Interferon-Induced Protein 44 and Interferon-Induced Protein 44-Like Restrict Replication of Respiratory Syncytial Virus.

Cellular intrinsic immunity, mediated by the expression of an array of interferon-stimulated antiviral genes, is a vital part of host defense. We have previously used a bioinformatic screen to identify two interferon-stimulated genes (ISG) with poorly characterized function, interferon-induced protein 44 (IFI44) and interferon-induced protein 44-like (IFI44L), as potentially being important in respiratory syncytial virus (RSV) infection. Using overexpression systems, CRISPR-Cas9-mediated knockout, and a knockout mouse model, we investigated the antiviral capability of these genes in the control of RSV replication. Overexpression of IFI44 or IFI44L was sufficient to restrict RSV infection at an early time postinfection. Knocking out these genes in mammalian airway epithelial cells increased levels of infection. Both genes express antiproliferative factors that have no effect on RSV attachment but reduce RSV replication in a minigenome assay. The loss of Ifi44 was associated with a more severe infection phenotype in a mouse model of infection. These studies demonstrate a function for IFI44 and IFI44L in controlling RSV infection.IMPORTANCE RSV infects all children under 2 years of age, but only a subset of children get severe disease. We hypothesize that susceptibility to severe RSV necessitating hospitalization in children without predefined risk factors is, in part, mediated at the antiviral gene level. However, there is a large array of antiviral genes, particularly in the ISG family, the mechanism of which is poorly understood. Having previously identified IFI44 and IFI44L as possible genes of interest in a bioinformatic screen, we dissected the function of these two genes in the control of RSV. Through a range of overexpression and knockout studies, we show that the genes are antiviral and antiproliferative. This study is important because IFI44 and IFI44L are upregulated after a wide range of viral infections, and IFI44L can serve as a diagnostic biomarker of viral infection.

The role of virus-specific CD8(+) cells in liver damage and viral control during persistent hepatitis B virus infection.

Hepatitis B virus (HBV) is a noncytopathic virus, and the recognition of infected hepatocytes by HBV-specific CD8 cells has been assumed to be the central mechanism causing both liver damage and virus control. To understand the role of cytotoxic T cells in the pathogenesis of HBV infection, we used functional assays that require T cell expansion in vitro and human histocompatibility leukocyte antigen (HLA)-peptide tetramers that allow direct ex vivo quantification of circulating and liver-infiltrating HBV-specific CD8 cells. Two groups of patients with persistent HBV infection were studied: one without liver inflammation and HBV replication, the other with liver inflammation and a high level of HBV replication. Contrary to expectation, a high frequency of intrahepatic HBV-specific CD8 cells was found in the absence of hepatic immunopathology. In contrast, virus-specific T cells were more diluted among liver infiltrates in viremic patients, but their absolute number was similar because of the massive cellular infiltration. Furthermore, inhibition of HBV replication was associated with the presence of a circulating reservoir of CD8(+) cells able to expand after specific virus recognition that was not detectable in highly viremic patients with liver inflammation. These results show that in the presence of an effective HBV-specific CD8 response, inhibition of virus replication can be independent of liver damage. When the HBV-specific CD8 response is unable to control virus replication, it may contribute to liver pathology not only directly but by causing the recruitment of nonvirus-specific T cells.

Paediatric antimicrobial stewardship programmes in the UK's regional children's hospitals.

A survey was conducted in UK regional children's hospitals with paediatric intensive care and paediatric infectious disease (PID) departments to describe the characteristics of paediatric antimicrobial stewardship (PAS) programmes. A structured questionnaire was sent to PAS coordinators. 'Audit and feedback' was implemented in 13 out of 17 centres. Microbiology-led services were more likely to implement antimicrobial restriction (75% vs 33% in PID-led services), to focus on broad-spectrum antibiotics, and to review patients with positive blood cultures. PID-led services were more likely to identify patients from e-prescribing or drug charts and review all antimicrobials. A PAS network has been established.

A critical role for tapasin in the assembly and function of multimeric MHC class I-TAP complexes.

