Satoko Matsunaga

Cyclosporin A and its analogs inhibit hepatitis B virus entry into cultured hepatocytes through targeting a membrane transporter, sodium taurocholate cotransporting polypeptide (NTCP).

Chronic hepatitis B virus (HBV) infection is a major public health problem worldwide. Although nucleos(t)ide analogs inhibiting viral reverse transcriptase are clinically available as anti‐HBV agents, emergence of drug‐resistant viruses highlights the need for new anti‐HBV agents interfering with other targets. Here we report that cyclosporin A (CsA) can inhibit HBV entry into cultured hepatocytes. The anti‐HBV effect of CsA was independent of binding to cyclophilin and calcineurin. Rather, blockade of HBV infection correlated with the ability to inhibit the transporter activity of sodium taurocholate cotransporting polypeptide (NTCP). We also found that HBV infection‐susceptible cells, differentiated HepaRG cells and primary human hepatocytes expressed NTCP, while nonsusceptible cell lines did not. A series of compounds targeting NTCP could inhibit HBV infection. CsA inhibited the binding between NTCP and large envelope protein in vitro. Evaluation of CsA analogs identified a compound with higher anti‐HBV potency, having a median inhibitory concentration <0.2 μM. Conclusion: This study provides a proof of concept for the novel strategy to identify anti‐HBV agents by targeting the candidate HBV receptor, NTCP, using CsA as a structural platform. (Hepatology 2014;59:1726–1737)

IRF8 inhibits C/EBPα activity to restrain mononuclear phagocyte progenitors from differentiating into neutrophils

Myeloid progenitors lose their potential to generate neutrophils when they adopt the mononuclear phagocyte lineage. The mechanism underlying this lineage restriction remains unknown. We here report that the protein expression of IRF8, an essential transcription factor for the development of dendritic cells (DCs) and monocytes, sharply increases at the monocyte-DC progenitor (MDP) stage and remains high in common monocyte progenitors (cMoPs). Irf8(-/-) MDPs and cMoPs accumulate but fail to efficiently generate their downstream populations, instead giving rise to neutrophils in vivo. IRF8 physically interacts with the transcription factor C/EBPα and prevents its binding to chromatin in MDPs and cMoPs, blocking the ability of C/EBPα to stimulate transcription and neutrophil differentiation. A partial inhibition of C/EBP activity in Irf8(-/-) haematopoietic progenitors alleviates the neutrophil overproduction in vivo. Thus, IRF8 not only bestows monocyte and DC differentiation potential upon mononuclear phagocyte progenitors but also restrains these progenitors from differentiating into neutrophils.

The phosphorylation of HIV-1 Gag by atypical protein kinase C facilitates viral infectivity by promoting Vpr incorporation into virions

BackgroundHuman immunodeficiency virus type 1 (HIV-1) Gag is the main structural protein that mediates the assembly and release of virus-like particles (VLPs) from an infected cell membrane. The Gag C-terminal p6 domain contains short sequence motifs that facilitate virus release from the plasma membrane and mediate incorporation of the viral Vpr protein. Gag p6 has also been found to be phosphorylated during HIV-1 infection and this event may affect virus replication. However, the kinase that directs the phosphorylation of Gag p6 toward virus replication remains to be identified. In our present study, we identified this kinase using a proteomic approach and further delineate its role in HIV-1 replication.ResultsA proteomic approach was designed to systematically identify human protein kinases that potently interact with HIV-1 Gag and successfully identified 22 candidates. Among this panel, atypical protein kinase C (aPKC) was found to phosphorylate HIV-1 Gag p6. Subsequent LC-MS/MS and immunoblotting analysis with a phospho-specific antibody confirmed both in vitro and in vivo that aPKC phosphorylates HIV-1 Gag at Ser487. Computer-assisted structural modeling and a subsequent cell-based assay revealed that this phosphorylation event is necessary for the interaction between Gag and Vpr and results in the incorporation of Vpr into virions. Moreover, the inhibition of aPKC activity reduced the Vpr levels in virions and impaired HIV-1 infectivity of human primary macrophages.ConclusionOur current results indicate for the first time that HIV-1 Gag phosphorylation on Ser487 is mediated by aPKC and that this kinase may regulate the incorporation of Vpr into HIV-1 virions and thereby supports virus infectivity. Furthermore, aPKC inhibition efficiently suppresses HIV-1 infectivity in macrophages. aPKC may therefore be an intriguing therapeutic target for HIV-1 infection.

Lyn Kinase Suppresses the Transcriptional Activity of IRF5 in the TLR-MyD88 Pathway to Restrain the Development of Autoimmunity

Interferon regulatory factor-5 (IRF5), a transcription factor critical for the induction of innate immune responses, contributes to the pathogenesis of the autoimmune disease systemic lupus erythematosus (SLE) in humans and mice. Lyn, a Src family kinase, is also implicated in human SLE, and Lyn-deficient mice develop an SLE-like disease. Here, we found that Lyn physically interacted with IRF5 to inhibit ubiquitination and phosphorylation of IRF5 in the TLR-MyD88 pathway, thereby suppressing the transcriptional activity of IRF5 in a manner independent of Lyns kinase activity. Conversely, Lyn did not inhibit NF-κB signaling, another major branch downstream of MyD88. Monoallelic deletion of Irf5 alleviated the hyperproduction of cytokines in TLR-stimulated Lyn(-/-) dendritic cells and the development of SLE-like symptoms in Lyn(-/-) mice. Our results reveal a role for Lyn as a specific suppressor of the TLR-MyD88-IRF5 pathway and illustrate the importance of fine-tuning IRF5 activity for the maintenance of immune homeostasis.

