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Molecular characterization of the model bacteriophage ΦX174 and biological implications in its complete refactoring

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posted on 24.11.2022, 02:33 authored by Bradley Wright

The bacteriophage ΦX174 is one of the best understood viral systems, so-much-so, the entire coding repertoire of the genome has been well characterised. In this thesis, I outline research that furthers our understanding of this model virus by characterising the molecular underpinnings of its infection of host Escherichia coli C122. I used a large-scale quantitative proteomics approach to temporally monitor the infection dynamics, revealing significant host proteome remodelling. Statistical analyses revealed pathways and host proteins of importance for infection fidelity and host infection response, in particular, the small heat shock proteins lbpA and lbpB. CRISPR-Cas9 mediated targeted gene knockout of these chaperones revealed unanticipated host growth phenotype loss under double knockout conditions, suggestive of their collective essentiality. Probing of their essentiality in ΦX174 infection revealed loss of infection fecundity under double knockout as demonstrated through infection centre assays.

I further explored viral biology with the use of an engineered ΦX174 strain that had been designed previously to exclude all instances of coding overlap within the genome. Coding overlap is a ubiquitous feature of viral genomes, and a phenomenon necessary to understand given our expanding ability to manipulate genetic code, where overlap may be inadvertently or deliberately interrupted. I found that the engineered ΦX174, despite maintaining coding potential, has significantly reduced progeny production, poorer capsid thermal stability, reduced attachment efficiency and reduced plaque sizes in comparison to the wild-type virus. Surprisingly, cellular lysis timing was maintained, and electron microscopy revealed no significant or obvious virion restructuring or deformities, thus alluding to genome integrity, packaging or ejection aberrations as the reasons for fidelity loss. To investigate further, I employed a targeted proteomics assay to temporally quantify viral protein production of the engineered and wild-type ΦX174 strains. The in vivo measurements of protein expression revealed deficiencies in the production of viral internal scaffolding protein B, and protein C, whose role is critical in viral genomic DNA packaging. I concluded that gene topology and cellular phenotype are critically linked, and emphasize the importance of natural sequence overlap for gene integrity.

The results of my work expand our knowledge of the biology of the model bacteriophage ΦX174, and importantly, contribute to furthering our understanding of the implications of coding overlap disruption and the roles model viruses and proteomics can play in exploring these pertinent questions for the future of synthetic biology.

History

Table of Contents

Chapter 1: Bacteriophage biology and research methods -- Chapter 2: The importance of studying gene overlap - reviewed -- Chapter 3: Quantifying molecular response to ΦX174 infection -- Chapter 4: Biological implications of gene modularisation in the virus ΦX174 -- Chapter 5: Conclusions and future perspectives -- Appendix A: Supplementary information: Chapter 3 -- Appendix B: Supplementary information: Chapter 4 -- Appendix C: Ethics approval -- Appendix D: References

Notes

A thesis submitted to Macquarie University in fulfilment of the requirements for the degree of Doctor of Philosophy

Awarding Institution

Macquarie University

Degree Type

Thesis PhD

Degree

Thesis PhD, Macquarie University, Department of Molecular Sciences, 2020

Department, Centre or School

Department of Molecular Sciences

Year of Award

2020

Principal Supervisor

Paul Jaschke

Rights

Copyright: The Author Copyright disclaimer: https://www.mq.edu.au/copyright-disclaimer

Language

English

Extent

212 pages

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