Newly assembled major histocompatibility complex (MHC) class I molecules, together with the endoplasmic reticulum chaperone calreticulin, interact with the transporter associated with antigen processing (TAP) through a molecule called tapasin. The molecular cloning of tapasin revealed it to be a transmembrane glycoprotein encoded by an MHC-linked gene. It is a member of the immunoglobulin superfamily with a probable cytoplasmic endoplasmic reticulum retention signal. Up to four MHC class I-tapasin complexes were found to bind to each TAP molecule. Expression of tapasin in a negative mutant human cell line (220) restored class I-TAP association and normal class I cell surface expression. Tapasin expression also corrected the defective recognition of virus-infected 220 cells by class I-restricted cytotoxic T cells, establishing a critical functional role for tapasin in MHC class I-restricted antigen processing.

Bacteremia in Childhood Life-Threatening Infections in Urban Gambia: EUCLIDS in West Africa.

BACKGROUND: The limited availability of microbiology services in sub-Saharan Africa impedes accurate diagnosis of bacterial pathogens and understanding of trends in prevalence and antibiotic sensitivities. We aimed to characterize bacteremia among hospitalized children in The Gambia and to identify factors associated with bacteremia and mortality. METHODS: We prospectively studied children presenting with suspected severe infection to 2 urban hospitals in The Gambia, between January 2013 and September 2015. Demographic and anthropometric data, clinical features, management, and blood culture results were documented. Urine screens for antibiotic activity were performed in a subset of participants. RESULTS: Of 411 children enrolled (median age, 29 months; interquartile range, 11-82), 79.5% (325 of 409) reported prehospital antibiotic use. Antimicrobial activity by urinary screen for antibiotic activity was detected in 70.8% (n = 80 of 113). Sixty-six bacterial pathogens were identified in 65 (15.8%) participants and Staphylococcus aureus predominated. Gram-positive organisms were more commonly identified than Gram-negative (P < .01). Antibiotic resistance against first-line antimicrobials (ampicillin and gentamicin) was common among Gram-negative bacteria (39%; range, 25%-100%). Factors significantly associated with bacteremia included the following: gender, hydration status, musculoskeletal examination findings, admission to the Medical Research Council The Gambia at London School of Hygiene & Tropical Medicine hospital, and meeting sepsis criteria. Those associated with increased mortality were presence of a comorbidity, clinical pallor, tachypnea, and altered consciousness. Tachycardia was associated with reduced mortality. CONCLUSIONS: The bacteremia rate in children with suspected childhood life-threatening infectious diseases in The Gambia is high. The pattern of pathogen prevalence and antimicrobial resistance has changed over time compared with previous studies illustrating the importance of robust bacterial surveillance programs in resource-limited settings.

TAPASIN, DAXX, RGL2, HKE2 and four new genes (BING 1, 3 to 5) form a dense cluster at the centromeric end of the MHC.

TAPASIN, a gene recently shown to be required for antigen presentation through MHC class I molecules, is located 180 kbp centromeric of HLA-DP in a region linked to several diseases, and associated with altered developmental phenotypes in the mouse. We present the genomic analysis of a 70 kbp gene-dense segment flanking the TAPASIN locus, including sequence, structure and preliminary characterisation of seven additional genes. BING1 is a Zn finger gene containing a POZ motif. BING3 is similar to myosin regulatory light chain. BING4 shows homologies only to hypothetical yeast and Caenorhabditis elegans proteins. BING5 is found within an intron of BING4 on the complementary strand, and encodes a molecule with no homologies to database proteins. Another three genes were identified whose full sequence was not previously known; namely, RGL2, DAXX (BING2) and HKE2. RGL2 encodes an effector of Ras, homologous to the mouse RalGDS protein, Rlf. DAXX encodes an effector of Fas that stimulates apoptosis through the Jun kinase (JNK) pathway. The location of DAXX is of interest given the linkage of autoimmune disease to the MHC and to apoptosis.

The hepatic glucagon receptor: a comparative study of the regulatory and structural properties.

