Soluble SLAMF7 is generated by alternative splicing in multiple myeloma cells.

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Notum deacylates octanoylated ghrelin.

OBJECTIVES: The only proteins known to be modified by O-linked lipidation are Wnts and ghrelin, and enzymatic removal of this post-translational modification inhibits ligand activity. Indeed, the Wnt-deacylase activity of Notum is the basis of its ability to act as a feedback inhibitor of Wnt signalling. Whether Notum also deacylates ghrelin has not been determined. METHODS: We used mass spectrometry to assay ghrelin deacylation by Notum and co-crystallisation to reveal enzyme-substrate interactions at the atomic level. CRISPR/Cas technology was used to tag endogenous Notum and assess its localisation in mice while liver-specific Notum knock-out mice allowed us to investigate the physiological role of Notum in modulating the level of ghrelin deacylation. RESULTS: Mass spectrometry detected the removal of octanoyl from ghrelin by purified active Notum but not by an inactive mutant. The 2.2 Å resolution crystal structure of the Notum-ghrelin complex showed that the octanoyl lipid was accommodated in the hydrophobic pocket of the Notum. The knock-in allele expressing HA-tagged Notum revealed that Notum was produced in the liver and present in the bloodstream, albeit at a low level. Liver-specific inactivation of Notum in animals fed a high-fat diet led to a small but significant increase in acylated ghrelin in the circulation, while no such increase was seen in wild-type animals on the same diet. CONCLUSIONS: Overall, our data demonstrate that Notum can act as a ghrelin deacylase, and that this may be physiologically relevant under high-fat diet conditions. Our study therefore adds Notum to the list of enzymes, including butyrylcholinesterase and other carboxylesterases, that modulate the acylation state of ghrelin. The contribution of multiple enzymes could help tune the activity of this important hormone to a wide range of physiological conditions.

Oseltamivir Is Effective against 1918 Influenza Virus Infection of Macaques but Vulnerable to Escape.

The 1918 influenza virus, subtype H1N1, was the causative agent of the most devastating pandemic in the history of infectious diseases. In vitro studies have confirmed that extreme virulence is an inherent property of this virus. Here, we utilized the macaque model for evaluating the efficacy of oseltamivir phosphate against the fully reconstructed 1918 influenza virus in a highly susceptible and relevant disease model. Our findings demonstrate that oseltamivir phosphate is effective in preventing severe disease in macaques but vulnerable to virus escape through emergence of resistant mutants, especially if given in a treatment regimen. Nevertheless, we conclude that oseltamivir would be highly beneficial to reduce the morbidity and mortality rates caused by a highly pathogenic influenza virus although it would be predicted that resistance would likely emerge with sustained use of the drug.IMPORTANCE Oseltamivir phosphate is used as a first line of defense in the event of an influenza pandemic prior to vaccine administration. Treatment failure through selection and replication of drug-resistant viruses is a known complication in the field and was also demonstrated in our study with spread of resistant 1918 influenza virus in multiple respiratory tissues. This emphasizes the importance of early treatment and the possibility that noncompliance may exacerbate treatment effectiveness. It also demonstrates the importance of implementing combination therapy and vaccination strategies as soon as possible in a pandemic situation.

Making, Exporting, and Modulating Wnts.

Wnt proteins activate a conserved signalling pathway that controls development and tissue homeostasis in all metazoans. The intensity of Wnt signalling must be tightly controlled to avoid diseases caused by excess or ectopic signalling. Over the years, many proteins dedicated to Wnt function have been identified, including Porcupine, which appends a palmitoleate moiety that is essential for signalling activity. This lipid inevitably affects subcellular trafficking and solubility, as well as providing a target for post-translational modulation. We review here the life history of Wnts, starting with progression through the secretory pathway, continuing with release and spread in the extracellular space, and finishing with the various proteins that dampen or inactivate Wnts in the extracellular space.

Godzilla-dependent transcytosis promotes Wingless signalling in Drosophila wing imaginal discs.

The apical and basolateral membranes of epithelia are insulated from each other, preventing the transfer of extracellular proteins from one side to the other. Thus, a signalling protein produced apically is not expected to reach basolateral receptors. Evidence suggests that Wingless, the main Drosophila Wnt, is secreted apically in the embryonic epidermis. However, in the wing imaginal disc epithelium, Wingless is mostly seen on the basolateral membrane where it spreads from secreting to receiving cells. Here we examine the apico-basal movement of Wingless in Wingless-producing cells of wing imaginal discs. We find that it is presented first on the apical surface before making its way to the basolateral surface, where it is released and allowed to interact with signalling receptors. We show that Wingless transcytosis involves dynamin-dependent endocytosis from the apical surface. Subsequent trafficking from early apical endosomes to the basolateral surface requires Godzilla, a member of the RNF family of membrane-anchored E3 ubiquitin ligases. Without such transport, Wingless signalling is strongly reduced in this tissue.

