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 Lyn's 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.

Streptococcus pneumoniae hijacks host autophagy by deploying CbpC as a decoy for Atg14 depletion.

Pneumococcal cell surface-exposed choline-binding proteins (CBPs) play pivotal roles in multiple infectious processes with pneumococci. Intracellular pneumococci can be recognized at multiple steps during bactericidal autophagy. However, whether CBPs are involved in pneumococci-induced autophagic processes remains unknown. In this study, we demonstrate that CbpC from S. pneumoniae strain TIGR4 activates autophagy through an interaction with Atg14. However, S. pneumoniae also interferes with autophagy by deploying CbpC as a decoy to cause autophagic degradation of Atg14 through an interaction with p62/SQSTM1. Thus, S. pneumoniae suppresses the autophagic degradation of intracellular pneumococci and survives within cells. Domain analysis reveals that the coiled-coil domain of Atg14 and residue Y83 of the dp3 domain in the N-terminal region of CbpC are crucial for both the CbpC-Atg14 interaction and the subsequent autophagic degradation of Atg14. Although homology modeling indicates that CbpC orthologs have similar structures in the dp3 domain, autophagy induction through Atg14 binding is an intrinsic property of CbpC. Our data provide novel insights into the evolutionary hijacking of host-defense systems by intracellular pneumococci.

Zika virus protease induces caspase-independent pyroptotic cell death by directly cleaving gasdermin D.

The association of Zika virus (ZIKV) infection with a congenital malformation in fetuses, neurological, and other systemic complications in adults have brought significant global health emergency. ZIKV targets nerve cells in the brain and causes cell death, such as pyroptosis, leading to neuroinflammation. Here we described a novel mechanism of pyroptosis caused by ZIKV protease. We found that ZIKV protease directly cleaved the GSDMD into N-terminal fragment (1-249) leading to pyroptosis in a caspase-independent manner, suggesting a direct mechanism of ZIKV-induced cell death and subsequent inflammation. Our findings might shed new light to explore the pathogenesis of ZIKV infections where ZIKV protease might be a suitable target for the development of antiviral agents.

Structure Revision of Protoaculeine B, a Post-translationally Modified N-Terminal Residue in the Peptide Toxin Aculeine B.

The structure of protoaculeine B, the N-terminal residue of the marine peptide toxin aculeine B, is revised to the cis-1,3-disubstituted tetrahydro-ß-carboline framework. We prepared two truncated model compounds that lack a long-chain polyamine using the one-step Pictet-Spengler reaction of tryptophan and compared their NMR, mass spectra, and chemical reactivity with those of the natural protoaculeine B. The synthetic models reproduced the profiles of the natural product well, which confirmed the appropriateness of the structure revision.

Pectenovarin, A New Ovarian Carotenoprotein from Japanese Scallop Mizuhopecten yessoensis.

The scallop Mizuhopecten yessoensis accumulates carotenoids in the ovary during the maturation stage. Its conspicuous pink color implies the presence of carotenoprotein. However, the carotenoprotein from the scallop ovary has never been isolated and characterized, probably due to its instability and complexity. Here, we developed an extraction and isolation procedure for the carotenoprotein by employing a basic buffer containing potassium bromide to facilitate its efficient extraction from the ovary, and we succeeded in obtaining the carotenoprotein, termed pectenovarin. The carotenoid composition of the pectenovarin was similar to that of the ovary. The N-terminal and internal amino acid sequences of pectenovarin showed a high similarity to those of vitellogenin, the precursor of egg yolk protein under analysis.

Biallelic Mutations in Nuclear Pore Complex Subunit NUP107 Cause Early-Childhood-Onset Steroid-Resistant Nephrotic Syndrome.

The nuclear pore complex (NPC) is a huge protein complex embedded in the nuclear envelope. It has central functions in nucleocytoplasmic transport, nuclear framework, and gene regulation. Nucleoporin 107 kDa (NUP107) is a component of the NPC central scaffold and is an essential protein in all eukaryotic cells. Here, we report on biallelic NUP107 mutations in nine affected individuals who are from five unrelated families and show early-onset steroid-resistant nephrotic syndrome (SRNS). These individuals have pathologically focal segmental glomerulosclerosis, a condition that leads to end-stage renal disease with high frequency. NUP107 is ubiquitously expressed, including in glomerular podocytes. Three of four NUP107 mutations detected in the affected individuals hamper NUP107 binding to NUP133 (nucleoporin 133 kDa) and NUP107 incorporation into NPCs in vitro. Zebrafish with nup107 knockdown generated by morpholino oligonucleotides displayed hypoplastic glomerulus structures and abnormal podocyte foot processes, thereby mimicking the pathological changes seen in the kidneys of the SRNS individuals with NUP107 mutations. Considering the unique properties of the podocyte (highly differentiated foot-process architecture and slit membrane and the inability to regenerate), we propose a "podocyte-injury model" as the pathomechanism for SRNS due to biallelic NUP107 mutations.

