Date of Award

Winter 12-15-2017

Author's School

Graduate School of Arts and Sciences

Author's Department

Biology & Biomedical Sciences (Molecular Microbiology & Microbial Pathogenesis)

Degree Name

Doctor of Philosophy (PhD)

Degree Type

Dissertation

Abstract

Influenza A viruses (IAV) are a major human and environmental pathogen. IAV successfully infects a diverse host range and adaptation of new viral strains to humans may cause pandemic events with high morbidity and mortality. As a member of the Orthomyxoviridae family, IAV inherently possesses a segmented genome, which enables a process of segment transmission between viruses following cellular co-infection, a process termed reassortment. The high rate of IAV mutation and continued co-circulation of diverse viral strains in divergent host species leads to the persistent prospect for emergence of new IAV with pandemic potential. Therefore, it is of great importance to understand the viral and host factors that restrict and promote the generation of emergent virus strains, their potential for pathogenesis, and discover novel mechanistic countermeasures against IAV, including improved vaccination and targeted therapeutic strategies.

Human and avian IAV co-circulate and occasionally co-infect the same host, leading to the potential for generation of novel genome constellations following reassortment. The specific host and viral molecular determinants that allow replication of reassortant progeny virus are not well defined. Here, I show that the viral genetic context and host cell in which reassortment occurs determine the potential for genetic diversity derived from multiple distantly related strains. Importantly, we identify single gene reassortants between a North American avian strain and the 2009 pandemic H1N1 virus that are capable of causing disease in mammals and replicate in a human cell line as well as induce the production of several pro-inflammatory cytokines linked to severe disease outcomes. Additionally, utilizing a different viral genetic background, I show that the reassortment potential is regulated by species and cell type specific differences in viral replication due to augmented viral polymerase function dependent on the identity of a single amino acid in the PA protein. Together, these studies provide evidence that context- dependent compatibility between both viral and host factors determine the possibility for generation of novel reassortant genome constellations and regulate their potential for replication and transmission in new host species.

Reassortment between IAV strains is likely dictated by the functional compatibility of vRNA segments bound by IAV nucleoprotein during genome packaging. I hypothesized that nucleoprotein (NP) scaffolds specific RNA elements that are required for genome packaging and interaction between viral RNA (vRNA) genome segments. Therefore, I sought to determine the functional consequences of genome architecture on genome packaging and for the first time determine the nucleotide-resolution landscape of NP-vRNA interactions in infected cells. We utilized Photoactivatable Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation (PAR-CLIP) coupled to next-generation sequencing to determine the specific interaction sites of vRNA bound by NP. We then interrogated the functional importance of regions of vRNA bound or unbound by NP and identified a number of potentially structured RNA features required for efficient genome packaging and virus propagation. These studies provide a framework for understanding the multifactorial restrictions of IAV reassortment and potential for generation of novel genome constellations with pandemic potential. Finally, these studies expand our understanding of how viral and host determinants shape the possible evolutionary trajectories of IAV through reassortment and required genetic elements needed for genome assembly.

Language

English (en)

Chair and Committee

Adrianus Boon

Committee Members

Gaya Amarasinghe, Michael Diamond, Sebla Kutluay, Deborah Lenschow,

Comments

Permanent URL: https://doi.org/10.7936/K7PG1R4J

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