The Role of Enhanced Glycoprotein Activity in Respiratory Syncytial Virus Pathogenesis Open Access

Lopez-Ona, Anne Hotard (2014)

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Respiratory syncytial virus (RSV) is the most important cause of lower respiratory tract infections in infants worldwide. To date, there is no vaccine for RSV, prophylaxis is limited to high risk infants or those with chronic heart or lung disease, and treatment is limited to symptom management. Severe RSV infections in infants are characterized by mucus secretion, epithelial cell sloughing, and immune cell infiltration in the lungs. Different RSV strains exhibit differential pathogenesis in the mouse model of RSV-induced disease, with chimeric strain A2-line19F presenting human-like disease in this model. We developed an improved recombination-mediated mutagenesis reverse genetics system based on RSV strain A2-line19F to generate mutants in both the attachment (G) and fusion (F) glycoproteins. We identified residues critical for the fusion activity of the line19-F protein, and hypothesized that a virus with these residues mutated would have reduced pathogenesis in the mouse model compared to the parental A2-line19F. While we demonstrated a correlation between fusion activity and early viral load in the mouse, we were unable to discern a correlation between fusion activity or viral load and mucus induction, an important parameter of RSV pathogenesis. We described an additional mutant of line19-F which enhanced the fusion activity of the protein. The virus expressing this F protein augmented pathogenesis in mice, highlighted by early weight loss, severe lung pathology, and high viral loads.

RSV strains in the BA clade contain a 60 nucleotide duplication in the G gene. We hypothesized that this duplication would boost the function of the G protein. We generated recombinant viruses expressing a consensus BA G protein with and without the duplication, and found that the duplicated region enhances virus binding to cells by a glycosaminoglycan dependent mechanism. Additionally, we discovered that the virus with the duplication exhibited higher viral loads in mice than the virus without the duplicated region. Based on these results, we concluded that the duplication gives RSV BA strains an advantage in virus infectivity and possibly in transmission. Taken together, we identified mutations in both major glycoproteins of RSV which enhanced protein activity and viral pathogenesis.

Table of Contents

Chapter 1: Introduction - An Overview of Respiratory Syncytial Virus 1

Chapter 2: A Stabilized Respiratory Syncytial Virus Reverse Genetics System Amenable to Recombination-Mediated Mutagenesis 10

Abstract 12

Introduction 13

Results 15

Discussion 23

Experimental Procedures 24

Acknowledgements 34

Chapter 3: Identification of Residues in the Human Respiratory Syncytial Virus Fusion Protein That Modulate Fusion Activity and Pathogenesis35

Abstract 37

Introduction 39

Materials and Methods 42

Results 49

Discussion 65

Acknowledgements 69

Chapter 4: Role of the 60 Nucleotide Duplication on the Respiratory Syncytial Virus Buenos Aires Strain Attachment Glycoprotein70

Abstract 72

Introduction 73

Materials and Methods 76

Results 81

Discussion 90

Acknowledgements 92

Chapter 5: Summary and Conclusions93


Figures and Tables

Chapter 2

Figure 1 - Model of RSV-BAC Recombination-Mediated Mutagenesis and Reverse Genetics 14

Figure 2 - Sequence-Optimized RSV Helper Plasmids Drive More Minigenome Activity than Wild-Type RSV Helper Plasmids 15

Figure 3 - A2-K-line19F Encoded mKate2 Serves as a Marker for RSV Infected

Cells 17

Figure 4 - In Vitro and In Vivo Growth Characteristics of A2-K-line19F and A2-RL-line19F 20

Figure 5 - Recombination-Mediated Mutagenesis Derived Mutant Viruses 22

Table 1 - Nucleotide Sequence Positions of Features in pSynkRSV-line19F 25

Chapter 3

Figure S1 - Amino Acid Alignment of RSV F 40

Figure 1 - Amino Acid Residues Unique to Line 19 F 50

Figure 2 - F Expression and Cell-Cell Fusion Activity 53

Figure 3 - Virus Replication in BEAS-2B Cells 55

Figure 4 - Lung Viral Load in BALB/c Mice 57

Figure 5 - Airway Mucus Induction in BALB/c Mice 59

Figure 6 - Pathogenesis of A2-line19F-K357T/Y371N 63

Chapter 4

Figure 1 - BA Strain Cloning Scheme 82

Figure 2 - Recombinant BA Strain Replication in BEAS-2B Cells 83

Figure 3 - Competitive Infection Ass ay 84

Figure 4 - Recombinant BA Strain Binding to BEAS-2B Cells 86

Figure 5 - Glycosaminoglycan Dependency of Recombinant BA Strains 88

Figure 6 - Recombinant BA Strain Lung Viral Load in BALB/c Mice 89

Chapter 5

Figure 1 - RSV A Strain F Protein Alignment 98

Figure 2 - A2-line19F-Y371N Replication in BHK-21 Cells and BALB/c Mice 101

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