Biomedical subjects

Hironori Nishitsuji

Publications and source records attributed to Hironori Nishitsuji.

8 recordsLinked to original sources

PubMed2025

Identification of glycogen synthase kinase 3alpha/beta as a host factor required for HBV transcription using high-throughput screening.

BACKGROUND AND AIMS: HBV leads to severe liver diseases, such as cirrhosis and HCC. Identification of host factors that regulate HBV replication can provide new therapeutic targets. The discovery of sodium taurocholate cotransporting polypeptide (NTCP) as an HBV entry receptor has enabled the establishment of hepatic cell lines for analyzing HBV infection and propagation. Using this new system, studies aimed at identifying host factors that regulate HBV propagation have increased. APPROACH AND RESULTS: We established an HBV-based-reporter gene expression system that mimics HBV replication from transcription to virus egress. Using this approach, we screened 1827 Food and Drug Administration-approved compounds and identified glycogen synthase kinase 3 (GSK3)alpha/beta inhibitors, including AZD1080, CHIR-98014, CHIR-98021, BIO, and AZD2858, as anti-HBV compounds. These compounds suppressed HBeAg and HBsAg production in HBV-infected human primary hepatocytes. Proteome analysis revealed that GSK3alpha/beta phosphorylated forkhead box K1/2 (FOXK1/2)s. A double-knockout of FOXK1/2 in HBV-infected HepG2-NTCP cells reduced HBeAg and HBsAg production. The rescue of FOXK2 expression, but not FOXK1 expression, in FOXK1/2-double-knockout cells restored HBeAg and HBsAg production. Importantly, phosphorylation of FOXK2 at Ser 424 is required for GSK3alpha/beta-mediated HBeAg and HBsAg production. We observed the binding of FOXK2 to HBV DNA in HepG2-NTCP cells. CONCLUSIONS: Our recombinant HBV-based screening system enables the discovery of new targets. Using our approach, we identified GSK3 inhibitors as potential anti-HBV agents.

Humans↗

PubMed2006

Suppression of human immunodeficiency virus type 1 replication by arginine deiminase of Mycoplasma arginini.

It was found previously that human immunodeficiency virus type 1 (HIV-1)-irrelevant CD8+ cytotoxic T lymphocytes (CTLs) from uninfected donors suppressed HIV-1 replication in a cell-contact-dependent manner. However, one of these CTL lines (CTL-3) also significantly suppressed HIV-1 replication through its supernatant. Here, the suppressive fraction from CTL-3 supernatant was purified and analysed by mass spectrometry. A protein band specific for the suppressive fraction was identified as arginine deiminase from Mycoplasma arginini, which catalyses the hydrolysis of arginine to citrulline. Addition of L-arginine or the use of antibiotics against mycoplasma restored supernatant-mediated but not cell-contact-dependent suppression of HIV-1 replication by CTL-3, clearly indicating that arginine deiminase of M. arginini in the supernatants suppressed HIV-1 replication, which is independent of CD8+ T-cell-mediated HIV-1 suppression via cell contact. Arginine deiminase is known to be a chemotherapeutic agent against arginine-requiring tumours and these results suggest that it also has potential application in antiviral therapy.

Cells, Cultured↗

PubMed2006

Effective suppression of human immunodeficiency virus type 1 through a combination of short- or long-hairpin RNAs targeting essential sequences for retroviral integration.

Small interfering RNA (siRNA) could provide a new therapeutic approach to treating human immunodeficiency virus type 1 (HIV-1) infection. For long-term suppression of HIV-1, emergence of siRNA escape variants must be controlled. Here, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNA) corresponding to conserved target sequences within the integrase (int) and the attachment site (att) genes, both of which are essential for HIV-1 integration. Compared to shRNA targeting of the HIV-1 transcription factor tat (shTat), shRNA against int (shIN) or the U3 region of att (shU3) showed a more potent inhibitory effect on HIV-1 replication in human CD4+ T cells. Infection with a high dose of HIV-1 resulted in the emergence of escape mutants during long-term culture. Of note, limited genetic variation was observed in the viruses resistant to shIN. A combination of shINs against wild-type and escape mutant sequences had a negative effect on their antiviral activities, indicating a potentially detrimental effect when administering multiple shRNA targeting the same region to combat HIV-1 variants. The combination of shIN and shU3 att exhibited the strongest anti-HIV-1 activity, as seen by complete abrogation of viral DNA synthesis and viral integration. In addition, a modified long-hairpin RNA spanning the 50 nucleotides in the shIN target region effectively suppressed wild-type and shIN-resistant mutant HIV-1. These results suggest that targeting of incoming viral RNA before proviral DNA formation occurs through the use of nonoverlapping multiple siRNAs is a potent approach to achieving sustained, efficient suppression of highly mutable viruses, such as HIV-1.