A new strategy to identify hepatitis B virus entry inhibitors by AlphaScreen technology targeting the envelope-receptor interaction.

Current anti-hepatitis B virus (HBV) agents have limited effect in curing HBV infection, and thus novel anti-HBV agents with different modes of action are in demand. In this study, we applied AlphaScreen assay to high-throughput screening of small molecules inhibiting the interaction between HBV large surface antigen (LHBs) and the HBV entry receptor, sodium taurocholate cotransporting polypeptide (NTCP). From the chemical screening, we identified that rapamycin, an immunosuppressant, strongly inhibited the LHBs-NTCP interaction. Rapamycin inhibited hepatocyte infection with HBV without significant cytotoxicity. This activity was due to impaired attachment of the LHBs preS1 domain to cell surface. Pretreatment of target cells with rapamycin remarkably reduced their susceptibility to preS1 attachment, while rapamycin pretreatment to preS1 did not affect its attachment activity, suggesting that rapamycin targets the host side. In support of this, a surface plasmon resonance analysis showed a direct interaction of rapamycin with NTCP. Consistently, rapamycin also prevented hepatitis D virus infection, whose entry into cells is also mediated by NTCP. We also identified two rapamycin derivatives, everolimus and temsirolimus, which possessed higher anti-HBV potencies than rapamycin. Thus, this is the first report for application of AlphaScreen technology that monitors a viral envelope-receptor interaction to identify viral entry inhibitors.

Production and characterization of monoclonal antibodies specific for major capsid VP1 protein of trichodysplasia spinulosa-associated polyomavirus.

Trichodysplasia spinulosa-associated polyomavirus (TSPyV), a newly identified polyomavirus, has been implicated as a causative agent of trychodysplasia spinulosa (TS), a rare proliferative skin disease in severely immunocompromised hosts. Diagnosis using mAbs is a promising tool with high specificity towards the specific antigen. However, thus far, no suitable mAbs for diagnosing TS disease have been identified. In this study, mAbs specific for VP1 of TSPyV were developed and characterized. Wheat germ cell-free synthesized VP1 protein of TSPyV was used to immunize BALB/c mice to generate hybridomas. Screening of the resultant hybridoma clones resulted in selection of five strongly positive clones that produce mAbs that react with the TSPyV-VP1 antigen. Epitope mapping and bioinformatic analysis showed that these mAbs recognized epitopes located within highly conserved C-terminal region of all clinical isolates of TSPyV-VP1. Further, all these mAbs were highly effective for immunofluorescence and immunoprecipitation analysis. Three of the five mAbs exhibited no cross-reactivity with VP1 of other related polyomaviruses. In addition, one of our mAbs (#14) provided immunohistochemical staining of skin tissue of TS disease. It can be concluded that three of the mAbs in this panel of anti-VP1 antibodies may provide a useful set of tools for studying TSPyV infection and making the specific diagnosis.

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.

MIP-1α level in nasopharyngeal aspirates at the first wheezing episode predicts recurrent wheezing.

BACKGROUND: Respiratory virus-induced wheezing, such as that induced by respiratory syncytial virus (RSV) and human rhinovirus, is an important risk factor for recurrent wheezing and childhood asthma. However, no biomarkers for predicting recurrent wheezing have been identified. OBJECTIVE: We searched for predictors of recurrent wheezing using nasopharyngeal aspirates obtained from patients during the first wheezing episode who were hospitalized with an acute lower respiratory tract illness. METHODS: We enrolled 82 infants during the first wheezing episode (median age, 5.0 months) who were hospitalized for acute lower respiratory tract illness between August 2009 and June 2012 and followed these patients for 2.5 years. Nasopharyngeal aspirates and blood samples were obtained on the first day of hospitalization. Viral genomes were identified by using RT-PCR and sequencing. Levels of 33 cytokines, tryptase, IgE, anti-RSV IgE, and anti-RSV IgG were measured by using ELISAs or the Bio-Plex multiplex assay. Predictors of recurrent wheezing were examined by using a stepwise logistic regression model with backward elimination. RESULTS: Sixty percent of the patients experienced recurrent wheezing episodes. One or more viruses were detected in the nasopharynxes of 93% of the patients during the first wheezing episode. IFN-γ, IL-2, IL-9, MIP-1α, and MIP-1ß levels were significantly higher among patients with recurrent wheezing than among those without recurrent wheezing (P < .05 or .01). The stepwise model demonstrated that the MIP-1α level (odds ratio, 7.72; 95% CI, 1.50-39.77; P = .015) was the strongest independent predictor of the occurrence of recurrent wheezing. CONCLUSION: An increased MIP-1α level in nasopharyngeal aspirates from patients with acute respiratory symptoms during the first wheezing episode caused by viral infections might predict recurrent wheezing.