Guanine nucleotide and Mg2+ ion regulation of [125I-Tyr10]monoiodoglucagon ([125I]MIG) binding to liver plasma membranes from chicken, rat, and rabbit was studied. It was found that [125I]MIG binding to chicken liver membranes was increased by the addition of Mg2+ ion, while binding to rat and rabbit liver membranes was unaffected. In the chicken liver membranes, the Mg2+ ion induced high affinity binding which was sensitive to guanine nucleotides, while the low affinity binding in the absence of Mg2+ ion was not. Maximal effects of Mg2+ ion were observed at 1 mM. Glucagon binding to rat liver membrane receptors was GTP sensitive regardless of whether Mg2+ ion was added. Glucagon binding to rabbit liver membranes was insensitive to both Mg2+ ions and GTP. This lack of GTP effect was not due to degradation of GTP; no effect of the nonhydrolyzable analog guanyl-5'-yl-imidodiphosphate was observable. Glucagon stimulation of rabbit liver adenylyl cyclase, however, was dependent on GTP, as was the case with all of the other liver adenylyl cyclases studied here. The Kact of GTP for the rabbit liver system was very similar to that for rat liver membranes. The glucagon receptor was covalently labeled with [125I]MIG using p-hydroxysuccinimidyl azidobenzoate and analyzed by sodium dodecyl sulfate-gel electrophoresis. In all cases, a major labeled band at 63,000 daltons was observed. The levels of glucagon receptor and stimulatory (Ns) and inhibitory (Ni) regulatory proteins of adenylyl cyclase were measured. The highest levels of glucagon receptor were measured in rat liver membranes, while the levels in chicken and rabbit membranes were 30-40% lower. Rabbit liver membrane had the highest levels of Ns, while rat liver membranes had 2-fold lower and chick liver membrane 4-fold lower levels than rabbit liver membranes. The levels of Ni was similar in the three systems. Thus, the ratio of Ns to glucagon receptor was highest in the rabbit. In the rat, this ratio was 3-fold lower than that in the rabbit. In the chicken membranes, the ratio was about 60% of that in the rat. These data suggest that the observed differences in effects of GTP on hormone binding can be explained by alterations in the ratio of the receptor and Ns proteins among the various species.

Genomic structure and domain organisation of the human Bak gene.

The Bcl-2 homologue, Bak, is a potent inducer of apoptosis. FISH data presented here located the gene to 6p21.3. Mapping was consistent with its location centromeric of the HSET locus and approximately 400kb from the MHC. The construction of a contig of genomic clones across the locus facilitated the sequencing of a PAC containing the gene. Comparison of the gene structure to functional and physical domains revealed a good agreement between the physical structure and the intron-exon organisation. The position of a single intron was conserved in comparison to other members of the Bcl-2 family, namely Bax, CED-9, Bcl-X and Bcl-2, but all other introns were displaced, consistent with a divergent phylogeny.

Structural analysis of the hepatic glucagon receptor. Identification of a guanine nucleotide-sensitive hormone-binding region.

[125I-Tyr10]Monoiodoglucagon [( 125I]MIG) was cross-linked to liver membrane glucagon receptors with hydroxysuccinimidyl-p-azidobenzoate, and the products were analyzed by sodium dodecyl sulfate-gel electrophoresis. Autoradiograms of the gel obtained after a 24-h exposure showed one major band at Mr = 63,000 that was sensitive to GTP and excess unlabeled glucagon. Exposure for 7 days showed labeling of an additional Mr = 33,000 species that was also sensitive to excess unlabeled glucagon. The Mr = 33,000 peptide can be obtained by subtilisin, trypsin, elastase, or Staphylococcus aureus V8 protease treatment of the [125I]MIG-occupied receptor in the membrane or in Lubrol-PX solution. In contrast, limited proteolysis of membranes containing vacant receptors results in labeling of a Mr = 24,000 peptide. The Mr = 24,000 peptide specifically binds [125I]MIG in a GTP-sensitive manner. The Mr = 33,000 peptide also retains GTP sensitivity since it releases bound [125I]MIG upon addition of GTP. Elastase treatment of the electroeluted Mr = 33,000 peptide yields the Mr = 24,000 and 15,000 fragments. The Mr = 15,000 peptide is the smallest fragment of the receptor as yet identified. Treatment of the Mr = 63,000 receptor with [125I]MIG cross-linked to it with endo-beta-N-acetylglucosaminidase F results in four distinct fragments with Mr values of 61,000, 56,000, 51,000, and 45,000; prolonged treatment resulted in the accumulation of the last two. Neither the Mr = 33,000 nor the Mr = 24,000 fragment appeared to be substrates for endo-beta-N-acetylglucosaminidase F. These data indicate that glucagon receptor is a glycoprotein of approximately 60,000 daltons which contains at least four N-linked glycans accounting for 18,000 daltons of its mass. Both its glucagon binding function and its capacity to interact with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans. Hormone occupancy of the receptor results in a conformational change so as to expose a region that is susceptible to proteolysis by proteases of varying specificities to yield a peptide of approximately 30,000 daltons that also does not contain N-linked glycans.

