Viral Determinants of Reliance on Multiple Infection in Influenza A Viruses Public

Abstract

Viral infections involving two or more virions in a single cell have been seen to impact infection outcomes for a diverse array of virus types. We previously found that influenza A/guinea fowl/Hong Kong/WF10/99 (H9N2) virus (GFHK99) displays a particularly high reliance on multiple infection in mammalian cells tied to the PA gene segment. This dissertation sought to uncover the viral processes underlying the high reliance phenotype of GFHK99. Herein, the experimental techniques and data collected in the evaluation of GFHK99 are described. PA 26K was found to suppress endonuclease activity and viral transcription, specifically within cells infected at low multiplicity. The model arising from this research postulates that sub-optimal activity of the GFHK99 endonuclease results in inefficient priming of viral transcription, an insufficiency which can be overcome with the introduction of additional viral templates to the cell. These findings add to the burgeoning field of collective cellular interactions of viruses and have implications for continued public health surveillance of H9N2 IAVs.

Table of Contents

Chapter 1: Introduction                                                                                         pg. 1

           Virus structure and lifecycle                                                                         pg. 1

           Fig. 1: The structure and proteins of an influenza A virion                           pg. 2

           Fig. 2: Antigenic drift and antigenic shift                                                     pg. 6

           IAV ecology and impact                                                                                pg. 8

           Interhost transmission                                                                                  pg. 14

           Fig. 3: Barriers to IAV spread                                                                                   pg. 14

           Reliance on multiple infection                                                                     pg. 18

           Fig. 4: The Lowen Lab multiple infection model                                         pg. 19

           Fig. 5: GFHK99 high reliance is linked to the PA gene segment                 pg. 20

           Dissertation aims                                                                                          pg. 21

           References                                                                                                     pg. 23

Chapter 2: Beneficial effects of cellular coinfection resolve

                  inefficiency in influenza A virus transcription                                   pg. 35

Introduction                                                                                                   pg. 36

Results                                                                                                           pg. 37

Fig 1. Replacement of the MaMN99 endonuclease region

with that of GFHK99 PA increases reliance on multiple infection.              pg. 38

 

Fig 2. Disrupting PA-X does not alter reliance on multiple infection

phenotype of GFHK99 and MaMN99 viruses.                                              pg. 41

 

Fig 3. PA 26K is associated with a higher reliance on multiple infection    pg. 42

than PA 26E.

 

Fig 4. Levels of PA protein are not decreased by PA 26K                           pg. 44

 

Fig 5. PA 26K confers lower endonuclease activity than PA 26E                pg. 46

 

Fig 6: Inhibition of cap-snatching increases reassortment                            pg. 47

                       

Discussion                                                                                                     pg. 47

Fig 7: Multiple infection allows a virus with an inefficient PA

endonuclease to replicate.                                                                              pg. 47

Methods                                                                                                         pg. 51

Tables                                                                                                            pg. 57

Table 1: Primers used for chimeric PA segment Gibson Assembly

Table 2: Primers for ΔPA-X site-directed mutagenesis

Table 3: Primers used for strand-specific qPCR of MaMN99

References                                                                                                     pg. 59

Chapter 3: Conclusions                                                                                           pg. 63

           References                                                                                                     pg. 69

About this Dissertation

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School	Laney Graduate School
Department	Biological and Biomedical Sciences
Subfield / Discipline	Immunology and Molecular Pathogenesis
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biology, Cell Biology, Virology
Mot-clé	pathogenesis sociovirology virus influenza collective interactions
Committee Chair / Thesis Advisor	Anice Lowen, Emory University
Committee Members	Arash Grakoui, Emory University John Altman, Emory University Terrence Tumpey, Emory University Dave Steinhauer, Emory University

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