Involvement of hepatitis C virus NS5A hyperphosphorylation mediated by casein kinase I-α in infectious virus production.

UNLABELLED: Nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) possesses multiple functions in the viral life cycle. NS5A is a phosphoprotein that exists in hyperphosphorylated and basally phosphorylated forms. Although the phosphorylation status of NS5A is considered to have a significant impact on its function, the mechanistic details regulating NS5A phosphorylation, as well as its exact roles in the HCV life cycle, are still poorly understood. In this study, we screened 404 human protein kinases via in vitro binding and phosphorylation assays, followed by RNA interference-mediated gene silencing in an HCV cell culture system. Casein kinase I-α (CKI-α) was identified as an NS5A-associated kinase involved in NS5A hyperphosphorylation and infectious virus production. Subcellular fractionation and immunofluorescence confocal microscopy analyses showed that CKI-α-mediated hyperphosphorylation of NS5A contributes to the recruitment of NS5A to low-density membrane structures around lipid droplets (LDs) and facilitates its interaction with core protein and the viral assembly. Phospho-proteomic analysis of NS5A with or without CKI-α depletion identified peptide fragments that corresponded to the region located within the low-complexity sequence I, which is important for CKI-α-mediated NS5A hyperphosphorylation. This region contains eight serine residues that are highly conserved among HCV isolates, and subsequent mutagenesis analysis demonstrated that serine residues at amino acids 225 and 232 in NS5A (genotype 2a) may be involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of virion production. These findings provide insight concerning the functional role of NS5A phosphorylation as a regulatory switch that modulates its multiple functions in the HCV life cycle. IMPORTANCE: Mechanisms regulating NS5A phosphorylation and its exact function in the HCV life cycle have not been clearly defined. By using a high-throughput screening system targeting host protein kinases, we identified CKI-α as an NS5A-associated kinase involved in NS5A hyperphosphorylation and the production of infectious virus. Our results suggest that the impact of CKI-α in the HCV life cycle is more profound on virion assembly than viral replication via mediation of NS5A hyperphosphorylation. CKI-α-dependent hyperphosphorylation of NS5A plays a role in recruiting NS5A to low-density membrane structures around LDs and facilitating its interaction with the core for new virus particle formation. By using proteomic approach, we identified the region within the low-complexity sequence I of NS5A that is involved in NS5A hyperphosphorylation and hyperphosphorylation-dependent regulation of infectious virus production. These findings will provide novel mechanistic insights into the roles of NS5A-associated kinases and NS5A phosphorylation in the HCV life cycle.

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

UNLABELLED: 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.

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

BACKGROUND: Human 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. RESULTS: A 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. CONCLUSION: Our 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.

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.

Isolation of novel prototype galectins from the marine ball sponge Cinachyrella sp. guided by their modulatory activity on mammalian glutamate-gated ion channels.

Here we report the bioactivity-guided isolation of novel galectins from the marine sponge Cinachyrella sp., collected from Iriomote Island, Japan. The lectin proteins, which we refer to as the Cinachyrella galectins (CchGs), were identified as the active principles in an aqueous sponge extract that modulated the function of mammalian ionotropic glutamate receptors. Aggregation of rabbit erythrocytes by CchGs was competed most effectively by galactosides but not mannose, a profile characteristic of members of the galectin family of oligosaccharide-binding proteins. The lectin activity was remarkably stable, with only a modest loss in hemagglutination after exposure of the protein to 100°C for 1 h, and showed little sensitivity to calcium concentration. CchG-1 and -2 appeared as 16 and 18 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, respectively, whereas matrix-assisted laser desorption ionization-time-of-flight-mass spectrometry indicated broad ion clusters centered at 16,216 and 16,423, respectively. The amino acid sequences of the CchGs were deduced using a combination of Edman degradation and cDNA cloning and revealed that the proteins were distant orthologs of animal prototype galectins and that multiple isolectins comprised the CchGs. One of the isolectins was expressed as a recombinant protein and exhibited physico-chemical and biological properties comparable with those of the natural lectins. The biochemical properties of the CchGs as well as their unexpected activity on mammalian excitatory amino acid receptors suggest that further analysis of these new members of the galectin family will yield further glycobiological and neurophysiological insights.

