not clear. Mitochondrial and glycolytic pathways play important roles in cellular energy metabolism to maintain biological functions. These metabolic pathways are affected by the influenza virus infection. In this study, we examined the relationship between the preventive effects of HKT against the influenza virus infection and cellular energy metabolism in mitochondria and glycolysis using Madin-Darby canine kidney cells and influenza A/PR/8/34 (H1N1) virus (IAV). METHODS: Mitochondrial and glycolytic metabolic pathways were evaluated on the basis of the oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), respectively, using the XF24 Extracellular Analyzer. RESULTS: The OCR/ECAR ratio in IAV-infected cells was lower than that in control cells. Cells that were treated with HKT before IAV infection showed a metabolic pattern similar to that in the control cells (increase in both OCR and ECAR). CONCLUSIONS: Our results suggest that HKT not only activates both mitochondrial and glycolytic energy metabolism in IAV-infected cells but also helps maintain metabolic homeostasis similar to that in noninfected cells.

The Preventive Effect of the Traditional Japanese Herbal Medicine, Hochuekkito, against Influenza A Virus via Autophagy in vitro.

BACKGROUND: Hochuekkito (HKT), a traditional Japanese herbal medicine, enhances the immunity of elderly or weak individuals. It is also known to have preventive effects against influenza clinically. However, the detailed mechanisms of the preventive effects have not been clarified. We examined the relationship between the preventive effects of HKT and autophagy, a known stress response and quality control mechanism, using Madin-Darby canine kidney cells and influenza A/PR/8/34 (H1N1) virus. METHODS: The effect of HKT on autophagy in influenza A virus (IAV)-infected cells was assessed by Western blotting and fluorescence microscopy using an RFP-GFP-LC3B sensor kit. RESULTS: In Western blotting, treatment with HKT before IAV infection (pre-HKT) tended to induce autophagy in IAV-infected cells at an early stage of infection, eventually suppressing IAV-induced autophagy. Moreover, several autolysosomes, indicative of normal autophagosome-lysosome fusion, were observed in Pre-HKT cells transduced with RFP-GFP-LC3B but not in untreated IAV-infected cells. CONCLUSIONS: These findings indicated that IAV-mediated inhibition of the fusion of autophagosomes with lysosomes was prevented by HKT treatment before infection. According to these results, we propose that this phenomenon is one of the preventive effects of HKT against IAV.

Fungus-Derived Neoechinulin B as a Novel Antagonist of Liver X Receptor, Identified by Chemical Genetics Using a Hepatitis C Virus Cell Culture System.

UNLABELLED: Cell culture systems reproducing virus replication can serve as unique models for the discovery of novel bioactive molecules. Here, using a hepatitis C virus (HCV) cell culture system, we identified neoechinulin B (NeoB), a fungus-derived compound, as an inhibitor of the liver X receptor (LXR). NeoB was initially identified by chemical screening as a compound that impeded the production of infectious HCV. Genome-wide transcriptome analysis and reporter assays revealed that NeoB specifically inhibits LXR-mediated transcription. NeoB was also shown to interact directly with LXRs. Analysis of structural analogs suggested that the molecular interaction of NeoB with LXR correlated with the capacity to inactivate LXR-mediated transcription and to modulate lipid metabolism in hepatocytes. Our data strongly suggested that NeoB is a novel LXR antagonist. Analysis using NeoB as a bioprobe revealed that LXRs support HCV replication: LXR inactivation resulted in dispersion of double-membrane vesicles, putative viral replication sites. Indeed, cells treated with NeoB showed decreased replicative permissiveness for poliovirus, which also replicates in double-membrane vesicles, but not for dengue virus, which replicates via a distinct membrane compartment. Together, our data suggest that LXR-mediated transcription regulates the formation of virus-associated membrane compartments. Significantly, inhibition of LXRs by NeoB enhanced the activity of all known classes of anti-HCV agents, and NeoB showed especially strong synergy when combined with interferon or an HCV NS5A inhibitor. Thus, our chemical genetics analysis demonstrates the utility of the HCV cell culture system for identifying novel bioactive molecules and characterizing the virus-host interaction machinery. IMPORTANCE: Hepatitis C virus (HCV) is highly dependent on host factors for efficient replication. In the present study, we used an HCV cell culture system to screen an uncharacterized chemical library. Our results identified neoechinulin B (NeoB) as a novel inhibitor of the liver X receptor (LXR). NeoB inhibited the induction of LXR-regulated genes and altered lipid metabolism. Intriguingly, our results indicated that LXRs are critical to the process of HCV replication: LXR inactivation by NeoB disrupted double-membrane vesicles, putative sites of viral replication. Moreover, NeoB augmented the antiviral activity of all known classes of currently approved anti-HCV agents without increasing cytotoxicity. Thus, our strategy directly links the identification of novel bioactive compounds to basic virology and the development of new antiviral agents.

