Glycovirology: Methods and Protocols

Preface
Contents
Contributors
Chapter 1: Comprehensive Cellular Glycan Profiling of Glycoproteins and Glycosphingolipids by Glycoblotting and BEP Methods
1 Introduction
2 Materials
2.1 Cultured Cell Disruption
2.2 Ethanol Precipitation
2.3 N-linked Glycan Preparation
2.4 GSL-Glycan and fOS Preparation
2.5 GAG Preparation
2.6 Glycoblotting for HPLC
2.7 2-AB-Labeled GAG Purification
2.8 Glycoblotting for MALDI-TOF MS
2.9 O-Linked Glycan Preparation, β-Elimination, and Labeling with Pyrazolone
2.10 PMP-Labeled O-Linked Glycan Purification
2.11 MALDI-TOF MS Analysis
2.12 HPLC Analysis
3 Methods
3.1 Cell Disruption
3.2 Ethanol Precipitation
3.3 N-Linked Glycan Preparation
3.4 GSL-Glycan Preparation
3.5 fOS Preparation
3.6 GAG Preparation
3.7 Glycoblotting for HPLC
3.8 2-AB-Labeled GAG Purification
3.9 Glycoblotting for MALDI-TOF MS
3.10 aoWR-Labeled Glycan Purification
3.11 O-Linked Glycan Preparation and β-Elimination Reaction
3.12 PMP-Labeled O-Linked Glycan Purification
3.13 MALDI-TOF MS Analysis
3.14 HPLC Analysis
4 Notes
References
Chapter 2: High-Performance Liquid Chromatography Mapping Method for the Structural Analysis of N-Glycans at the Molecular, Ce...
1 Introduction
2 Materials
2.1 Oligosaccharide Samples Are Prepared From Glycoproteins
2.2 Fluorescent Labeling with 2-Aminopyridine
2.3 Glycan Separation by a DEAE Column
2.4 Glycan Separation by an ODS Column
2.5 Glycan Separation by an Amide Column
3 Methods
3.1 Oligosaccharide Samples Are Prepared From Glycoproteins
3.2 Fluorescent Labeling with 2-Aminopyridine
3.3 Purification of 2-PA-Labeled Oligosaccharide
3.4 Glycan Separation by a DEAE Column with Anion-Exchange Chromatography
3.5 Glycan Separation by an ODS Column with Reverse-Phase Chromatography
3.6 Glycan Separation by an Amide Column with Normal-Phase Chromatography
3.7 Identification of the Glycan Structure
4 Notes
References
Chapter 3: Detection of Sialic Acids on the Cell Surface Using Flow Cytometry
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Cell Staining and Analysis
3 Method
3.1 Cell Preparation
3.2 Cell Staining and Analysis
4 Notes
References
Chapter 4: Lectin Staining to Detect Human and Avian Influenza Virus Receptors in the Airway of Nonhuman Primates
1 Introduction
2 Materials
2.1 Critical Equipment
2.2 Reagents
2.3 Other Materials
3 Methods
3.1 Preparation of the Negative Control
3.2 Preparation of Blocking Buffer
3.3 Preparation of Tissue Blocks
3.4 Preparation of Tissue Slides
3.5 Lectin Staining
4 Notes
References
Chapter 5: Preparation of Glycan Arrays Using Glycopeptides Derived From Biomaterials
1 Introduction
2 Materials
2.1 Preparation of Glycopeptides From Biomaterials
2.2 Preparation of Glycan Array
2.3 Detection of Influenza Virus Using Glycan Array
3 Methods
3.1 Preparation of Glycopeptides From Biomaterials
3.2 Preparation of Glycan Array for Influenza Virus Detection
3.3 Binding Assay Using the Neoglycoprotein
4 Notes
References
Chapter 6: Glycan Profiling of Viral Glycoproteins with the Lectin Microarray
1 Introduction
2 Materials
3 Methods
3.1 Preparation of Antibodies
3.2 Procedure for Antibody-Overlay Method
3.2.1 Immunoprecipitation
3.2.2 LMA
3.3 Procedure for Direct-Labeling Method
3.3.1 Immunoprecipitation
3.3.2 LMA
3.4 Data Processing and Interpretation of the Results
3.5 Compatibility of Antibodies with LMA
3.6 Application of LMA to Immunologically ``Recessive´´ Glycoproteins in Viruses
4 Notes
References
Chapter 7: Preparation of Recombinant PA Endonuclease Domain Protein of Influenza A Virus and Its Application for Glycobiology...
