Natural Influenza Isolates and Pseudorevertant Mutants Have Species Specific Receptor Binding Profiles and Recognize a Subset of Sialic Acid Derivatives Open Access

Abstract

Influenza A virus is a single-stranded, negative-sense RNA virus, responsible for significant seasonal morbidity and mortality every year. Influenza encodes the surface hemagglutinin glycoprotein (HA), a multifunctional protein that is responsible for receptor binding, and closely linked to transmission, host adaptation, and pathogenicity. Binding to sialic acid is critical to viral infectivity, and can be affected by several well-characterized amino acids within the receptor binding site. We passaged a non-agglutinating mutant virus in mice and MDCK cells to isolate pseudorevertant viruses with mutations outside of the receptor binding site that significantly affected receptor binding. Despite being fully infectious in mice and MDCK cells, these mutant viruses did not recognize the classical sialic acid receptors present on glycan microarrays available from the Consortium for Functional Glycomics. Rather these mutants bound to a subset of sialic acid derivatives present in human serum and other mammalian tissues. Additionally, we sought to determine if the receptor binding patterns of early pandemic H1N1 isolates differed from both a prior seasonal strain and a related swine strain incapable of sustained human-to-human transmission. Interestingly, the strains bound highly similar receptors, indicating that many circulating swine strains might have the necessary receptor specificity to cause human infection, but additional factors are necessary for efficient transmission among humans. Together, these results utilize glycan microarray technology to broadly explore the influenza receptor binding of laboratory and mammalian species, and begin to describe binding to a new subset of biologically relevant sialic acid derivatives.

Table of Contents

Table of Contents

Introduction...1

Influenza Background...1
Influenza Gene Segments...10
Influenza Life Cycle...17
Sialic Acid as a Receptor...36
Vaccines and Antivirals...38

Chapter 2 - "Comparison of the receptor binding properties of contemporary swine isolates and early human pandemic H1N1 isolates (Novel 2009 H1N1)...43

Abstract...45
Introduction...46
Results...49
Discussion...70
Materials and Methods...77
References...81

Chapter 3 - Analysis of influenza virus hemagglutinin receptor binding mutants with limited receptor recognition properties and conditional replication characteristics...97

Abstract...99
Introduction...101
Materials and Methods...107
Results...111
Discussion...128
References...138

Chapter 4 - Discussion and Future Results...152

References...171

List of Figures and Tables

Introduction:

Figure 1 - Structure of A/Aichi/2/1968 hemagglutinin...17

Figure 2 - Structural differences between Neu5Ac and Neu5Gc...20

Figure 3 - Influenza HA interaction with sialic acid in α2,3 and α2,6 conformation...21

Chapter 2:

Figure 1 - Schematic diagram of the HA receptor binding site and major structural characteristics...51

Table 1 - Sequence alignment of the receptor binding site of strains examined on glycan microarrays...52

Table 2 - Agglutination of erythrocytes by purified influenza strains...52

Figure 2 - Phylogenetic tree of influenza isolates used in this paper...54

Figure 3 - Binding to CFG glycan microarrays by contemporary swine isolates...56

Figure 4 - Binding to CFG glycan microarrays by control and human Influenza isolates...57

Figure 5 - The top five glycans bound by purified swine isolate viruses on the CFG microarray...58

Figure 6 - Comparison of glycans bound on CFG microarrays...59

Figure 7 - Fluorescent binding and neuraminidase activity assays of influenza isolates...60

Chapter 3:

Figure 1 - Structural locations of mutations found in pseudorevertant viruses...105

Figure 2 - In vitro and in vivo viral titers in 3 dpi mouse lungs and MDCK cells...114

Table 1 - Agglutination of erythrocytes from different species by pseudorevertant viruses...118

Table 2 - Neuraminidase activity of pseudorevertant virses on chicken and guinea pig erythrocytes...118

Figure 3 - Fluorescent binding assays of pseudorevertant viruses...120

Figure 4 - Binding by pseudorevertant viruses to CFG microarrays...123

Figure 5 - Structures of the five glycans with the highest binding levels with Y98F/K238N HA...124

Table 3 - Glycans bound significantly by pseudorevertant viruses on sialo- derivative glycan microarrays...126

Figure 6 - Binding to sialoderivative glycan microarrays by pseudorevertant viruses...127

Discussion:

Figure 1 - Sialidase activity of influenza virus on MDCK cells after 3 hr incubation at 37°C...158

Figure 2 - PAGE analysis reveals the loss of an N-linked glycosylation site...161

Figure 3 - Binding of human and swine isolates to sialo-derivative glycan microarrays...166

Figure 4 - The activity of V. cholera neuraminidase on sialo-derivative glycan microarrays...168

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	Biology, Virology
Keyword	Receptor Binding Virology Influenza
Committee Chair / Thesis Advisor	Steinhauer, David, Emory University
Committee Members	Speck, Sam, Emory University Donis, Ruben O., Emory University Compans, Richard W, Emory University Cummings, Richard, Emory University

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