Preclinical efficacy of the synthetic retinoid ST1926 for treating adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) is an aggressive neoplasm caused by human T-cell leukemia virus type 1 (HTLV-1). The HTLV-1 oncoprotein Tax plays an important role in ATL pathogenesis. ATL carries a poor prognosis due to chemotherapy resistance, stressing the need for alternative therapies. Here, we investigate the preclinical efficacy of the synthetic retinoid ST1926 in ATL and peripheral T-cell lymphomas. Clinically achievable concentrations of ST1926 induced a dramatic inhibition of cell proliferation in malignant T-cell lines and primary ATL cells with minimal effect on resting or activated normal lymphocytes. ST1926 induced apoptosis, DNA damage, and upregulation of p53 proteins in malignant T cells, whereas it caused an early downregulation of Tax proteins in HTLV-1-positive cells. In murine ATL, oral treatment with ST1926 prolonged survival and reduced leukemia cell infiltration, white blood cell counts, and spleen mass. In spleens of ST1926-treated animals, p53 and p21 proteins were upregulated, poly (ADP-ribose) polymerase was cleaved, and Tax transcripts were reduced. These results highlight the promising use of ST1926 as a targeted therapy for ATL.

A Kampo (traditional Japanese herbal) medicine, Hochuekkito, pretreatment in mice prevented influenza virus replication accompanied with GM-CSF expression and increase in several defensin mRNA levels.

A Kampo medicine, Hochuekkito (TJ-41), with an influenza virus-preventing effect had life-extending effectiveness, and immunological responses other than interferon (IFN)-α release were examined. TJ-41 (1 g/kg) was given to C57BL/6 male mice orally once a day for 2 weeks. Mice were then intranasally infected with influenza virus. After infection, virus titers and various parameters, mRNA levels and protein expression, for immunoresponses in the bronchoalveolar lavage fluid or removed lung homogenate, were measured by plaque assay, quantitative RT-PCR and ELISA. IFN-α and -ß levels of TJ-41-treated mice were higher than those of the control. Toll-like receptor TLR7 and TLR9 mRNAs were elevated after infection, but retinoic acid-inducible gene (RIG-1) family mRNA levels, RIG-1, melanoma differentiation-associated gene 5 and Leishmania G protein 2 showed no response in either TJ-41 or control groups. Interferon regulatory transcription factor (IRF)-3 mRNA levels to stimulate type I (α/ß) IFN were increased, but IRF-7 did not change. Only granulocyte-macrophage colony-stimulating factor (GM-CSF) after Hochuekkito treatment was significantly elevated 2 and 3 days after infection. The mRNA levels of 7 defensins after infection increased compared to preinfection values. The key roles of TJ-41 were not only stimulation of type I IFN release but also GM-CSF-derived anti-inflammation activity. Furthermore, defensin (antimicrobial peptide) mRNA levels increased by infection and were further enhanced by TJ-41 treatment. Defensin might prevent influenza virus replication.

Identification of a varicella-zoster virus replication inhibitor that blocks capsid assembly by interacting with the floor domain of the major capsid protein.