1 Introduction
2 Materials
2.1 LB Medium
2.2 TBG M9 Medium
2.3 Buffer A - 500 mM NaCl
2.4 Buffer 1 for Washing the Resin
2.5 Buffer 2 for Washing the Resin
2.6 Buffer 3 (50 ml) for Washing the Resin
2.7 Buffer 4 (40 ml) for Protein Elution
2.8 50% Glycerol
2.9 Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.9.1 Separating Gel, 20 ml (for 12% Gel)
2.9.2 Stacking Gel, 5 ml
3 Methods
3.1 Preparation of E. coli Competent Cells
3.2 Construction of Expression Vector
3.3 Transformation of E. coli with Plasmid DNA
3.4 Preparation of E. coli Glycerol Stock for Protein Expression
3.5 Expression of Recombinant 6x-Histidine-Tagged PA Endonuclease Protein in E. coli
3.6 Purification of 6x-Histidine-Tagged Influenza A Virus PA Endonuclease Domain Protein
3.6.1 Washing and Equilibration of the Ni2+-Agarose Resin with Buffer A
3.6.2 Purification of 6x-His-Tagged Influenza A Virus PA Endonuclease Domain Protein with Ni2+-Agarose Using a Stepwise Tube M...
3.6.3 Further Purification of Influenza A Virus PA Endonuclease Domain Protein Using Anion-Exchange Column Chromatography
3.7 Endonuclease Activity Assay of the N-Terminal Domain Protein of PA Subunit of Influenza A RNA-Dependent RNA Polymerase for...
4 Notes
References
Chapter 8: Receptor-Binding Specificity of Influenza Viruses
1 Introduction
2 Materials
2.1 Preparation of Plasmids Containing a cDNA Encoding the HA or NA Genes of Influenza Viruses
2.2 Generation of PB2-KO Viruses
2.3 Virus Purification and Hemagglutination Assay
2.4 Biolayer Interferometry
3 Methods
3.1 Preparation of Plasmids Containing a cDNA Encoding the HA or NA Genes of Influenza Viruses
3.2 Generation of PB2-KO Viruses
3.3 Virus Purification and Hemagglutination Assay
3.4 Biolayer Interferometry
4 Notes
References
Chapter 9: Functional Analysis of Sulfatide in Influenza A Virus Infection and Replication
1 Introduction
2 Materials
2.1 Chemicals and Buffers
2.2 Cells
2.3 Media
2.4 Proteins and Viruses
2.5 Instruments
3 Methods
3.1 TLC Virus-Binding Assay to Glycolipids
3.2 Solid-Phase Virus-Binding Assay to Glycolipids
3.3 Binding Kinetics of Sulfatide with HA by a QCM
3.4 Comparison of Virus Replication Between Sulfatide-Enriched and Parent Cells
3.5 Suppression of Nuclear Export of vRNP Complexes in Sulfatide Knockdown Cells
3.6 A Relationship Between Nuclear Export of vRNP Complexes and AIF-Mediated Apoptosis in Sulfatide-Enriched Cells Infected wi...
3.7 Requirement of PB1-F2 on Progeny Virus Production in Sulfatide-Enriched Cells Infected with Influenza A Virus
3.8 Differentiation and Sulfatide Staining of Primary Cultured Human Bronchial Epithelial Cells
4 Notes
References
Chapter 10: Quantification of Receptor Association, Dissociation, and NA-Dependent Motility of Influenza A Particles by Biolay...
