Entero virus 71 (EV71) causes hand, foot, and mouth disease (HFMD) and occasionally leads to severe neurological complications and even death. Scavenger receptor class B member 2 (SCARB2) is a functional receptor for EV71, that mediates viral attachment, internalization, and uncoating. However, the exact binding site of EV71 on SCARB2 is unknown. In this study, we generated a monoclonal antibody (mAb) that binds to human but not mouse SCARB2. It is named JL2, and it can effectively inhibit EV71 infection of target cells. Using a set of chimeras of human and mouse SCARB2, we identified that the region containing residues 77-113 of human SCARB2 contributes significantly to JL2 binding. The structure of the SCARB2-JL2 complex revealed that JL2 binds to the apical region of SCARB2 involving α-helices 2, 5, and 14. Our results provide new insights into the potential binding sites for EV71 on SCARB2 and the molecular mechanism of EV71 entry.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; chemistry ; genetics ; metabolism ; Binding Sites ; Cell Line ; Crystallography, X-Ray ; Enterovirus A, Human ; drug effects ; genetics ; growth & development ; immunology ; Fibroblasts ; drug effects ; virology ; Gene Expression ; HEK293 Cells ; Humans ; Immunoglobulin Fab Fragments ; chemistry ; genetics ; metabolism ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; immunology ; Mice ; Models, Molecular ; Protein Binding ; Protein Conformation, alpha-Helical ; Protein Conformation, beta-Strand ; Protein Interaction Domains and Motifs ; Receptors, Scavenger ; chemistry ; genetics ; immunology ; Receptors, Virus ; chemistry ; genetics ; immunology ; Recombinant Fusion Proteins ; chemistry ; genetics ; immunology ; Sequence Alignment ; Sequence Homology, Amino Acid ; Sf9 Cells ; Spodoptera ; Thermodynamics

Boiled silkworm pupa is a traditional food in Asia, and patients with silkworm pupa food allergy are common in these regions. Still now only one allergen from silkworm, arginine kinase, has been identified. The purpose of this study was to identify novel food allergens in silkworm pupa by analyzing a protein extract after heat treatment. Heat treated extracts were examined by proteomic analysis. A 27-kDa glycoprotein was identified, expressed in Escherichia coli, and purified. IgE reactivity of the recombinant protein was investigated by ELISA. High molecular weight proteins (above 100 kDa) elicited increased IgE binding after heat treatment compared to that before heat treatment. The molecular identities of these proteins, however, could not be determined. IgE reactivity toward a 27-kDa glycoprotein was also increased after heating the protein extract. The recombinant protein was recognized by IgE antibodies from allergic subjects (33.3%). Glycation or aggregation of protein by heating may create new IgE binding epitopes. Heat stable allergens are shown to be important in silkworm allergy. Sensitization to the 27-kDa glycoprotein from silkworm may contribute to elevation of IgE to silkworm.
Adolescent ; Adult ; Allergens/*chemistry/*immunology ; Amino Acid Sequence ; Animals ; Bombyx/*chemistry/genetics/growth & development/*immunology ; Epitopes/immunology ; Female ; Food Hypersensitivity/etiology ; Glycoproteins/*chemistry/genetics/*immunology ; Hot Temperature ; Humans ; Immunoglobulin E/immunology ; Male ; Molecular Sequence Data ; Molecular Weight ; Proteomics ; Pupa/chemistry/immunology ; Recombinant Proteins/biosynthesis/chemistry/immunology ; Sequence Alignment