DS-5670a, a novel mRNA-encapsulated lipid nanoparticle vaccine against severe acute respiratory syndrome coronavirus 2: Results from a phase 2 clinical study.

BACKGROUND: DS-5670a is a vaccine candidate for coronavirus disease 2019 (COVID-19) harnessing a novel modality composed of messenger ribonucleic acid (mRNA) encoding the receptor-binding domain (RBD) from the spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) encapsulated in lipid nanoparticles. Here, we report the safety, immunogenicity, and pharmacokinetic profile of DS-5670a from a phase 2 clinical trial in healthy adults who were immunologically naïve to SARS-CoV-2. METHODS: The study consisted of an open-label, uncontrolled, dose-escalation part and a double-blind, randomized, uncontrolled, 2-arm, parallel-group part. A total of 80 Japanese participants were assigned to receive intramuscular DS-5670a, containing either 30 or 60 µg of mRNA, as two injections administered 4 weeks apart. Safety was assessed by characterization of treatment-emergent adverse events (TEAEs). Immunogenicity was assessed by neutralization titers against SARS-CoV-2, anti-RBD immunoglobulin (Ig)G levels, and SARS-CoV-2 spike-specific T cell responses. Plasma pharmacokinetic parameters of DS-5670a were also evaluated. RESULTS: Most solicited TEAEs were mild or moderate with both the 30 and 60 µg mRNA doses. Four participants (10 %) in the 60 µg mRNA group developed severe redness at the injection site, but all cases resolved without treatment. There were no serious TEAEs and no TEAEs leading to discontinuation. Humoral immune responses in both dose groups were greater than those observed in human convalescent serum; the 60 µg mRNA dose produced better responses. Neutralization titers were found to be correlated with anti-RBD IgG levels (specifically IgG1). DS-5670a elicited antigen-specific T helper 1-polarized cellular immune responses. CONCLUSIONS: The novel mRNA-based vaccine candidate DS-5670a provided favorable immune responses against SARS-CoV-2 with a clinically acceptable safety profile. Confirmatory trials are currently ongoing to evaluate the safety and immunogenicity of DS-5670a as the primary vaccine and to assess the immunogenicity when administered as a heterologous or homologous booster. TRIAL REGISTRY: https://jrct.niph.go.jp/latest-detail/jRCT2071210086.

NMR study of xenotropic murine leukemia virus-related virus protease in a complex with amprenavir.

Xenotropic murine leukemia virus-related virus (XMRV) is a virus created through recombination of two murine leukemia proviruses under artificial conditions during the passage of human prostate cancer cells in athymic nude mice. The homodimeric protease (PR) of XMRV plays a critical role in the production of functional viral proteins and is a prerequisite for viral replication. We synthesized XMRV PR using the wheat germ cell-free expression system and carried out structural analysis of XMRV PR in a complex with an inhibitor, amprenavir (APV), by means of NMR. Five different combinatorially (15)N-labeled samples were prepared and backbone resonance assignments were made by applying Otting's method, with which the amino acid types of the [(1)H, (15)N] HSQC resonances were automatically identified using the five samples (Wu et al., 2006) [14]. A titration experiment involving APV revealed that one APV molecule binds to one XMRV PR dimer. For many residues, two distinct resonances were observed, which is thought to be due to the structural heterogeneity between the two protomers in the APV:XMRV PR=1:2 complex. PR residues at the interface with APV have been identified on the basis of chemical shift perturbation and identification of the intermolecular NOEs by means of filtered NOE experiments. Interestingly, chemical shift heterogeneity between the two protomers of XMRV PR has been observed not only at the interface with APV but also in regions apart from the interface. This indicates that the structural heterogeneity induced by the asymmetry of the binding of APV to the XMRV PR dimer is transmitted to distant regions. This is in contrast to the case of the APV:HIV-1 PR complex, in which the structural heterogeneity is only localized at the interface. Long-range transmission of the structural change identified for the XMRV PR complex might be utilized for the discovery of a new type of drug.