Cell Line↗

PubMed2006

Identification of a novel human immunodeficiency virus type 1 integrase interactor, Gemin2, that facilitates efficient viral cDNA synthesis in vivo.

Retroviral integrase (IN) catalyzes the integration of viral cDNA into a host chromosome. Additional roles have been suggested for IN, including uncoating, reverse transcription, and nuclear import of the human immunodeficiency virus type 1 (HIV-1) genome. However, the underlying mechanism is largely unknown. Here, using a yeast two-hybrid system, we identified a survival motor neuron (SMN)-interacting protein 1 (Gemin2) that binds to HIV-1 IN. Reduction of Gemin2 with small interfering RNA duplexes (siGemin2) dramatically reduced HIV-1 infection in human primary monocyte-derived macrophages and also reduced viral cDNA synthesis. In contrast, siGemin2 did not affect HIV-1 expression from the integrated proviral DNA. Although Gemin2 was undetectable in cell-free viral particles, coimmunoprecipitation experiments using FLAG-tagged Gemin2 strongly suggested that Gemin2 interacts with the incoming viral genome through IN. Further experiments reducing SMN or other SMN-interacting proteins suggested that Gemin2 might act on HIV-1 either alone or with unknown proteins to facilitate efficient viral cDNA synthesis soon after infection. Thus, we provide the evidence for a novel host protein that binds to HIV-1 IN and facilitates viral cDNA synthesis and subsequent steps that precede integration in vivo.

Cells, Cultured↗

PubMed2004

Inducible-costimulator-mediated suppression of human immunodeficiency virus type 1 replication in CD4(+) T lymphocytes.

We investigated the effects of signaling through CD28 family molecules on human immunodeficiency virus type 1 (HIV-1) replication in vitro. A monoclonal antibody (mAb) specific for inducible costimulator (ICOS) suppressed both X4 and R5 HIV-1 replication in CD4(+) peripheral blood mononuclear cells (PBMC). This suppression was not attributable to reduced cell growth or viability. CD28 mAb showed variable effects and also suppressed HIV-1 replication when immobilized. Replication of pseudotype viruses with HIV-1-but not with vesicular stomatitis virus G-envelope was efficiently suppressed in CD4(+) PBMC treated with ICOS or CD28 mAbs. However, CD4, CXCR4, and CCR5 expression on the surface was not down-regulated. Moreover, HIV-1 replication in CD4(+) PBMC was suppressed by a soluble form of human B7-H2, a ligand of ICOS, but was enhanced by soluble B7-1, a ligand for CD28. These findings suggest that natural or artificial ligands for ICOS potentially suppress HIV-1 replication mainly at the entry stages.

Animals↗

PubMed2004

Expression of small hairpin RNA by lentivirus-based vector confers efficient and stable gene-suppression of HIV-1 on human cells including primary non-dividing cells.