As part of our ongoing influenza surveillance program in South China, 19 field strains of H9N2 subtype avian influenza viruses (AIVs) were isolated from dead or diseased chicken flocks in Guangdong province, South China, between 2012 and 2013. Hemagglutinin (HA) genes of these strains were sequenced and analyzed and phylogenic analysis showed that 12 of the 19 isolates belonged to the lineage h9.4.2.5, while the other seven belonged to h9.4.2.6. Specifically, we found that all of the viruses isolated in 2013 belonged to lineage h9.4.2.5. The lineage h9.4.2.5 viruses contained a PSRSSRdownward arrowGLF motif at HA cleavage site, while the lineage h9.4.2.6 viruses contained a PARSSRdownward arrowGLF at the same position. Most of the isolates in lineage h9.4.2.5 lost one potential glycosylation site at residues 200-202, and had an additional one at residues 295-297 in HA1. Notably, 19 isolates had an amino acid exchange (Q226L) in the receptor binding site, which indicated that the viruses had potential affinity of binding to human like receptor. The present study shows the importance of continuing surveillance of new H9N2 strains to better prepare for the next epidemic or pandemic outbreak of H9N2 AIV infections in chicken flocks.
Animals ; *Chickens ; China ; Hemagglutinin Glycoproteins, Influenza Virus/chemistry/*genetics/metabolism ; Influenza A Virus, H9N2 Subtype/*genetics/metabolism ; Influenza in Birds/virology ; Phylogeny ; Poultry Diseases/*virology ; Sequence Analysis, RNA/veterinary

SCARB2 (scavenger receptor class B, member 2) is a lysosomal membrane glucoprotein, which is encoded by SCARB2 gene. It takes vital parts in the physiological and pathological processes including the transportation of beta-glucocerebrosidase to the lysosome, infection of EV71 and load-induced cardiac myocyte hypertrophy. This article has reviewed the molecular structure and functions of SCARB2 gene and its protein, as well as their relationship with diseases.
Hand, Foot and Mouth Disease ; genetics ; Humans ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; physiology ; Myoclonic Epilepsies, Progressive ; genetics ; Parkinson Disease ; genetics ; Receptors, Scavenger ; chemistry ; genetics ; physiology

Uch37 is a de-ubiquitinating enzyme that is activated by Rpn13 and involved in the proteasomal degradation of proteins. The full-length Uch37 was shown to exhibit low iso-peptidase activity and is thought to be auto-inhibited. Structural comparisons revealed that within a homo-dimer of Uch37, each of the catalytic domains was blocking the other's ubiquitin (Ub)-binding site. This blockage likely prevented Ub from entering the active site of Uch37 and might form the basis of auto-inhibition. To understand the mode of auto-inhibition clearly and shed light on the activation mechanism of Uch37 by Rpn13, we investigated the Uch37-Rpn13 complex using a combination of mutagenesis, biochemical, NMR, and small-angle X-ray scattering (SAXS) techniques. Our results also proved that Uch37 oligomerized in solution and had very low activity against the fluorogenic substrate ubiquitin-7-amino-4-methylcoumarin (Ub-AMC) of de-ubiquitinating enzymes. Uch37Δ(Hb,Hc,KEKE), a truncation removal of the C-terminal extension region (residues 256-329) converted oligomeric Uch37 into a monomeric form that exhibited iso-peptidase activity comparable to that of a truncation-containing the Uch37 catalytic domain only. We also demonstrated that Rpn13C (Rpn13 residues 270-407) could disrupt the oligomerization of Uch37 by sequestering Uch37 and forming a Uch37-Rpn13 complex. Uch37 was activated in such a complex, exhibiting 12-fold-higher activity than Uch37 alone. Time-resolved SAXS (TR-SAXS) and FRET experiments supported the proposed mode of auto-inhibition and the activation mechanism of Uch37 by Rpn13. Rpn13 activated Uch37 by forming a 1:1 stoichiometric complex in which the active site of Uch37 was accessible to Ub.
Binding Sites ; Catalytic Domain ; Chromatography, Gel ; Crystallography, X-Ray ; Humans ; Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Nuclear Magnetic Resonance, Biomolecular ; Protein Binding ; Protein Conformation ; Protein Multimerization ; Scattering, Small Angle ; Ubiquitin Thiolesterase ; chemistry ; genetics ; metabolism ; Ultracentrifugation