Galectin-9 restricts hepatitis B virus replication via p62/SQSTM1-mediated selective autophagy of viral core proteins.

Autophagy has been linked to a wide range of functions, including a degradative process that defends host cells against pathogens. Although the involvement of autophagy in HBV infection has become apparent, it remains unknown whether selective autophagy plays a critical role in HBV restriction. Here, we report that a member of the galectin family, GAL9, directs the autophagic degradation of HBV HBc. BRET screening revealed that GAL9 interacts with HBc in living cells. Ectopic expression of GAL9 induces the formation of HBc-containing cytoplasmic puncta through interaction with another antiviral factor viperin, which co-localized with the autophagosome marker LC3. Mechanistically, GAL9 associates with HBc via viperin at the cytoplasmic puncta and enhanced the auto-ubiquitination of RNF13, resulting in p62 recruitment to form LC3-positive autophagosomes. Notably, both GAL9 and viperin are type I IFN-stimulated genes that act synergistically for the IFN-dependent proteolysis of HBc in HBV-infected hepatocytes. Collectively, these results reveal a previously undescribed antiviral mechanism against HBV in infected cells and a form of crosstalk between the innate immune system and selective autophagy in viral infection.

Production and partial purification of membrane proteins using a liposome-supplemented wheat cell-free translation system.

BACKGROUND: Recently, some groups have reported on cell-free synthesis of functional membrane proteins (MPs) in the presence of exogenous liposomes (liposomes). Previously, we reported synthesis of a functional AtPPT1 plant phosphate transporter that was associated with liposomes during translation. However, it is unclear whether or not lipid/MP complex formation is common to all types of MPs in the wheat cell-free system. RESULTS: AtPPT1 was synthesized using a wheat cell-free system with or without liposomes. AtPPT1 synthesized with liposomes showed high transport activity, but the activity of AtPPT1 synthesized without liposomes was less than 10% activity of that with liposomes. To test whether co-translational association with liposomes is observed in the synthesis of other MPs, we used 40 mammalian MPs having one to 14 transmembrane domains (TMDs) and five soluble proteins as a control. The association rate of all 40 MPs into liposomes was more than 40% (mean value: 59%), while that of the five soluble proteins was less than 20% (mean value: 12%). There were no significant differences in association rate among MPs regardless of the number of TMDs and synthesis yield. These results indicate that the wheat cell-free system is a highly productive method for lipid/MP complex formation and is suitable for large-scale preparation. The liposome association of green fluorescent protein (GFP)-fusion MPs were also tested and recovered as lipid/MP complex after floatation by Accudenz density gradient ultracentrifugation (DGU). Employment of GFP-MPs revealed optimal condition for Accudenz floatation. Using the optimized Accudenz DGU condition, P2RX4/lipid complexes were partially purified and detected as a major band by Coomassie Brilliant Blue (CBB)-staining after SDS-PAGE. CONCLUSION: Formation of lipid/AtPPT1 complex during the cell-free synthesis reaction is critical for synthesis of a functional MP. The lipid/MP complex during the translation was observed in all 40 MPs tested. At least 29 MPs, as judged by their higher productivity compared to GFP, might be suitable for a large-scale preparation. MPs synthesized by this method form lipid/MP complexes, which could be readily partially purified by Accudenz DGU. Wheat cell-free protein synthesis in the presence of liposomes will be a useful method for preparation of variety type of MPs.

Isolation, amino acid sequence and biological activities of novel long-chain polyamine-associated peptide toxins from the sponge Axinyssa aculeata.

A novel family of functionalized peptide toxins, aculeines (ACUs), was isolated from the marine sponge Axinyssa aculeate. ACUs are polypeptides with N-terminal residues that are modified by the addition of long-chain polyamines (LCPA). Aculeines were present in the sponge extract as a complex mixture with differing polyamine chain lengths and peptide structures. ACU-A and B, which were purified in this study, share a common polypeptide chain but differ in their N-terminal residue modifications. The amino acid sequence of the polypeptide portion of ACU-A and B was deduced from 3' and 5' RACE, and supported by Edman degradation and mass spectral analysis of peptide fragments. ACU induced convulsions upon intracerebroventricular (i.c.v.) injection in mice, and disrupted neuronal membrane integrity in electrophysiological assays. ACU also lysed erythrocytes with a potency that differed between animal species. Here we describe the isolation, amino acid sequence, and biological activity of this new group of cytotoxic sponge peptides.