RNA interference (RNAi) is a sequence-specific RNA degradation process mediated by short double-stranded RNAs termed small interfering RNAs. Here, we describe the lentivirus-based vector small interfering RNA system expressing. As a pilot study, we generated constructs expressing small hairpin RNA (shRNA) specific for luciferase gene (shLuc) or green fluorescence protein (shGFP) under the control of human H1 RNA polymerase III promoter. The effect of the shRNA was evaluated against HIV-1 infection in a single-round or multiple-round infectious system using an HIV-1 molecular clone carrying the luc or GFP gene. In the single-round infectious system, cells transduced with shLuc by lentiviral vector significantly reduced (approximately 90% reduction) viral gene expression after challenge infection at a multiplicity of infection of 10. These transduced cells continued to resist against at least four sequentially repeated challenge infections. Importantly, this efficient antiviral activity persisted over 35 days in culture. In a multiple-round infectious system using a replication-competent HIV-1 molecular clone carrying the GFP gene, we also observed that a lentiviral vector expressing shGFP could inhibit HIV-1 replication for at least 1 week. The profound effect of lentiviral shRNA was also observed in human primary monocyte-derived macrophages. Thus, shRNA introduced through the lentiviral vector can be useful for efficient and stable gene suppression in human cells including primary non-dividing cells. Moreover, quantitative analysis of viral cDNA synthesis on challenge infection showed that viral genomic RNAs packaged in incoming virus core might not be targeted by shLuc. Instead, the degradation of transcripts from integrated proviral DNAs might be a major cause of the profound reduction in HIV-1 gene expression by shRNA in our system.

Cell Division↗

PubMed2004

Evaluation of the functional involvement of human immunodeficiency virus type 1 integrase in nuclear import of viral cDNA during acute infection.

Nuclear import of viral cDNA is a critical step for establishing the proviral state of human immunodeficiency virus type 1 (HIV-1). The contribution of HIV-1 integrase (IN) to the nuclear import of viral cDNA is controversial, partly due to a lack of identification of its bona fide nuclear localization signal. In this study, to address this putative function of HIV-1 IN, the effects of mutations at key residues for viral cDNA recognition (PYNP at positions 142 to 145, K156, K159, and K160) were evaluated in the context of viral replication. During acute infection, some mutations (N144Q, PYNP>KL, and KKK>AAA) severely reduced viral gene expression to less than 1% the wild-type (WT) level. None of the mutations affected the synthesis of viral cDNA. Meanwhile, the levels of integrated viral cDNA produced by N144Q, PYNP>KL, and KKK>AAA mutants were severely reduced to less than 1% the WT level. Quantitative PCR analysis of viral cDNA in nuclei and fluorescence in situ hybridization analysis showed that these mutations significantly reduced the level of viral cDNA accumulation in nuclei. Further analysis revealed that IN proteins carrying the N144Q, PYNP>KL, and KKK>AAA mutations showed severely reduced binding to viral cDNA but kept their karyophilic properties. Taken together, these results indicate that mutations that reduced the binding of IN to viral cDNA resulted in severe impairment of virus infectivity, most likely by affecting the nuclear import of viral cDNA that proceeds integration. These results suggest that HIV-1 IN may be one of the critical constituents for the efficient nuclear import of viral cDNA.

Active Transport, Cell Nucleus↗

PubMed2004

Repression of tax expression is associated both with resistance of human T-cell leukemia virus type 1-infected T cells to killing by tax-specific cytotoxic T lymphocytes and with impaired tumorigenicity in a rat model.

Human T-cell leukemia virus type 1 (HTLV-1) causes adult T-cell leukemia (ATL). Although the viral transactivation factor, Tax, has been known to have apparent transforming ability, the exact function of Tax in ATL development is still not clear. To understand the role of Tax in ATL development, we introduced short-interfering RNAs (siRNAs) against Tax in a rat HTLV-1-infected T-cell line. Our results demonstrated that expression of siRNA targeting Tax successfully downregulated Tax expression. Repression of Tax expression was associated with resistance of the HTLV-1-infected T cells to Tax-specific cytotoxic-T-lymphocyte killing. This may be due to the direct effect of decreased Tax expression, because the Tax siRNA did not alter the expression of MHC-I, CD80, or CD86. Furthermore, T cells with Tax downregulation appeared to lose the ability to develop tumors in T-cell-deficient nude rats, in which the parental HTLV-1-infected cells induce ATL-like lymphoproliferative disease. These results indicated the importance of Tax both for activating host immune response against the virus and for maintaining the growth ability of infected cells in vivo. Our results provide insights into the mechanisms how the host immune system can survey and inhibit the growth of HTLV-1-infected cells during the long latent period before the onset of ATL.

Animals↗