Notum deacylates Wnt proteins to suppress signalling activity.

Signalling by Wnt proteins is finely balanced to ensure normal development and tissue homeostasis while avoiding diseases such as cancer. This is achieved in part by Notum, a highly conserved secreted feedback antagonist. Notum has been thought to act as a phospholipase, shedding glypicans and associated Wnt proteins from the cell surface. However, this view fails to explain specificity, as glypicans bind many extracellular ligands. Here we provide genetic evidence in Drosophila that Notum requires glypicans to suppress Wnt signalling, but does not cleave their glycophosphatidylinositol anchor. Structural analyses reveal glycosaminoglycan binding sites on Notum, which probably help Notum to co-localize with Wnt proteins. They also identify, at the active site of human and Drosophila Notum, a large hydrophobic pocket that accommodates palmitoleate. Kinetic and mass spectrometric analyses of human proteins show that Notum is a carboxylesterase that removes an essential palmitoleate moiety from Wnt proteins and thus constitutes the first known extracellular protein deacylase.

Competitive incorporation of homologous gene segments of influenza A virus into virions.

By using two reporter protein-encoding virus-like RNAs derived from identical viral RNA (vRNA) segments, we assessed their incorporation efficiency into single progeny virions. Most plaques formed by the recombinant viruses that were generated in cells positive for both reporter genes expressed only one or the other protein. These results suggest that two virus-like RNAs encoding different reporter proteins compete for incorporation into virions, and individual influenza virions incorporate single, but not multiple, copies of homologous vRNA segments.

Replication-incompetent influenza A viruses that stably express a foreign gene.

A biologically contained influenza A virus that stably expresses a foreign gene can be effectively traced, used to generate a novel multivalent vaccine and have its replication easily assessed, all while satisfying safety concerns regarding pathogenicity or reversion. This study generated a PB2-knockout (PB2-KO) influenza virus that harboured the GFP reporter gene in the coding region of its PB2 viral RNA (vRNA). Replication of the PB2-KO virus was restricted to a cell line stably expressing the PB2 protein. The GFP gene-encoding PB2 vRNA was stably incorporated into progeny viruses during replication in PB2-expressing cells. The GFP gene was expressed in virus-infected cells with no evidence of recombination between the recombinant PB2 vRNA and the PB2 protein mRNA. Furthermore, other reporter genes and the haemagglutinin and neuraminidase genes of different virus strains were accommodated by the PB2-KO virus. Finally, the PB2-KO virus was used to establish an improved assay to screen neutralizing antibodies against influenza viruses by using reporter gene expression as an indicator of virus infection rather than by observing cytopathic effect. These results indicate that the PB2-KO virus has the potential to be a valuable tool for basic and applied influenza virus research.

Effect of an asparagine-to-serine mutation at position 294 in neuraminidase on the pathogenicity of highly pathogenic H5N1 influenza A virus.

Like the histidine-to-tyrosine substitution at position 274 in neuraminidase (NA H274Y), an asparagine-to-serine mutation at position 294 in this protein (NA N294S) confers oseltamivir resistance to highly pathogenic H5N1 influenza A viruses. However, unlike viruses with the NA H274Y mutation, the properties of viruses possessing NA N294S are not well understood. Here, we assessed the effect of the NA N294S substitution on the replication and pathogenicity of human H5N1 viruses and on the efficacy of the NA inhibitors oseltamivir and zanamivir in mouse and ferret models. Although NA N294S-possessing H5N1 viruses were attenuated in mice and ferrets compared to their oseltamivir-sensitive counterparts, one of the infected ferrets died from systemic infection, demonstrating the potential lethality in ferrets of oseltamivir-resistant H5N1 viruses with the NA N294S substitution. The efficacy of oseltamivir, but not that of zanamivir, against an NA N294S-possessing virus was substantially impaired both in ferrets and in vitro. These results demonstrate the considerable pathogenicity of NA N294S substitution-possessing H5N1 viruses and underscore the importance of monitoring the emergence of the NA N294S mutation in circulating H5N1 viruses.

Characterization of oseltamivir-resistant 2009 H1N1 pandemic influenza A viruses.

