Influenza A Virus Hemagglutinin: An Examination of Divergent Subtypes with Respect to Stability and Receptor Binding Properties Público

Abstract

The hemagglutinin (HA) of influenza A viruses mediates the processes associated with viral entry: binding to cell surface receptors and facilitating fusion of the virion membrane with the host endosomal membrane. The HA structure incorporates two functional domains related to these activities, the receptor binding site in the globular head and the fusion peptide and pocket in the stem region. These sites have specific characteristics associated with subtype and host, and the differences often define species barriers. In this work, we have characterized the effects of mutations in the stem region of HA for strains representing each structural group, as well as avian and human hosts. We found that for the conserved residue 58, a lysine to isoleucine change increased the acid stability of most HAs, lowering the pH of fusion. A mutation at conserved residue 112, from aspartic acid to glycine, decreased the acid stability, raising the pH of fusion for all HA subtypes. The effects of both mutations were consistent across structural and host classifications, correlating with the conserved nature of both residues. Future studies are planned to examine the effects of mutations at group specific positions. We began our study of receptor binding by characterizing the broad receptor specificity of a range of influenza viruses again differing in subtype and host. The receptor specificity is linked to residues within the receptor binding pocket of HA, though their positions are altered between group 1 and group 2 subtypes. Avian viruses preferentially recognize α2,3-Sialic acid terminating glycans and mammalian viruses recognize α2,6-Sialic acid. Our results using the Consortium for Functional Glycomics glycan microarray corroborate these statements, though they reveal a general over-simplification in that other structural determinants beyond the terminating sialic acid moiety are important for Influenza A virus binding. To identify the receptors used in natural infection, we developed the Pig Lung Shotgun Microarray incorporating N-glycans isolated from swine lung tissue. Eight endogenous receptors were identified and characterized. Continuation of this work involves expanding our repertoire of natural arrays to include human lung glycans and examining the role of neuraminidase in entry as the substrate specificity overlaps with that of HA.

Table of Contents

ABSTRACT

ACKNOWLEDGMENTS

TABLE OF CONTENTS

LIST OF FIGURES AND TABLES

INTRODUCTION 1

The Virus Life Cycle and Management of Infection 3

Receptor Binding and Entry 3

Transcription 7

Translation 8

Assembly and Budding 9

Vaccines and Antivirals 10

Subtypes, Reassortment, and Pandemic Events 12

The Role of the Host: Carbohydrate Interaction, Interspecies Transmission, And Pathogenicity 16

Carbohydrates Recognized by Influenza A Virus 16

Getting to The Surface- Mucins and Virion Morphology 18 Presentation of Receptors in Avian, Swine, and Human Hosts 21

Interspecies Transmission 26

Pathogenicity 29

Functional Balance of HA and NA 30

Dissertation Overview 32

INFLUENZA HEMAGGLUTININ (HA) STEM REGION MUTATIONS THAT STABILIZE OR DESTABILIZE THE STRUCTURES OF MULTIPLE HA SUBTYPES 34

Abstract 35

Importance 36

Introduction 36

Materials and Methods 40

Results 45

Discussion 55

Acknowledgments 61

SHOTGUN GLYCOMICS OF PIG LUNG IDENTIFIES NATURAL ENDOGENOUS RECEPTORS FOR INFLUENZA VIRUSES 62

Abstract 63

Significance Statement 64

Introduction 65

Results 70

Discussion 85

Experimental Methods 92

Acknowledgments 97 Supplementary Material 98

CONCLUSIONS AND FUTURE STUDIES 116

APPENDIX: ANALYSIS OF FUSION PEPTIDE POCKET RESIDUES OF INFLUENZA A VIRUS HEMAGGLUTININ AND THEIR EFFECT ON THE pH STABILITY OF THE PROTEIN 125

Abstract 126

Introduction 127

Materials and Methods 129

Results and Discussion 134

Acknowledgments 142

REFERENCES 146

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Microbiology & Molecular Genetics
Degree	PhD
Submission	Dissertation
Language	English
Research Field	Chemistry, Biochemistry Biology, Molecular Biology, Virology
Palavra-chave	HA Fusion HA Receptor Binding Influenza A Virus
Committee Chair / Thesis Advisor	Steinhauer, David, Emory University
Committee Members	Moore, Martin L, Emory University Katz, Jackie, Centers for Disease Control and Prevention Cummings, Richard, Emory University Lowen, Anice Carmen, Emory University

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