Kenji Takeuchi

A Tryptophan-Rich Motif in the Human Parainfluenza Virus Type 2 V Protein Is Critical for the Blockade of Toll-Like Receptor 7 (TLR7)- and TLR9-Dependent Signaling

ABSTRACT Plasmacytoid dendritic cells (pDCs) do not produce alpha interferon (IFN-α) unless viruses cause a systemic infection or overcome the first-line defense provided by conventional DCs and macrophages. We show here that even paramyxoviruses, whose infections are restricted to the respiratory tract, have a V protein able to prevent Toll-like receptor 7 (TLR7)- and TLR9-dependent IFN-α induction specific to pDCs. Mutational analysis of human parainfluenza virus type 2 demonstrates that the second Trp residue of the Trp-rich motif (Trp-X3-Trp-X9-Trp) in the C-terminal domain unique to V, a determinant for IRF7 binding, is critical for the blockade of TLR7/9-dependent signaling.

Inhibition of hepatitis C virus replication through adenosine monophosphate‐activated protein kinase‐dependent and ‐independent pathways

Persistent infection with hepatitis C virus (HCV) is closely correlated with type 2 diabetes. In this study, replication of HCV at different glucose concentrations was investigated by using J6/JFH1‐derived cell‐adapted HCV in Huh‐7.5 cells and the mechanism of regulation of HCV replication by AMP‐activated protein kinase (AMPK) as an energy sensor of the cell analyzed. Reducing the glucose concentration in the cell culture medium from 4.5 to 1.0 g/L resulted in suppression of HCV replication, along with activation of AMPK. Whereas treatment of cells with AMPK activator 5‐aminoimidazole‐4‐carboxamide 1‐β‐D‐ribofuranoside (AICAR) suppressed HCV replication, compound C, a specific AMPK inhibitor, prevented AICARs effect, suggesting that AICAR suppresses the replication of HCV by activating AMPK in Huh‐7.5 cells. In contrast, compound C induced further suppression of HCV replication when the cells were cultured in low glucose concentrations or with metformin. These results suggest that low glucose concentrations and metformin have anti‐HCV effects independently of AMPK activation.

Human metapneumovirus M2-2 protein inhibits viral transcription and replication

M2-2 protein of human metapneumovirus (HMPV) is encoded by one of two overlapping open reading frames within M2 mRNA. The precise function of HMPV M2-2 protein remains unknown. We here examined effect of M2-2 protein on HMPV transcription and replication using a minigenome construct and monitoring luciferase reporter gene expression. The minigenome assays demonstrated that M2-2 protein inhibited both transcription and RNA replication. The inhibitory function of M2-2 protein was completely abrogated by removal of eight or four amino acids from its N- or C-terminus, respectively, demonstrating importance of both short terminal sequences for maintaining its functional structure. Immunoprecipitation experiments revealed interaction of M2-2 protein with L protein, which might be involved in inhibition of HMPV transcription and replication. Prior accumulation of intracellular M2-2 protein severely restrained HMPV from replicating. Thus inherent viral control of the M2-2 gene expression in infected cells seems to be essential for efficient HMPV replication.

Dectin-1-mediated signaling leads to characteristic gene expressions and cytokine secretion via spleen tyrosine kinase (Syk) in rat mast cells.

Background: β-Glucan receptor Dectin-1 in dendritic cells and macrophages plays important roles in antifungal immunity. Results: Dectin-1 is expressed in rat mast cells, and its tyrosine phosphorylation induces characteristic gene expression of transcription factors and cytokines through protein-tyrosine kinase Syk. Conclusion: Dectin-1 functions in rat mast cells. Significance: Dectin-1-mediated signaling in mast cells may contribute to antifungal immunity. Dectin-1 recognizes β-glucan and plays important roles for the antifungal immunity through the activation of spleen tyrosine kinase (Syk) in dendritic cells or macrophages. Recently, expression of Dectin-1 was also identified in human and mouse mast cells, although its physiological roles were largely unknown. In this report, rat mast cell line RBL-2H3 was analyzed to investigate the molecular mechanism of Dectin-1-mediated activation and responses of mast cells. Treatment of cells with Dectin-1-specific agonist curdlan induced tyrosine phosphorylation of cellular proteins and the interaction of Dectin-1 with the Src homology 2 domain of Syk. These responses depended on tyrosine phosphorylation of the hemi-immunoreceptor tyrosine-based activation motif in the cytoplasmic tail of Dectin-1, whereas they were independent of the γ-subunit of high-affinity IgE receptor. DNA microarray and real-time PCR analyses showed that Dectin-1-mediated signaling stimulated gene expression of transcription factor Nfkbiz and inflammatory cytokines, such as monocyte chemoattractant protein-1, IL-3, IL-4, IL-13, and tumor necrosis factor (TNF)-α. The response was abrogated by pretreatment with Syk inhibitor R406. These results suggest that Syk is critical for Dectin-1-mediated activation of mast cells, although the signaling differs from that triggered by FcϵRI activation. In addition, these gene expressions induced by curdlan stimulation were specifically observed in mast cells, suggesting that Dectin-1-mediated signaling of mast cells offers new insight into the antifungal immunity.

Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase

Background: HCV NS5A regulates viral RNA replication and virus particle assembly. Results: Phosphorylation of NS5A by c-Abl is required for efficient production of infectious HCV particles but not for viral RNA replication. Conclusion: HCV particle assembly involves tyrosine phosphorylation of NS5A. Significance: This study provides the first evidence for the importance of NS5A tyrosine phosphorylation in the HCV life cycle. Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr330 (Tyr2306 in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr330.

HCV NS5A Protein Containing Potential Ligands for Both Src Homology 2 and 3 Domains Enhances Autophosphorylation of Src Family Kinase Fyn in B Cells

Hepatitis C virus (HCV) infects B lymphocytes and induces mixed cryoglobulinemia and B cell non-Hodgkins lymphoma. The molecular mechanism for the pathogenesis of HCV infection-mediated B cell disorders remains obscure. To identify the possible role for HCV nonstructural 5A (NS5A) protein in B cells, we generated the stable B cell lines expressing Myc-His tagged NS5A. Immunoprecipitation study in the presence or absence of pervanadate (PV) implied that NS5A was tyrosine phosphorylated by pervanadate (PV) treatment of the cells. Therefore we examined pull-down assay by using glutathione S-transferase (GST)-fusion proteins of various Src homology 2 (SH2) domains, which associates with phosphotyrosine within a specific amino acid sequence. The results showed that NS5A specifically bound to SH2 domain of Fyn from PV-treated B cells in addition to Src homology 3 (SH3) domain. Substitution of Arg176 to Lys in the SH2 domain of Fyn abrogated this interaction. Deletion mutational analysis demonstrated that N-terminal region of NS5A was not required for the interaction with the SH2 domain of Fyn. Tyr334 was identified as a tyrosine phosphorylation site in NS5A. Far-western analysis revealed that SH2 domain of Fyn directly bound to NS5A. Fyn and NS5A were colocalized in the lipid raft. These results suggest that NS5A directly binds to the SH2 domain of Fyn in a tyrosine phosphorylation-dependent manner. Lastly, we showed that the expression of NS5A in B cells increased phosphorylation of activation loop tyrosine in the kinase domain of Fyn. NS5A containing ligand for both SH2 and SH3 domains enhances an aberrant autophosphorylation and kinase activity of Fyn in B cells.

Enhancement of B-cell receptor signaling by a point mutation of adaptor protein 3BP2 identified in human inherited disease cherubism

Tyrosine phosphorylation of adaptor protein c‐Abl‐Src homology 3 (SH3) domain‐binding protein‐2 (3BP2, also referred to SH3BP2) positively regulates the B‐cell antigen receptor (BCR)‐mediated signal transduction, leading to the activation of nuclear factor of activated T cells (NFAT). Here we showed the effect of the proline to arginine substitution of 3BP2 in which is the most common mutation in patients with cherubism (P418R) on B‐cell receptor signaling. Comparing to the wild type, overexpression of the mutant form of 3BP2 (3BP2‐P416R, corresponding to P418R in human protein) enhanced BCR‐mediated activation of NFAT. 3BP2‐P416R increased the signaling complex formation with Syk, phospholipase C‐γ2 (PLC‐γ2), and Vav1. In contrast, 3BP2‐P416R could not change the association with the negative regulator 14‐3‐3. Loss of the association mutant that was incapable to associate with 14‐3‐3 could not mimic BCR‐mediated NFAT activation in Syk‐deficient cells. Moreover, BCR‐mediated phosphorylation of extracellular signal regulated kinase (ERK) and c‐Jun N‐terminal kinase (JNK) was not affected by P416R mutation. These results showed that P416R mutation of 3BP2 causes the gain of function in B cells by increasing the interaction with specific signaling molecules.

