Mutational Analysis of Non-essential Genes of Respiratory Syncytial Virus Open Access

Abstract

Respiratory syncytial virus (RSV) is the most important pathogen underlying acute lower respiratory tract infections in infants. Currently there are no vaccines or antivirals. Understanding the functions of RSV virulence genes should provide new insights into development of vaccines and antivirals. Two non-structural genes of RSV (NS1 and NS2) suppress the host immune responses. Deletion of either or both NS genes generated virus that was either under-attenuated or over-attenuated for an effective and safe vaccine candidate for infants and young children. We used a codon deoptimization strategy to reduce the expression of NS genes based on human codon usage bias, resulting in dNSh virus. dNSh was attenuated in BEAS-2B and primary normal human bronchial epithelial cells cultured at air-liquid interface, but not in Vero or HEp-2 cells. In BALB/c mice, dNSh exhibited 10-fold lower viral load yet induced higher levels of RSV-neutralizing antibodies than the A2 non deoptimized virus. Both viruses induced the same protection efficacy against challenge strains A/1997/12-35 and A2-line19F. Upon codon deoptimization, the NS target protein, STAT2, was degraded to a lesser extent than infection with non-deoptimized virus. dNSh induced less NF-κB activation, suggesting less pro inflammatory potential. Serial passages in BEAS-2B did not generate nucleotide changes in the deoptimized regions. RSV strain differences are an important factor in pathogenesis. The two surface glycoproteins, attachment protein G and fusion protein F, play major functions during infection. Previous studies indicated G protein was not required for infection, based on the fact that G-null virus still replicates efficiently in several cell-lines. We re-evaluated the functions of the G gene for a clinical isolate A2001/2-20 (2-20) in comparison to the lab strain A2. We generated recombinant viruses containing G and F or no G protein from either A2 (kRSV-A2G-A2F and kRSV-GstopA2F) or 2-20 strains (kRSV-2-20G-2-20F and kRSV-Gstop2-20F). We quantified the contribution of G to virus binding, entry kinetics, infectivity, and replication in vitro. Removal of 2-20 G from the virus had more deleterious effects than removal of A2 G in all the above processes. Overall, the 2-20 strain F had a higher dependence on its G protein than did the A2 strain.

Table of Contents

CHAPTER 1: INTRODUCTION - AN OVERVIEW OF RESPIRATORY SYNCYTIAL VIRUS BIOLOGY...1

1.1 RSV EPIDEMIOLOGY, CLASSIFICATION...2

1.2 RSV STRUCTURE...3

1.3 RSV LIFE CYCLE AND REVERSE GENETICS SYSTEM...6

1.4 RSV CELL CULTURE SYSTEM...7

1.5 RSV STRAIN DIFFERENCES...8

1.6 RSV AND THE HOST INNATE IMMUNITY...9

1.7 RSV AND THE HOST ADAPTIVE IMMUNITY...10

1.8 RSV VACCINES AND ANTIVIRALS...11

CHAPTER 2...14

Refining the balance of attenuation and immunogenicity of respiratory syncytial virus by targeted codon deoptimization of virulence genes...14

ABSTRACT...16

IMPORTANCE...17

INTRODUCTION...18

RESULTS...20

DISCUSSION...31

MATERIALS AND METHODS...35

ACKNOWLEDGMENTS...40

CHAPTER 3...41

Functional Differences for the Attachment Glycoprotein of Respiratory Syncytial Virus Clinical Isolate A2001/2-20...42

ABSTRACT...44

INTRODUCTION...45

MATERIALS AND METHODS...48

RESULTS...54

DISCUSSION...66

ACKNOWLEDGMENTS...70

CHAPTER 4: DISCUSSION...71

REFERENCE...83

CHAPTER 2

Fig 1. Nucleotide sequence alignment of RSV A2 strain NS1 and NS2 with human codon deoptimized NS1 and NS2...21

Fig 2. Generation of recombinant RSV with codon deoptimized NS1 and NS2...22

Fig 3. Expression of NS1 and NS2 proteins during RSV infection in cell lines...23

Fig 4. Growth kinetics of kRSV-A2 and kRSV-dNSh in vitro...25

Fig 5. Attenuation, efficacy, and immunogenicity...27

Fig 6. STAT2 degradation and NF-κB activation...29

CHAPTER 3

Fig 1. Schematic design of the recombinant viruses and quantification of surface glycoproteins in purified virions...55

Fig 2. Greater contribution of 2-20 G than A2 G to binding to the cell...57

Fig 3. Entry kinetics in BEAS-2B cells...59

Fig 4. Infectivity in BEAS-2B, CHO-K1, and pgsD-677 cell lines...61

Fig 5. Contribution of G protein to virus in vitro growth kinetics...63

Fig 6. Cell-to-cell fusion activity by dual-split protein fusion assay...65

CHAPTER 4

Fig 1. F protein sequence comparison of A2 strain with several clinical isolates...81

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 Biology, Microbiology
Keyword	Respiratory Syncytial Virus Vaccine Non-essential Gene Glycoprotein
Committee Chair / Thesis Advisor	Moore, Martin L, Emory University
Committee Members	Plemper, Richard, Georgia State University Steinhauer, David, Emory University Steel, John, Emory University Anderson, Larry J, Emory University

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