Characterization of the hepatic glucagon receptor.

The hepatic glucagon receptor was covalently labeled with [125I-Tyr10]-monoiodoglucagon by use of the heterobifunctional crosslinker hydroxysuccinimidyl-p-azidobenzoate and analyzed by SDS-gel electrophoresis. The autoradiogram of the gel showed one band at Mr = 63,000 that was sensitive to excess unlabeled glucagon and GTP. The labeled receptor was solubilized with Lubrol-PX and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f degrees = 1.8 and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees for 15 min prior to the addition of [125I-Tyr10] permitted us to identify the high molecular weight form (Mr approximately equal to 113,000) by direct SDS-gel electrophoretic analysis. Limited elastase treatment of the hormone-occupied receptor results in the appearance of a Mr = 33,000 fragment, that retains guanine nucleotide sensitivity. Elastase treatment of vacant receptors results in a Mr = 24,000 fragment that binds hormone in a GTP-sensitive manner. The Mr = 24,000 fragment is contained within the Mr = 33,000 fragment. The Mr = 63,000 receptor upon treatment with endo-beta-N-acetylglucosamine F for 4 h yields four fragments of apparent Mr = 61,000, 56,000, 51,000, and 45,000; 24 h treatment results in the accumulation of the last two fragments. Neither Mr = 33,000 and 24,000 fragment appear to be substrates for endo-beta-N-acetylglucosaminidase F. These data allow us to conclude that the hepatic glucagon receptor in the membrane is a dimer of approximately 60,000 dalton hormone binding subunit which is a glycoprotein containing at least four N-linked glycans accounting for 18,000 daltons of its mass. Both the hormone binding function and the capacity for the interaction with the stimulatory regulator of adenylyl cyclase are contained within a fragment of only approximately 21,000 daltons that does not contain any N-linked glycans.

Glucagon receptor-mediated activation of Gs is accompanied by subunit dissociation.

The effect of the glucagon receptor on the activation of the stimulatory GTP-binding protein of adenylyl cyclase (Gs) in the native rat liver membrane environment was studied. The activated state of Gs was assessed by its ability to reconstitute the cyc- S49 cell membrane adenylyl cyclase. The Gs protein was activated by saturating concentrations of guanosine 5'-thiotriphosphate (GTP gamma S) or guanyl-5'-yl imidodiphosphate in a hormone-dependent manner at 0.4 mM Mg2+ in native membranes or in membranes that had been treated with 1 mM N-ethylmaleimide to eliminate the catalytic activity of adenylyl cyclase. At 50 mM Mg2+, Gs was fully activated by GTP gamma S in the absence of hormone. The unactivated Gs protein migrates around 4 S, whereas activated Gs migrates around 2 S on sucrose density gradients. When pure Gs is analyzed on sucrose density gradients, it is found that the unactivated protein migrates at 4.1 S. Gs was activated by saturating concentrations of GTP gamma S and Mg2+, and the alpha subunit of Gs was chromatographically purified. The resolved alpha subunit of Gs that is capable of stimulating the cyc- adenylyl cyclase migrates at 2.1 S. From these data, we conclude that activation of Gs results in the dissociation of this protein in the membrane environment and that the hormone-occupied receptor promotes this dissociation process under conditions where Mg2+ ions are limiting.