Association of 3BP2 with SHP-1 regulates SHP-1-mediated production of TNF-α in RBL-2H3 cells

Adaptor protein 3BP2, a c‐Abl Src homology 3 (SH3) domain‐binding protein, is tyrosine phosphorylated and positively regulates mast cell signal transduction after the aggregation of the high affinity IgE receptor (FcεRI). Overexpression of the Src homology 2 (SH2) domain of 3BP2 results in the dramatic suppression of antigen‐induced degranulation in rat basophilic leukemia RBL‐2H3 cells. Previously, a linker for activation of T cells (LAT) was identified as one of the 3BP2 SH2 domain‐binding protein. In this report, to further understand the functions of 3BP2 in FcεRI‐mediated activation of mast cell, we explored the protein that associates with the SH2 domain of 3BP2 and found that SH2 domain‐containing phosphatase‐1 (SHP‐1) inducibly interacts with the SH2 domain of 3BP2 after the aggregation of FcεRI. The phosphorylation of Tyr564 in the carboxy (C)‐terminal tail region of SHP‐1 is required for the direct interaction of SHP‐1 to the SH2 domain of 3BP2. The expression of the mutant form of SHP‐1 which was unable to interact with 3BP2 resulted in the significant reduction in SHP‐1‐mediated tumor necrosis factor‐α (TNF‐α) production without any effects on the degranulation in antigen‐stimulated RBL‐2H3 cells. These findings suggest that 3BP2 directly interacts with Tyr564‐phosphorylated form of SHP‐1 and positively regulates the function of SHP‐1 in FcεRI‐mediated signaling in mast cells.

Tyrosine phosphorylation of 3BP2 is indispensable for the interaction with VAV3 in chicken DT40 cells

Adaptor protein c-Abl SH3 domain-binding protein-2 (3BP2) is known to play regulatory roles in immunoreceptor-mediated signal transduction. We have previously demonstrated that Tyr(174), Tyr(183) and Tyr(446) in mouse 3BP2 are predominantly phosphorylated by Syk, and the phosphorylation of Tyr(183) and the Src homology 2 (SH2) domain of mouse 3BP2 are critical for B cell receptor (BCR)-induced activation of nuclear factor of activated T cells (NFAT) in human B cells. In this report, we have shown that Syk, but not Abl family protein-tyrosine kinases, is critical for BCR-mediated tyrosine phosphorylation of 3BP2 in chicken DT40 cells. Mutational analysis showed that Tyr(174), Tyr(183) and Tyr(426) of chicken 3BP2 are the major phosphorylation sites by Syk and the SH2 domain of 3BP2 is critical for tyrosine phosphorylation. In addition, phosphorylation of Tyr(426) is required for the inducible interaction with the SH2 domain of Vav3. Moreover, the expression of the mutant form of 3BP2 in which Tyr(426) was substituted to Phe resulted in the reduction in BCR-mediated Rac1 activation, when compared with the case of wild-type. Altogether, these data suggest that 3BP2 is involved in the activation of Rac1 through the regulation of Vav3 by Syk-dependent phosphorylation of Tyr(426) following BCR stimulation.

Inhibition of the gamma interferon response by a Sendai virus C protein mutant with no STAT1-binding ability

Sendai virus C protein interacts with the signal transducer and activator of transcription (STAT) 1. This interaction is believed to be essential for the Sendai virus inhibition of the interferon (IFN) response. We here analyzed CF170S (a C protein mutant with the F170S mutation) with no STAT1‐binding ability. CF170S lacked the ability to inhibit the IFN‐α response, but retained the ability to inhibit the IFN‐γ response. IFN‐γ stimulation caused STAT1 phosphorylation, formation of the gamma‐activated factor capable of binding to a gamma‐activated sequence DNA probe, and STAT1 nuclear translocation, even in the presence of CF170S. These results suggest that C protein has the STAT1‐binding‐independent anti‐IFN‐γ mechanism, which targets processes after the STAT1 nuclear translocation event.