Unlike the well-established picture for the entry of enveloped viruses, the mechanism of cellular entry of non-enveloped eukaryotic viruses remains largely mysterious. Picornaviruses are representative models for such viruses, and initiate this entry process by their functional receptors. Here we present the structural and functional studies of SCARB2, a functional receptor of the important human enterovirus 71 (EV71). SCARB2 is responsible for attachment as well as uncoating of EV71. Differences in the structures of SCARB2 under neutral and acidic conditions reveal that SCARB2 undergoes a pivotal pH-dependent conformational change which opens a lipid-transfer tunnel to mediate the expulsion of a hydrophobic pocket factor from the virion, a pre-requisite for uncoating. We have also identified the key residues essential for attachment to SCARB2, identifying the canyon region of EV71 as mediating the receptor interaction. Together these results provide a clear understanding of cellular attachment and initiation of uncoating for enteroviruses.
Acids ; chemistry ; Amino Acid Sequence ; Animals ; Capsid Proteins ; chemistry ; genetics ; metabolism ; Enterovirus A, Human ; genetics ; metabolism ; physiology ; HEK293 Cells ; Host-Pathogen Interactions ; Humans ; Hydrogen-Ion Concentration ; Lysosome-Associated Membrane Glycoproteins ; chemistry ; genetics ; metabolism ; Molecular Docking Simulation ; Molecular Sequence Data ; Protein Binding ; Protein Conformation ; Protein Interaction Mapping ; Protein Structure, Tertiary ; RNA, Viral ; genetics ; metabolism ; Receptors, Scavenger ; chemistry ; genetics ; metabolism ; Sequence Homology, Amino Acid ; Sf9 Cells ; Static Electricity ; Virion ; genetics ; metabolism ; Virus Attachment

Chronic granulomatous disease (CGD) is a rare genetic disease, which is caused by defects in the NADPH oxidase complex (gp91phox, p22phox, p40phox, p47phox, and p67phox) of phagocytes. This defect results in impaired production of superoxide anions and other reactive oxygen species (ROS), which are necessary for killing bacterial and fungal microorganisms and leads to recurrent, life-threatening bacterial and fungal infections and granulomatous inflammation. The dihydrorhodamine (DHR) flow cytometry assay is a useful diagnostic tool for CGD that can detect absent or reduced NADPH oxidase activity in stimulated phagocytes. We report a patient with X-linked CGD carrying a novel mutation of the CYBB gene whose chimerism status following hematopoietic stem cell transplantation (HSCT) has been rapidly determined using the DHR assay. The level of DHR activity correlates well with short tandem repeat PCR analysis. Considering the advantages of this simple, rapid, and cost-effective procedure, serial measurement of DHR assay would facilitate the rapid determination of a patient's engraftment status, as a supplementary monitoring tool of chimerism status following HSCT.
Base Sequence ; *Chimerism ; DNA Mutational Analysis ; Flow Cytometry ; Granulomatous Disease, Chronic/*diagnosis/*enzymology/genetics/surgery ; *Hematopoietic Stem Cell Transplantation ; Homozygote ; Humans ; Infant, Newborn ; Male ; Membrane Glycoproteins/chemistry/*genetics ; Mutation ; NADPH Oxidase/chemistry/*genetics ; Polymerase Chain Reaction ; Rhodamines/chemistry/metabolism

The distribution of glycosylation sites in HA proteins was various among H5 subtype avian influenza viruses (AIVs), however, the role of glycosylation sites to the virus is still unclear. In this study, avian influenza H5N1 viruses with deletion of the glycosylation sites in HA were constructed and rescued by site direct mutation and reverse genetic method, and their biological characteristics and virulence were determined. The result showed that the mutants were confirmed to be corrected by HA gene sequencing and Western blot analysis. The EID50 and TCID50 tested in SPF chick embryo and MDCK cells of a mutant rSdelta158 with deletion of glycosylation site at position 158 were slight lower than that of wild type rescued virus rS, and the plaque diameter of rSdelta158 was significant smaller than that of rS. The EID50 and TCID50 of mutants rSdelta169 and rSdelta290 with deletion of glycosylation sites at position 169 and 290, respectively, were slight higher than that of wild type rescued virus rS, the plaque diameters of rSdelta169 and rSdelta290 were similar as that of rS, but the plaque numbers of rSdelta169 and rSdelta290 were 10-fold higher than that to rS. On the other hand, the rSdelta158, rSdelta169 and rSdelta290 showed similar growth rate in chicken embryo fibroblast as rS. All viruses remained high pathogenicity to SPF chickens. Therefore, the growth of AIV can be affected by changes of glycosylation sites in HA protein, by which the effect is variable in different cells.
Amino Acid Motifs ; Animals ; Cell Line ; Chick Embryo ; Chickens ; Glycosylation ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; metabolism ; Influenza A Virus, H5N1 Subtype ; chemistry ; genetics ; growth & development ; metabolism ; Influenza in Birds ; virology ; Poultry Diseases ; virology