Influenza viruses resistant to antiviral drugs emerge frequently. Not surprisingly, the widespread treatment in many countries of patients infected with 2009 pandemic influenza A (H1N1) viruses with the neuraminidase (NA) inhibitors oseltamivir and zanamivir has led to the emergence of pandemic strains resistant to these drugs. Sporadic cases of pandemic influenza have been associated with mutant viruses possessing a histidine-to-tyrosine substitution at position 274 (H274Y) in the NA, a mutation known to be responsible for oseltamivir resistance. Here, we characterized in vitro and in vivo properties of two pairs of oseltaimivir-sensitive and -resistant (possessing the NA H274Y substitution) 2009 H1N1 pandemic viruses isolated in different parts of the world. An in vitro NA inhibition assay confirmed that the NA H274Y substitution confers oseltamivir resistance to 2009 H1N1 pandemic viruses. In mouse lungs, we found no significant difference in replication between oseltamivir-sensitive and -resistant viruses. In the lungs of mice treated with oseltamivir or even zanamivir, 2009 H1N1 pandemic viruses with the NA H274Y substitution replicated efficiently. Pathological analysis revealed that the pathogenicities of the oseltamivir-resistant viruses were comparable to those of their oseltamivir-sensitive counterparts in ferrets. Further, the oseltamivir-resistant viruses transmitted between ferrets as efficiently as their oseltamivir-sensitive counterparts. Collectively, these data indicate that oseltamivir-resistant 2009 H1N1 pandemic viruses with the NA H274Y substitution were comparable to their oseltamivir-sensitive counterparts in their pathogenicity and transmissibility in animal models. Our findings highlight the possibility that NA H274Y-possessing oseltamivir-resistant 2009 H1N1 pandemic viruses could supersede oseltamivir-sensitive viruses, as occurred with seasonal H1N1 viruses.

The HA and NS genes of human H5N1 influenza A virus contribute to high virulence in ferrets.

Highly pathogenic H5N1 influenza A viruses have spread across Asia, Europe, and Africa. More than 500 cases of H5N1 virus infection in humans, with a high lethality rate, have been reported. To understand the molecular basis for the high virulence of H5N1 viruses in mammals, we tested the virulence in ferrets of several H5N1 viruses isolated from humans and found A/Vietnam/UT3062/04 (UT3062) to be the most virulent and A/Vietnam/UT3028/03 (UT3028) to be avirulent in this animal model. We then generated a series of reassortant viruses between the two viruses and assessed their virulence in ferrets. All of the viruses that possessed both the UT3062 hemagglutinin (HA) and nonstructural protein (NS) genes were highly virulent. By contrast, all those possessing the UT3028 HA or NS genes were attenuated in ferrets. These results demonstrate that the HA and NS genes are responsible for the difference in virulence in ferrets between the two viruses. Amino acid differences were identified at position 134 of HA, at positions 200 and 205 of NS1, and at positions 47 and 51 of NS2. We found that the residue at position 134 of HA alters the receptor-binding property of the virus, as measured by viral elution from erythrocytes. Further, both of the residues at positions 200 and 205 of NS1 contributed to enhanced type I interferon (IFN) antagonistic activity. These findings further our understanding of the determinants of pathogenicity of H5N1 viruses in mammals.

In vitro and in vivo characterization of new swine-origin H1N1 influenza viruses.

Influenza A viruses cause recurrent outbreaks at local or global scale with potentially severe consequences for human health and the global economy. Recently, a new strain of influenza A virus was detected that causes disease in and transmits among humans, probably owing to little or no pre-existing immunity to the new strain. On 11 June 2009 the World Health Organization declared that the infections caused by the new strain had reached pandemic proportion. Characterized as an influenza A virus of the H1N1 subtype, the genomic segments of the new strain were most closely related to swine viruses. Most human infections with swine-origin H1N1 influenza viruses (S-OIVs) seem to be mild; however, a substantial number of hospitalized individuals do not have underlying health issues, attesting to the pathogenic potential of S-OIVs. To achieve a better assessment of the risk posed by the new virus, we characterized one of the first US S-OIV isolates, A/California/04/09 (H1N1; hereafter referred to as CA04), as well as several other S-OIV isolates, in vitro and in vivo. In mice and ferrets, CA04 and other S-OIV isolates tested replicate more efficiently than a currently circulating human H1N1 virus. In addition, CA04 replicates efficiently in non-human primates, causes more severe pathological lesions in the lungs of infected mice, ferrets and non-human primates than a currently circulating human H1N1 virus, and transmits among ferrets. In specific-pathogen-free miniature pigs, CA04 replicates without clinical symptoms. The assessment of human sera from different age groups suggests that infection with human H1N1 viruses antigenically closely related to viruses circulating in 1918 confers neutralizing antibody activity to CA04. Finally, we show that CA04 is sensitive to approved and experimental antiviral drugs, suggesting that these compounds could function as a first line of defence against the recently declared S-OIV pandemic.