1 Introduction
2 Materials
2.1 Glycan Receptors
2.2 Biolayer Interferometry Materials
2.2.1 Octet Equipment
2.2.2 General Materials
3 Methods
3.1 Expression and Purification of Biotinylated Glycoprotein Receptors
3.2 Virus Preparation
3.2.1 Virus Production
3.2.2 Captocore Core Virus Purification (for Details, Also See GE Application Note 29-0003-34 AA)
3.2.3 NanoSight NS300 Virus Particle Counting
3.3 Quantification of Virus Association Rate (Fig. 1)
3.3.1 Maximum Association Rate (See Fig. 1a, b)
3.3.2 Receptor Density-Dependent Association Rate (See Fig. 1c, d)
3.4 Virus Dissociation (Quantifying the HA/NA Balance and Virus Motility) (Fig. 2)
3.4.1 Virus Dissociation Rate After Different Levels of Association (Fig. 2a-c)
3.4.2 Dissociation Rate Comparison of Different Viruses (Fig. 2d, e)
3.4.3 Virus Association in Presence of NA Activity (Fig. 2f) (See Note 17)
3.5 Inhibitor Assays of Virus Association and Dissociation (Fig. 3)
3.5.1 Virus Dissociation Inhibitors (Fig. 3a-c) (See Note 19)
3.5.2 Virus Binding Inhibitors (See Note 20)
4 Notes
References
Chapter 11: Receptor-Binding Assay for Avian Influenza Viruses
1 Introduction
2 Materials
2.1 Preparation of the Viruses
2.2 Reverse Genetics
2.3 Glycan-Binding Assay
3 Methods
3.1 Preparation of the Viruses
3.2 Reverse Genetics of Avian Influenza Viruses
3.2.1 Cloning of Viral Genes into the pHW2000 Vector
3.2.2 Rescue and Propagation of Viruses
3.3 Glycan-Binding Assay of Avian Influenza Viruses
3.3.1 Selection of Sialylglycoconjugates
3.3.2 Glycan-Binding Assay
3.4 Data Processing and Interpretation of the Results
3.5 Point to Note for the Glycan-Binding Assay of Avian Influenza Viruses
4 Notes
References
Chapter 12: Characterization of Influenza Virus Binding to Receptors on Isolated Cell Membranes
1 Introduction
2 Materials
2.1 Preparation of Viruses
2.2 Preparation of Epithelial Cell Membranes
2.3 Preparation of Peroxidase-Labeled Fetuin (Fet-HRP)
2.4 Preparation of Desialylated BSA (See Note 4)
2.5 Binding of Influenza Viruses to Cell Membranes
2.5.1 Stock Solutions
2.5.2 Working Solutions Freshly Prepared from Stock Solutions
2.5.3 Materials and Equipment
3 Methods
3.1 Preparation of Viruses
3.1.1 Inactivation (See Note 7)
3.1.2 Concentration
3.2 Preparation of Epithelial Cell Membranes
3.3 Preparation of Peroxidase-Labeled Fetuin (Fet-HRP) (See Note 12)
3.4 Preparation of Desialylated BSA (BSA-NA) (See Note 4)
3.5 Determination of Working Dilution of Fet-HRP
3.6 Binding of IV to Cell Membranes Using Dilution Series and Scatchard Plots
3.6.1 Binding Steps
3.6.2 Data Analysis
3.7 Binding of IV to Cell Membranes Using Standard Virus Concentration
4 Notes
References
Chapter 13: Characterization of Human Parainfluenza Virus Receptor Using Terminal Sialic Acid Linkage-Modified Cells
1 Introduction
2 Materials
2.1 Chemicals and Buffers
2.2 Enzymes and Plasmids
2.3 Cells
2.4 Media
2.5 Viruses and Antibodies
2.6 Instruments
3 Methods
3.1 Preparation of Sialic Acid Linkage-Modified hRBC
3.1.1 Preparation of Normal hRBC
3.1.2 Preparation of AUSD-Treated hRBC
3.1.3 Preparation of 23SD-Treated hRBC
3.1.4 Preparation of ST3-Resialylated hRBC
3.1.5 Preparation of ST6-Resialylated hRBC
3.2 Hemagglutination Assay
3.3 Hemadsorption Assay
3.4 Virus Infectivity Assay for Sialidase-Treated or Sialyltransferase-Treated Cells
3.4.1 Sialidase Treatment of LLC-MK2 Cells
3.4.2 Resialylation of Lec2 Cells
3.4.3 Resialylation of A549 Cells
3.4.4 Virus Infection and Staining of Infected Cells
4 Notes
References
Chapter 14: Solid-Phase Binding Assay for Ganglioside Binding of Human Respiroviruses
1 Introduction
2 Materials
2.1 Chemicals and Buffers
2.2 Equipment
2.3 Cells
2.4 Media
2.5 Proteins and Viruses
2.6 Gangliosides
2.7 Instruments
3 Methods
3.1 Preparation of Ganglioside-Coated Microtiter Plates
3.2 Solid-Phase Binding Assay for Respiroviruses
4 Notes
References
Chapter 15: Evaluation of the Glycan-Binding and Esterase Activities of Hemagglutinin-Esterase-Fusion Glycoprotein from Influe...