To study the prevalence and variation of influenza A(H3N2) viruses, the antigenic and genetic characteristics of influenza A(H3N2) viruses circulating in Mainland China during April 2011 to March 2012 were analyzed. The results showed that influenza A(H3N2) viruses increased gradually since 2012 and became the dominant strain since March. The viruses were antigenically closely related to the vaccine strain A/PER/16/09 (87.2%) and the representative virus A/FJ/196/09 (76.0%) in Mainland China. The genetic characteristics analysis results showed that recently isolated viruses belonged to the Vic/208 clade, and most of the low reaction strains also fell into the same clade. Crystal structure analysis of HA protein found that, compared with the vaccine strain A/PER/16/09, the recently isolated viruses had amino acid substitutions in the antigenic site A, B and C areas, in addition to gaining potential glycosylation sites at the amino acid position of 45 of HA and 367 of NA. Although the majority of circulating influenza A (H3N2) viruses in 2011-2012 season in Mainland China were antigeniclly matched by current influenza vaccine strain and the selected representative viruses, low reaction strains have increased since 2012, therefore it is necessary to strengthen the surveillance on the variation of influenza virus and to provide solid information for the vaccine strain selection.
Amino Acid Sequence ; China ; epidemiology ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Humans ; Influenza A Virus, H3N2 Subtype ; classification ; genetics ; isolation & purification ; physiology ; Influenza, Human ; epidemiology ; virology ; Models, Molecular ; Molecular Sequence Data ; Phylogeny

In June 2013, the first human H6N1 influenza virus infection was confirmed in Taiwan. However, the origin and molecular characterization of this virus, A/Taiwan/2/2013 (H6N1), have not been well studied thus far. In the present report, we performed phylogenetic and coalescent analyses of this virus and compared its molecular profile/characteristics with other closely related strains. Molecular characterization of H6N1 revealed that it is a typical avian influenza virus of low pathogenicity, which might not replicate and propagate well in the upper airway in mammals. Phylogenetic analysis revealed that the virus clusters with A/chicken/Taiwan/A2837/2013 (H6N1) in seven genes, except PB1. For the PB1 gene, A/Taiwan/2/2013 was clustered with a different H6N1 lineage from A/chicken/Taiwan/ A2837/2013. Although a previous study demonstrated that the PB2, PA, and M genes of A/Taiwan/2/2013 might be derived from the H5N2 viruses, coalescent analyses revealed that these H5N2 viruses were derived from more recent strains than that of the ancestor of A/Taiwan/2/2013. Therefore, we propose that A/Taiwan/2/2013 is a reassortant from different H6N1 lineages circulating in chickens in Taiwan. Furthermore, compared to avian isolates, a single P186L (H3 numbering) substitution in the hemagglutinin H6 of the human isolate might increase the mammalian receptor binding and, hence, this strain's pathogenicity in humans. Overall, human infection with this virus seems an accidental event and is unlikely to cause an influenza pandemic. However, its co-circulation and potential reassortment with other influenza subtypes are still worthy of attention.
Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Hemagglutinin Glycoproteins, Influenza Virus ; chemistry ; genetics ; Humans ; Influenza A Virus, H5N2 Subtype ; genetics ; physiology ; Influenza A virus ; genetics ; isolation & purification ; physiology ; Influenza, Human ; epidemiology ; virology ; Laboratories ; Models, Molecular ; Molecular Sequence Data ; Phylogeny ; Poultry ; virology ; Protein Conformation ; Taiwan ; epidemiology ; Viral Proteins ; genetics