Identification of a new GTP-binding protein. A Mr = 43,000 substrate for pertussis toxin.

In purified preparations of human erythrocyte GTP-binding proteins, we have identified a new substrate for pertussis toxin, which has an apparent molecular mass of 43 kDa by silver and Coomassie Blue staining. Pertussis toxin-catalyzed ADP-ribosylation of the 43-kDa protein is inhibited by Mg2+ ion and this inhibition is relieved by the co-addition of micromolar amounts of guanine nucleotides. GTP affects the ADP-ribosylation with a K value of 0.8 microM. Addition of a 10-fold molar excess of purified beta gamma subunits (Mr = 35,000 beta; and Mr = 7,000 gamma) of other GTP-binding proteins results in a significant decrease in the pertussis toxin-mediated ADP-ribosylation of the 43-kDa protein. Treatment of the GTP-binding proteins with guanosine 5'-O-(thiotriphosphate) and 50 mM MgCl2 resulted in shifting of the 43-kDa protein from 4 S to 2 S on sucrose density gradients. Immunoblotting analysis of the 43-kDa protein with the antiserum A-569, raised against a peptide whose sequence is found in the alpha subunits of all of the known GTP-binding, signal-transducing proteins (Mumby, S. M., Kahn, R. A., Manning, D. R., and Gilman, A. G. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 265-259) showed that the 43-kDa protein is specifically recognized by the common peptide antiserum. A pertussis toxin substrate of similar molecular weight was observed in human erythrocyte membranes, bovine brain membranes, membranes made from the pituitary cell line GH4C1, in partially purified GTP-binding protein preparations of rat liver, and in human neutrophil membranes. Treatment of neutrophils with pertussis toxin prior to preparation of the membranes resulted in abolishment of the radiolabeling of this protein. From these data, we conclude that we have found a new pertussis toxin substrate that is a likely GTP-binding protein.

The hepatic glucagon receptor. Solubilization, characterization, and development of an affinity adsorption assay for the soluble receptor.

The hepatic glucagon receptor was covalently labeled with [125I-Try10]monoiodoglucagon [( 125I]MIG) by use of the heterobifunctional cross-linker hydroxysuccinimidyl p-azidobenzoate. Labeling of the Mr = 63,000 peptide was sensitive to glucagon and GTP at concentrations at which they affect [125I]MIG binding to the receptor. The labeled receptor was solubilized with Lubrol-PX, and the hydrodynamic characteristics of the receptor were determined. The molecular parameters of the solubilized receptor are: S20,w = 4.3 +/- 0.1, Stokes radius = 6.3 +/- 0.1 nm, frictional coefficient f/f0 = 1.8, and a calculated Mr = 119,000. Incubation of liver membranes at 32 degrees C for 15 min prior to the addition of [125I]MIG permitted us to identify the high molecular weight form (Mr = approximately 113,000) of the receptor by direct sodium dodecyl sulfate-gel electrophoretic analysis. The Mr = 63,000 peptide can be adsorbed to wheat germ lectin-Sepharose. The glycoprotein nature of the receptor has been utilized to develop an assay for the detergent-solubilized receptor that uses wheat germ lectin-Sepharose as a solid matrix to adsorb the [125I] MIG-receptor complex. The free hormone remains in the liquid phase and is removed in the supernatant after low speed centrifugation. 3-[(3-Cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS) solubilizes receptors with retention of [125I]MIG binding activity. [125I]MIG binding to the CHAPS-solubilized receptor is specifically affected by unlabeled glucagon. Interaction of [125I]MIG with the soluble receptor is insensitive to the presence of GTP. IC50 for glucagon using the soluble receptor was 33-70 nM, irrespective of the presence or absence of GTP, while when the membrane-bound receptor was used, the IC50 in the absence of GTP was 2-4 nM and in the presence of GTP was 35-80 nM. These data allow us to conclude that the hepatic glucagon receptor in the membrane and in the nondenaturing detergent solution is a dimer of the Mr = 63,000 hormone-binding subunit and a glycoprotein. The soluble receptor does not display any functional interaction with the stimulatory regulator.