1 Introduction
2 Materials
2.1 Protein Expression and Purification
2.2 Glycan Microarray
2.3 MDCK Cell-Binding Assay
2.4 Hemagglutination Assay
2.5 Solid-Phase Lectin Binding Assay
2.6 Immunofluorescence of Tissue Sections
2.7 Esterase Enzymatic Activity Assay
3 Methods
3.1 HEF Protein Expression and Purification
3.1.1 Generation of Recombinant Bacmid
Transforming DH10Bac E. Coli
Analyzing Recombinant Bacmid DNA
3.1.2 Amplification of Recombinant Baculovirus
3.1.3 Production and Purification of HEF Protein
3.2 Glycan Microarray
3.3 MDCK Cell-Binding Assay
3.4 Hemagglutination Assay
3.5 Solid-Phase Lectin Binding Assay
3.6 Immunofluorescence of Tissue Sections
3.7 Esterase Enzymatic Activity Assay
4 Notes
References
Chapter 16: Roles of Glycans and Non-glycans on the Epithelium and in the Immune System in H1-H18 Influenza A Virus Infections
1 Introduction
2 Receptors on the Epithelium May Play Roles as Decoys, Coreceptors, or Actual Receptors for H1-H16 Viruses Depending on Their...
2.1 Sialyl Glycans Determine Host and Tissue Tropism of H1-H16 IAVs
2.2 Other Negatively Charged Glycans May Act as Decoys or Coreceptors for IAV
2.3 Annexins A5 and A1 May Be Additional Receptors Enhancing IAV Entry
3 Receptors in the Immune System May Act as Decoys, Coreceptors, or Potential Receptors for H1-H16 IAV Entry into a Cell
3.1 Acidic Surfactant Phospholipids, Sialyl Glycans on Surfactant Proteins, and the Carbohydrate Recognition Domain (CRD) of S...
3.2 Sialyl Glycans on Mucins Play a Critical Role as Decoys to Trap IAVs
3.3 Sialyl Glycans and C-Type Lectins on Macrophages and Dendritic Cells May Be Used as Receptors for IAV Infection
3.4 Sialyl Glycans on Natural Killer Cells May Be Used as Receptors for IAV Infection
4 MHC Class II HLA-DR May Be a Receptor and/or an Internalization Mediator of H17-H18 Viruses
5 Concluding Remarks
References
Chapter 17: Roles of Sialyl Glycans in HCoV-OC43, HCoV-HKU1, MERS-CoV and SARS-CoV-2 Infections
1 Introduction
2 Roles of Sialyl Glycans in HCoV-OC43 and HCoV-HKU1 Infections
3 Roles of Sialyl Glycans in MERS-CoV Infection
4 Roles of Sialyl Glycans in SARS-CoV-2 Infection
4.1 Do Sialic Acids Play a Role in Restriction of SARS-CoV-2 Infection?
4.2 Do Sialic Acids Play a Role in Enhancement of SARS-CoV-2 Infection and Transmission?
4.2.1 S Glycoproteins Produced in the Presence of Kifunensine Prefer Gal6S, Neu5Ac9NAc, and Neu4,5Ac2
4.2.2 S1-NTD Prefers Neu5Ac
4.2.3 S1-CTD (RBD) Prefers Gangliosides
4.3 Are Sialic Acids not Involved in Syncytium Formation of SARS-CoV-2?
5 Concluding Remarks
References
Chapter 18: Enzymatic Substrates and Fluorescence Imaging of Influenza Virus Sialidase
1 Introduction
2 Materials
2.1 Chemicals and Buffers
2.2 Cells
2.3 Media
2.4 Proteins and Viruses
2.5 Primers
2.6 Instruments
3 Methods
3.1 Live Imaging of Virus-Infected Cells or Drug-Resistant Virus-Infected Cells
3.2 Selective Imaging of Drug-Resistant Virus-Infected Cells
3.3 Highly Efficient Isolation of a Drug-Resistant Virus
3.4 Live Imaging and Immunostaining of NA-Expressed Cells by Using the New Sialidase Fluorogenic Probe and Anti-NA Monoclonal ...
