The Role of Enhanced Glycoprotein Activity in Respiratory Syncytial Virus Pathogenesis Pubblico
Lopez-Ona, Anne Hotard (2014)
Abstract
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
References106
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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