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.

PIM kinases facilitate lentiviral evasion from SAMHD1 restriction via Vpx phosphorylation.

Lentiviruses have evolved to acquire an auxiliary protein Vpx to counteract the intrinsic host restriction factor SAMHD1. Although Vpx is phosphorylated, it remains unclear whether such phosphorylation indeed regulates its activity toward SAMHD1. Here we identify the PIM family of serine/threonine protein kinases as the factors responsible for the phosphorylation of Vpx and the promotion of Vpx-mediated SAMHD1 counteraction. Integrated proteomics and subsequent functional analysis reveal that PIM family kinases, PIM1 and PIM3, phosphorylate HIV-2 Vpx at Ser13 and stabilize the interaction of Vpx with SAMHD1 thereby promoting ubiquitin-mediated proteolysis of SAMHD1. Inhibition of the PIM kinases promotes the antiviral activity of SAMHD1, ultimately reducing viral replication. Our results highlight a new mode of virus-host cell interaction in which host PIM kinases facilitate promotion of viral infectivity by counteracting the host antiviral system, and suggest a novel therapeutic strategy involving restoration of SAMHD1-mediated antiviral response.

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.

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.

Development of a cell-based assay to identify hepatitis B virus entry inhibitors targeting the sodium taurocholate cotransporting polypeptide.

Sodium taurocholate cotransporting polypeptide (NTCP) is a major entry receptor of hepatitis B virus (HBV) and one of the most attractive targets for anti-HBV drugs. We developed a cell-mediated drug screening method to monitor NTCP expression on the cell surface by generating a HepG2 cell line with tetracycline-inducible expression of NTCP and a monoclonal antibody that specifically detects cell-surface NTCP. Using this system, we screened a small molecule library for compounds that protected against HBV infection by targeting NTCP. We found that glabridin, a licorice-derived isoflavane, could suppress viral infection by inducing caveolar endocytosis of cell-surface NTCP with an IC50 of ~40 µM. We also found that glabridin could attenuate the inhibitory effect of taurocholate on type I interferon signaling by depleting the level of cell-surface NTCP. These results demonstrate that our screening system could be a powerful tool for discovering drugs targeting HBV entry.

Chemical array system, a platform to identify novel hepatitis B virus entry inhibitors targeting sodium taurocholate cotransporting polypeptide.

Current anti-hepatitis B virus (HBV) agents including interferons and nucleos(t)ide analogs efficiently suppress HBV infection. However, as it is difficult to eliminate HBV from chronically infected liver, alternative anti-HBV agents targeting a new molecule are urgently needed. In this study, we applied a chemical array to high throughput screening of small molecules that interacted with sodium taurocholate cotransporting polypeptide (NTCP), an entry receptor for HBV. From approximately 30,000 compounds, we identified 74 candidates for NTCP interactants, and five out of these were shown to inhibit HBV infection in cell culture. One of such compound, NPD8716, a coumarin derivative, interacted with NTCP and inhibited HBV infection without causing cytotoxicity. Consistent with its NTCP interaction capacity, this compound was shown to block viral attachment to host hepatocytes. NPD8716 also prevented the infection with hepatitis D virus, but not hepatitis C virus, in agreement with NPD8716 specifically inhibiting NTCP-mediated infection. Analysis of derivative compounds showed that the anti-HBV activity of compounds was apparently correlated with the affinity to NTCP and the capacity to impair NTCP-mediated bile acid uptake. These results are the first to show that the chemical array technology represents a powerful platform to identify novel viral entry inhibitors.

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.

The tumour suppressor APC promotes HIV-1 assembly via interaction with Gag precursor protein.

Diverse cellular proteins and RNAs are tightly regulated in their subcellular localization to exert their local function. Here we report that the tumour suppressor adenomatous polyposis coli protein (APC) directs the localization and assembly of human immunodeficiency virus (HIV)-1 Gag polyprotein at distinct membrane components to enable the efficient production and spread of infectious viral particles. A proteomic analysis and subsequent biomolecular interaction assay reveals that the carboxyl terminus of APC interacts with the matrix region of Gag. Ectopic expression of APC, but not its familial adenomatous polyposis-related truncation mutant, prominently enhances HIV-1 production. Conversely, the depletion of APC leads to a significant decrease in membrane targeting of viral components, resulting in the severe loss of production of infectious virions. Furthermore, APC promotes the directional assembly of viral components at virological synapses, thereby facilitating cell-to-cell viral transmission. These findings reveal an unexpected role of APC in the directional spread of HIV-1.