4 Notes
References
Chapter 19: Assays for Determining the Sialidase Activity of Influenza Viruses and Monitoring Influenza Virus Susceptibility t...
1 Introduction
2 Materials
2.1 Equipment
2.2 Reagents
2.3 NA Inhibitors
2.4 Reference Viruses
3 Methods
3.1 4-MU Standard Curve
3.2 NA Activity Assay
3.3 NA Inhibition Assay
4 Notes
References
Chapter 20: Synthesis and Neuraminidase Inhibitory Activity of Sialic Acid Analogues with Fluoro, Phosphono, and Sulfo Functio...
Abbreviations
1 Introduction
2 Materials
2.1 Synthesis of Fluoro-, Phosphono-, and Sulfo-Sialic Acids
2.2 Fluorometric NA Inhibition Assay
2.3 Influenza Viral Replication Inhibition Assay
3 Methods
3.1 Synthesis of 2α,3ax-Difluoro-2-Deoxy-Neu5Ac (A)
3.1.1 Protection of Neu5Ac
3.1.2 Deacetoxylation of 3 (Method a)
3.1.3 Deacetoxylation of 3 (Method b)
3.1.4 β-Difluorination of 4
3.1.5 Hydrolysis of 5 to Give 2β,3eq-Difluoro-2-Deoxy-Neu5Ac (B)
3.1.6 Hydroxyfluorination of 4
3.1.7 Fluorine Substitution of 6ax
3.1.8 Hydrolysis of 7 to Give 2α,3ax-Difluoro-2-Deoxy-Neu5Ac (A)
3.2 Synthesis of 2α-Phosphono-2-Decarboxy-2-Deoxy-Neu5Ac (C)
3.2.1 Protection and Decarboxylation of Neu5Ac
3.2.2 Introduction of Phosphono to Form 10
3.2.3 Hydrolysis of 10 to Give 2α-Phosphono-2-Decarboxy-2-Deoxy-Neu5Ac (C)
3.3 Synthesis of 2α-Sulfo-2-Decarboxy-2-Deoxy-Neu5Ac (D)
3.3.1 Introduction of Acetylsulfanyl Group to Form 11
3.3.2 Oxidation of 11 with Oxone to Form 12
3.3.3 Hydrolysis to Give 2α-Sulfo-2-Decarboxy-2-Deoxy-Neu5Ac (D)
3.4 Fluorometric NA Inhibition Assay with 4-Methylumbelliferyl-Neu5Ac
3.5 Assay for Inhibition of Influenza Viral Replication
4 Notes
References
Chapter 21: Influenza A Virus Neuraminidase Inhibitors
1 Introduction
2 Overview of Influenza Virus NA
3 N-Acetylneuraminic Acid (Neu5Ac)-Based Derivatives
3.1 Neu5Ac2en (2,3-Didehydro-2-Deoxy-N-Acetylneuraminic Acid or DANA) and FANA (2,3-Didehydro-2-Deoxy-N-Trifluoroacetyl-neuram...
3.2 Structure-Based Designed Inhibitors-Zanamivir, Oseltamivir, Laninamivir Octanoate, Peramivir, and cPro-GUN
3.3 Oseltamivir-Based Inhibitors
4 Covalent NA Inhibitors
5 Sulfo-Sialic Acid Analogs
6 N-Acetyl-6-Sulfo-β-d-Glucosaminide-Based Inhibitors
7 Inhibitors Targeting the 150-Loop of Group 1 NAs
8 Conjugation Inhibitors
9 Acylhydrazone Derivatives
10 Monoclonal Antibodies (mAbs)
11 PVP-I
12 Natural Compounds
13 Future Perspectives
References
Index