RuvB-like protein 2 is a suppressor of influenza A virus polymerases.

In pro- and eukaryotic cells, RuvB-like protein 2 (RBL2) resolves Holliday junction recombination intermediates. Here, we identified RBL2 as a suppressor of influenza A virus replication. Human RBL2 appears to interfere with the oligomerization of the viral nucleoprotein, a critical step in the assembly of viral replication complexes.

Mutational analysis of conserved amino acids in the influenza A virus nucleoprotein.

The nucleoprotein (NP), which has multiple functions during the virus life cycle, possesses regions that are highly conserved among influenza A, B, and C viruses. To better understand the roles of highly conserved NP amino acids in viral replication, we conducted a comprehensive mutational analysis. Using reverse genetics, we attempted to generate 74 viruses possessing mutations at conserved amino acids of NP. Of these, 48 mutant viruses were successfully rescued; 26 mutants were not viable, suggesting a critical role of the respective NP amino acids in viral replication. To identify the step(s) in the viral life cycle that is impaired by these NP mutations, we examined viral-genome replication/transcription, NP localization, and incorporation of viral-RNA segments into progeny virions. We identified 15 amino acid substitutions in NP that inhibited viral-genome replication and/or transcription, resulting in significant growth defects of viruses possessing these substitutions. We also found several NP mutations that affected the efficient incorporation of multiple viral-RNA (vRNA) segments into progeny virions even though a single vRNA segment was incorporated efficiently. The respective conserved amino acids in NP may thus be critical for the assembly and/or incorporation of sets of eight vRNA segments.

Mitogen-activated protein kinase-activated kinase RSK2 plays a role in innate immune responses to influenza virus infection.

Viral infections induce signaling pathways in mammalian cells that stimulate innate immune responses and affect cellular processes, such as apoptosis, mitosis, and differentiation. Here, we report that the ribosomal protein S6 kinase alpha 3 (RSK2), which is activated through the "classical" mitogen-activated protein kinase pathway, plays a role in innate immune responses to influenza virus infection. RSK2 functions in the regulation of cell growth and differentiation but was not known to play a role in the cellular antiviral response. We have found that knockdown of RSK2 enhanced viral polymerase activity and growth of influenza viruses. Influenza virus infection stimulates NK-kappaB- and beta interferon-dependent promoters. This stimulation was reduced in RSK2 knockdown cells, suggesting that RSK2 executes its effect through innate immune response pathways. Furthermore, RSK2 knockdown suppressed influenza virus-induced phosphorylation of the double-stranded RNA-activated protein kinase PKR, a known antiviral protein. These findings establish a role for RSK2 in the cellular antiviral response.

Establishment of canine RNA polymerase I-driven reverse genetics for influenza A virus: its application for H5N1 vaccine production.

In the event of a new influenza pandemic, vaccines whose antigenicities match those of circulating strains must be rapidly produced. Here, we established an alternative reverse genetics system for influenza virus using the canine polymerase I (PolI) promoter sequence that works efficiently in the Madin-Darby canine kidney cell line, a cell line approved for human vaccine production. Using this system, we were able to generate H5N1 vaccine seed viruses more efficiently than can be achieved with the current system that uses the human PolI promoter in African green monkey Vero cells, thus improving pandemic vaccine production.

Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima.

The gene encoding carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima (tm0053) was cloned. The recombinant protein (EST53) was overexpressed in Escherichia coli without its NH(2)-terminal hydrophobic region, and with a C-terminal hexahistidine sequence. The enzyme was purified to homogeneity by heat treatment, followed by Ni(2+) affinity chromatography, and then characterized. Among the p-nitrophenyl esters tested, the best substrate was p-nitrophenyl decanoate with K (m) and k (cat) values of 3.1 muM and 10.8 s(-1), respectively, at 60 degrees C and pH 7.5. The addition of O,O'-bis(2-aminoethyl)ethyleneglycol-N,N,N',N'-tetraacetic acid decreased the esterase activity, indicating that EST53 is dependent on the presence of Ca(2+) ion. In addition, its activity was inhibited by the addition of phenylmethylsulfonyl fluoride and diethyl pyrocarbonate, indicating that it contains serine and histidine residues, which play key roles in the catalytic mechanism. EST53 shows a relatively high degree of similarity to Burkholderia lipases that belong to family I.2 of the lipolytic enzymes, whereas the local sequence around the pentapeptide of EST53 is most similar to those of Bacillus lipases belonging to family I.4.