Paraquat Toxicity Induced by Voltage-dependent Anion Channel 1 Acts as an NADH-dependent Oxidoreductase

Paraquat (PQ), a herbicide used worldwide, causes fatal injury to organs upon high dose ingestion. Treatments for PQ poisoning are unreliable, and numerous deaths have been attributed inappropriate usage of the agent. It is generally speculated that a microsomal drug-metabolizing enzyme system is responsible for PQ toxicity. However, recent studies have demonstrated cytotoxicity via mitochondria, and therefore, the cytotoxic mechanism remains controversial. Here, we demonstrated that mitochondrial NADH-dependent PQ reductase containing a voltage-dependent anion channel 1 (VDAC1) is responsible for PQ cytotoxicity. When mitochondria were incubated with NADH and PQ, superoxide anion (O2̇̄) was produced, and the mitochondria ruptured. Outer membrane extract oxidized NADH in a PQ dose-dependent manner, and oxidation was suppressed by VDAC inhibitors. Zymographic analysis revealed the presence of VDAC1 protein in the oxidoreductase, and the direct binding of PQ to VDAC1 was demonstrated using biotinylated PQ. VDAC1-overexpressing cells showed increased O2̇̄ production and cytotoxicity, both of which were suppressed in VDAC1 knockdown cells. These results indicated that a VDAC1-containing mitochondrial system is involved in PQ poisoning. These insights into the mechanism of PQ poisoning not only demonstrated novel physiological functions of VDAC protein, but they may facilitate the development of new therapeutic approaches.

ASK1 restores the antiviral activity of APOBEC3G by disrupting HIV-1 Vif-mediated counteraction

APOBEC3G (A3G) is an innate antiviral restriction factor that strongly inhibits the replication of human immunodeficiency virus type 1 (HIV-1). An HIV-1 accessory protein, Vif, hijacks the host ubiquitin–proteasome system to execute A3G degradation. Identification of the host pathways that obstruct the action of Vif could provide a new strategy for blocking viral replication. We demonstrate here that the host protein ASK1 (apoptosis signal-regulating kinase 1) interferes with the counteraction by Vif and revitalizes A3G-mediated viral restriction. ASK1 binds the BC-box of Vif, thereby disrupting the assembly of the Vif–ubiquitin ligase complex. Consequently, ASK1 stabilizes A3G and promotes its incorporation into viral particles, ultimately reducing viral infectivity. Furthermore, treatment with the antiretroviral drug AZT (zidovudine) induces ASK1 expression and restores the antiviral activity of A3G in HIV-1-infected cells. This study thus demonstrates a distinct function of ASK1 in restoring the host antiviral system that can be enhanced by AZT treatment.

Development of oligomannose-coated liposome-based nasal vaccine against human parainfluenza virus type 3

Human parainfluenza viruses (HPIVs) are the etiologic agents of lower respiratory infections and pneumonia in infants, young children and immunocompromised hosts. The overarching goal for the prevention of HPIV infection is the development of an effective vaccine against HPIVs. In the present study, we investigated the effectiveness of oligomannose-coated liposomes (OMLs) as an antigen-delivery system in combination with a synthetic double-stranded RNA analog for the induction of mucosal and systematic immunity against HPIV3. Full-length hemagglutinin-neuraminidase (HN) protein was synthesized using the wheat germ cell-free protein production system and then encapsulated into OML to serve as the antigen. Intranasal administration of the HN-filling OML (OML-HN) with the synthetic double-stranded RNA adjuvant, polyriboinosinic-polyribocytidylic acid [poly(I:C)] generated significant viral-specific systemic and mucosal immune responses as evidenced by the prominent induction of serum IgG and nasal wash IgA, respectively. On the other hand, no significant immune responses were observed in mice immunized with OML-HN without the adjuvant. Furthermore, serum from mice immunized with OML-HN plus poly(I:C) significantly suppressed viral infection in cell culture model. Our results provide the first evidence that intranasal co-administration of OML-encapsulated HN with the poly(I:C) adjuvant augments the viral-specific immunity against HPIV3.