Molecular dissection of HBV evasion from restriction factor tetherin: A new perspective for antiviral cell therapy.

Viruses have evolved various strategies to escape from the innate cellular mechanisms inhibiting viral replication and spread. Extensive evidence has highlighted the ineffectiveness of interferon (IFN) therapy against chronic hepatitis B virus (HBV) infection, implying the existence of mechanisms by which HBV evades IFN-induced antiviral responses. In our current study, we demonstrate that HBV surface protein (HBs) plays a crucial role in counteracting the IFN-induced antiviral response mediated by tetherin (also known as BST-2). The type I IFN treatment of HBV-producing cells marginally but significantly inhibited the release of HBsAg and viral DNA, but this release was recovered by the knockdown of tetherin. HBs can interact with tetherin via its fourth transmembrane domain thereby inhibiting its dimerization and antiviral activity. The expression of a tetherin mutant devoid of the HBs-binding domain promoted a prominent restriction of HBV particle production that eventually resulted in the alleviation of caspase-1-mediated cytotoxicity and interleukin-1ß secretion in induced pluripotent stem cell (iPSC)-derived hepatocytes. Our current results thus reveal a previously undescribed molecular link between HBV and tetherin during the course of an IFN-induced antiviral response. In addition, strategies to augment the antiviral activity of tetherin by impeding tetherin-HBs interactions may be viable as a therapeutic intervention against HBV.

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.

Protoaculeine B, a putative N-terminal residue for the novel peptide toxin aculeines.

A new polyamine-modified indole derivative protoaculeine B (1) was isolated from Okinawan marine sponge Axinyssa aculeata. The structure of 1 was assigned on the basis of spectral data along with chemical transformations. Because the structure of 1 greatly inferred the N-terminal amino acid for highly modified peptide toxin aculeines, the probable structure for aculeine B was proposed on the basis of high-resolution mass spectral analysis.

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.

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.

Molecular and enzymatic characterization of XMRV protease by a cell-free proteolytic analysis.

Xenotropic murine leukemia virus-related virus (XMRV) is a virus generated under artificial conditions by the recombination of 2 murine leukemia virus (MLV) proviruses, PreXMRV-1 and PreXMRV-2, during the in vivo passage of human prostate cancer cells in athymic nude mice. The molecular etiology of XMRV infection has not been characterized and its implication in human prostate cancer progression remains equivocal. As a step toward resolving this issue we developed an in vitro enzymatic assay system to characterize XMRV protease (PR)-mediated cleavage of host-cell proteins. Enzymatically-active XMRV PR protein was synthesized using a wheat-germ cell-free system. By monitoring cleavage activity of XMRV PR by AlphaScreen and 2-color immunoblot analyses, we revealed that the catalytic activity of XMRV PR is selectively blocked by the HIV PR inhibitor, Amprenavir, and identified several human tumor suppressor proteins, including PTEN and BAX, to be substrates of XMRV PR. This system may provide an attractive means for analyzing the function of retrovirus proteases and provide a technology platform for drug screening.

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.

Long-chain polyamines (LCPAs) from marine sponge: possible implication in spicule formation.

Two distinct marine organisms, diatoms and sponges, deposit dissolved silicates to construct highly architectural and species-specific body supports. Several factors such as proteins, long-chain polyamines (LCPAs), or polypeptides modified with LCPAs are known to be involved in this process. The LCPAs contained in the silica walls of diatoms are thought to play pivotal roles in the silica deposition. In sponges, however, a protein called silicatein and several other proteins have been reported to be the factors involved in the silica deposition. However, no other factors involved in this process have been reported. We have identified the LCPAs from the marine sponge Axinyssa aculeata and present here some evidence that sponge-derived LCPAs can deposit silica and that the LCPA derivatives are associated with spicules. The results indicate a common chemistry between sponges and diatoms, the two major players in the biological circulation of silicon in the marine environment. A wide variety of organisms are known to utilize silica in their biological processes. Polyamines or other functional molecules might be involved, in combination with proteins, in their biosilicification process.