The ligand affinity of proteins measured by isothermal denaturation kinetics.

An isothermal denaturation kinetic method was developed for identifying potential ligands of proteins and measuring their affinity. The method is suitable for finding ligands specific toward proteins of unknown function and for large-scale drug screening. It consists of analyzing the kinetics of isothermal denaturation of the protein-with and without the presence of potential specific ligands-as measured by long-wavelength fluorescent dyes whose quantum yield increases when bound to hydrophobic regions exposed upon unfolding of the proteins. The experimental procedure was developed using thymidylate kinase and stromelysin as target proteins. The kinetics of thermal unfolding of both of these enzymes were consistent with a pathway of two consecutive first-order rate-limiting steps. Reflecting the stabilizing effect of protein/ligand complexes, the presence of specific ligands decreased the value of the rate constants of both steps in a dose-dependent manner. The dependence of the rate constants on ligand concentration obeyed a simple binding isotherm, the analysis of which yielded an accurate equilibrium constant for ligand binding. The method was validated by comparing its results with those obtained under the same conditions by steady-state fluorescence spectroscopy, circular dichroism, and uv spectrophotometry: The corresponding rate constants were comparable for each of the analytical detection methods.

Rat tapasin: cDNA cloning and identification as a component of the class I MHC assembly complex.

During the assembly of major histocompatibility complex (MHC) class I molecules transient associations are formed with the endoplasmic reticulum resident chaperones calnexin and calreticulin, ERp57 oxidoreductase, and also with tapasin, the latter mediating binding of the class I molecules to the transporter associated with antigen processing (TAP). We report here the isolation of a cDNA encoding rat tapasin from a DA (RT1av1) library. The cDNA encodes a proline-rich (11.3%) polypeptide of 464 residues with a potential ER-retention KK motif at its COOH-terminus, and a predicted molecular mass of 48 kDa. Matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry of peptides derived from in-gel tryptic digestion of a TAP-associated protein match regions of the predicted translation product. A species of the correct molecular mass and predicted pl was also identified in association with radiolabelled immunoprecipitates of the rat TAP complex analysed by two-dimensional gel electrophoresis. This confirms rat tapasin as a component of the rat MHC class I assembly complex.

A structural variation for MurB: X-ray crystal structure of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB).

The X-ray crystal structure of the substrate free form of Staphylococcus aureus UDP-N-acetylenolpyruvylglucosamine reductase (MurB) has been solved to 2.3 A resolution with an R-factor of 20.3% and a free R-factor of 22.3%. While the overall fold of the S. aureus enzyme is similar to that of the homologous Escherichia coli MurB X-ray crystal structure, notable distinctions between the S. aureus and E. coli MurB protein structures occur in residues involved in substrate binding. Analysis of available MurB sequences from other bacteria suggest that the S. aureus MurB structure is representative of a distinct structural class of UDP-N-acetylenolpyruvylglucosamine reductases including Bacillus subtilis and Helicobacter pylori that are characterized by a modified mechanism for substrate binding.

Crystallization and preliminary X-ray analysis of UDP-N-acetylenolpyruvylglucosamine reductase (MurB) from Staphylococcus aureus.

UDP-N-acetylenolpyruvylglucosamine reductase (MurB) is an essential enzyme in the bacterial cell-wall biosynthetic pathway, making it a potential therapeutic target for novel antibiotics. Diffraction-quality crystals of both the native and Se-methionine-expressed MurB from Staphylococcus aureus have been prepared by sitting-drop vapour diffusion from solutions containing polyethylene glycol (PEG) 8000, ammonium sulfate, sodium cacodylate pH 6.5 and dimethyl sulfoxide (DMSO). Crystals belong to the cubic space group I2(1)3, with unit-cell parameters a = b = c = 178.99 A. X-ray data from these crystals were collected at the Advanced Photon Source 17-ID beamline and were used to solve the MurB structure to 2.3 A resolution.