Interferon-Induced SCYL2 Limits Release of HIV-1 by Triggering PP2A-Mediated Dephosphorylation of the Viral Protein Vpu

HIV-infected cells dephosphorylate and inactivate a viral factor to restrict viral spreading. Belt and Braces Antiviral Approach In the ongoing arms race between pathogens and their hosts, each side generates factors with which to thwart the other. In the case of HIV-1 infection, cells produce type I interferon (IFN), which induces production of tetherin (also known as BST2), which traps new virus particles at the plasma membrane, preventing their release. For its part, HIV-1 produces the protein Vpu, which causes the degradation of tetherin, thus promoting viral release. Miyakawa et al. found that another IFN-inducible host protein, SCYL2, antagonized the function of Vpu, enabling tetherin to fulfill its role as a viral restriction factor. Vpu requires phosphorylation at two serine residues to function normally, and SCYL2 recruited the phosphatase PP2A to Vpu to render it inactive. Together, these data suggest the existence of a host back-up system to restrict viral spreading. Human cells respond to infection by retroviruses through the actions of proteins that inhibit the spread of viruses to other cells. One example is bone marrow stromal cell antigen 2 (BST2; also known as tetherin), which is an interferon (IFN)–inducible protein that restricts the release of progeny virions from infected cells. The HIV-1 accessory protein Vpu (viral protein U) causes degradation of BST2, and phosphorylation of Vpu at residues Ser52 and Ser56 is required for this function. We report that the host protein SCY1-like protein 2 (SCYL2) mediates the dephosphorylation of Vpu, antagonizing Vpu function and facilitating BST2-dependent restriction of HIV-1 release. SCYL2 reduced the number of virus particles released from cells infected with wild-type HIV-1, but not a strain lacking vpu, in a BST2-dependent manner. SCYL2 stimulated the dephosphorylation of Vpu on Ser52 and Ser56 by recruiting protein phosphatase 2A (PP2A) to Vpu. Conversely, depletion of SCYL2 resulted in enhanced phosphorylation of Vpu and increased viral particle release. Moreover, SCYL2 was produced in response to type I IFN and contributed to IFN-mediated viral restriction. Together, these results suggest that SCYL2 serves as a regulatory factor for Vpu, reducing the extent of Vpu phosphorylation and consequently enhancing BST2-mediated viral restriction.

Mutations in genes encoding polycomb repressive complex 2 subunits cause Weaver syndrome

Weaver syndrome (WS) is a rare congenital overgrowth disorder caused by heterozygous mutations in EZH2 (enhancer of zeste homolog 2) or EED (embryonic ectoderm development). EZH2 and EED are core components of the polycomb repressive complex 2 (PRC2), which possesses histone methyltransferase activity and catalyzes trimethylation of histone H3 at lysine 27. Here, we analyzed eight probands with clinically suspected WS by whole‐exome sequencing and identified three mutations: a 25.4‐kb deletion partially involving EZH2 and CUL1 (individual 1), a missense mutation (c.707G>C, p.Arg236Thr) in EED (individual 2), and a missense mutation (c.1829A>T, p.Glu610Val) in SUZ12 (suppressor of zeste 12 homolog) (individual 3) inherited from her father (individual 4) with a mosaic mutation. SUZ12 is another component of PRC2 and germline mutations in SUZ12 have not been previously reported in humans. In vitro functional analyses demonstrated that the identified EED and SUZ12 missense mutations cause decreased trimethylation of lysine 27 of histone H3. These data indicate that loss‐of‐function mutations of PRC2 components are an important cause of WS.

Anti-interleukin-5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin-5 levels

Atherosclerotic diseases, such as coronary artery disease and peripheral artery disease, are systemic disorders and among the leading causes of mortality and morbidity throughout the world. However, the exact pathophysiological mechanisms underlying the development of atherosclerosis remain unknown; currently, atherosclerosis is thought to involve an inflammatory process. Systemic inflammatory reactions and accumulation of immune cells in atherosclerotic lesions in situ are considered essential. We have comprehensively analyzed autoantibodies in patients with atherosclerosis by means of a newly developed high‐throughput autoantibody analysis system. A wide range of autoantibodies was found in sera from patients with atherosclerosis. After we statistically analyzed the titers of each autoantibody with conventional techniques, the results underwent text‐mining analyses based on natural language processing. Combinatory analysis revealed a close association between anti‐interleukin (IL)‐5 antibody and atherosclerosis. Titers of anti‐IL‐5 antibodies and serum IL‐5 concentrations were also closely associated with other risk factors, such as low‐density lipoprotein cholesterol, serum creatinine, fasting plasma glucose, gender, and age, suggesting that suppressed IL‐5 function mediated by autoantibodies in patients with atherosclerosis plays an important role in the disease process. To validate the clinical significance of these findings, we computed the specificity and sensitivity of titers of anti‐IL‐5 autoantibodies for human atherosclerosis. When antibody titers of 1.49 were assumed to predict the presence of atherosclerosis, the sensitivity was 95.0% and the specificity 91.0%, with an area under the curve of 0.940. Our results provide important clues to understanding the role of autoantibody‐mediated immune reactions in human atherosclerosis and suggest novel therapeutic opportunities for management of the disease.—Ishigami, T., Abe, K., Aoki, I., Minegishi, S., Ryo, A., Matsunaga, S., Matsuoka, K., Takeda, H., Sawasaki, T., Umemura, S., and Endo, Y., Anti‐interleukin‐5 and multiple autoantibodies are associated with human atherosclerotic diseases and serum interleukin‐5 levels. FASEB J. 27, 3437–3445 (2013). www.fasebj.org