A novel anti-varicella-zoster virus compound, a derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one (coded as 35B2), was identified from a library of 9,600 random compounds. This compound inhibited both acyclovir (ACV)-resistant and -sensitive strains. In a plaque reduction assay under conditions in which the 50% effective concentration of ACV against the vaccine Oka strain (V-Oka) in human fibroblasts was 4.25 µM, the 50% effective concentration of 35B2 was 0.75 µM. The selective index of the compound was more than 200. Treatment with 35B2 inhibited neither immediate-early gene expression nor viral DNA synthesis. Twenty-four virus clones resistant to 35B2 were isolated, all of which had a mutation(s) in the amino acid sequence of open reading frame 40 (ORF40), which encodes the major capsid protein (MCP). Most of the mutations were located in the regions corresponding to the "floor" domain of the MCP of herpes simplex virus 1. Treatment with 35B2 changed the localization of MCP in the fibroblasts infected with V-Oka but not in the fibroblasts infected with the resistant clones, although it did not affect steady-state levels of MCP. Overexpression of the scaffold proteins restored the normal MCP localization in the 35B2-treated infected cells. The compound did not inhibit the scaffold protein-mediated translocation of MCP from the cytoplasm to the nucleus. Electron microscopic analysis demonstrated the lack of capsid formation in the 35B2-treated infected cells. These data indicate the feasibility of developing a new class of antivirals that target the herpesvirus MCPs and inhibit normal capsid formation by a mechanism that differs from those of the known protease and encapsidation inhibitors. Further biochemical studies are required to clarify the precise antiviral mechanism.

Innate sensors of influenza virus: clues to developing better intranasal vaccines.

Mucosal immunity acquired by natural infection with influenza viruses at the respiratory tract is more effective and cross-protective against subsequent variant virus infection than systemic immunity induced by parenteral immunization with inactivated vaccines. To develop an effective influenza vaccine, it is beneficial to mimic the process of natural infection that bridges innate and adaptive immune systems. The innate immune system that recognizes influenza virus infection consists of several classes of pattern-recognition receptors, including the Toll-like receptors, the retinoic acid-inducible gene-I-like receptors and the NOD-like receptors. Here, we review our current understanding of the mechanism of innate recognition of influenza and how the signals emanating from the innate sensors control adaptive immunity. Further, we discuss the potential roles of these receptors in developing intranasal influenza vaccines.

Distribution of PrP(Sc) in cattle with bovine spongiform encephalopathy slaughtered at abattoirs in Japan.

Three 80- to 95-month-old Holstein dairy cattle infected naturally with the agent of bovine spongiform encephalopathy (BSE) and slaughtered at abattoirs in Japan were examined for the distribution of disease-specific and protease-resistant prion protein (PrP(Sc)) by immunohistochemistry (IHC) and Western blot (WB) analyses. The cattle showed no clinical signs or symptoms relevant to BSE but were screened as positive by enzyme-linked immunosorbent assay, a rapid test for BSE. This positive result was confirmed by IHC or WB in a specimen of the medulla oblongata. Histopathologically, these cattle showed no vacuolation in tissue sections from the central nervous system except for the medulla oblongata. Both IHC and WB analyses revealed PrP(Sc) accumulation in the brain, spinal cord, satellite and ganglionic cells of the dorsal root ganglia, and the myenteric plexus of the distal ileum. In addition, small amounts of PrP(Sc) were detected in the peripheral nerves of 2 cattle by WB. No PrP(Sc) was demonstrated by either method in the Peyer's patches of the distal ileum; lymphoid tissues including the palatine tonsils, lymph nodes, and spleen; or other tissues. The distribution of PrP(Sc) accumulation in the preclinical stage was different between naturally infected cattle and cattle inoculated experimentally with the BSE agent.

Topoisomerase I and ATP activate cDNA synthesis of human immunodeficiency virus type 1.

Replication of human immunodeficiency virus type 1 (HIV-1) is regulated at reverse transcription. Cellular topoisomerase I has been reported to be carried into HIV-1 virions and enhance cDNA synthesis in vitro, suggesting that topoisomerase I expressed in virus producer cells regulates reverse transcription. Here, by employing both indicator cell assay and endogenous reverse transcription (ERT) assay, we show that topoisomerase I and adenosine triphosphate (ATP) enhanced cDNA synthesis of HIV-1. In addition, topoisomerase I mutants, R488A and K532A, lacking enzymatic activity, attenuated the efficiency of cDNA synthesis and resulted in inhibition of the infectivity of HIV-1, suggesting that the activity of topoisomerase I lacking in these mutants is indispensable for the cDNA synthesis in the HIV-1 replication process. Furthermore, ATP could dissociate topoisomerase I from the topoisomerase I-RNA complex and enhance cDNA synthesis in vitro. These findings suggest that cellular topoisomerase I and ATP play a pivotal role in the synthesis of cDNA of HIV-1.