Dissecting mechanisms of intersegment epistasis and its role in influenza A virus biology.

Diefenbacher, Meghan

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https://hdl.handle.net/2142/116064 Copy

Description

Title

Dissecting mechanisms of intersegment epistasis and its role in influenza A virus biology.

Author(s)

Diefenbacher, Meghan

Issue Date

2022-07-10

Director of Research (if dissertation) or Advisor (if thesis)

Brooke, Christopher B

Doctoral Committee Chair(s)

Brooke, Christopher B

Committee Member(s)

• Whitaker, Rachel
• Kehl-Fie, Thomas
• Blanke, Steven

Department of Study

Microbiology

Discipline

Microbiology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Influenza A virus
• gene segment replication
• intersegment epistasis

Abstract

Influenza A virus (IAV) is a significant respiratory pathogen that causes hundreds of thousands of deaths worldwide each year. The IAV genome is divided into eight negative-sense, single-stranded RNA segments. Genetic interactions within and between the eight segments and their encoded proteins, otherwise known as intersegment epistasis, play an important role in modulating IAV replication and evolution. However, the current understanding of the extent and nature of these epistatic interactions as well as their mechanisms remains incomplete. Here, I aim to investigate mechanisms by which intersegment epistasis modulates IAV replication and the genomic structure of IAV populations. The eight gene segments are replicated and transcribed in the context of viral ribonucleoprotein complexes (vRNPs) in which each segment is encapsidated along its length by the viral nucleoprotein (NP) and is associated with a single copy of the viral RNA dependent-RNA polymerase (RdRp). Each segment exhibits its own unique level and temporal pattern of expression, however, the exact mechanisms underlying the independent regulation of individual gene segment expression are not well understood. I previously demonstrated that a single substitution in NP (F346S) selectively modulates neuraminidase (NA) gene segment expression while leaving other segments largely unaffected. The specificity of this effect for the NA segment is viral strain specific and depends on the UTR sequence of the NA segment. While the NP:F346S substitution did not significantly affect the ability of NP to bind to and oligomerize along the viral RNAs, it specifically decreased the ability of NP to promote NA segment vRNA synthesis. In addition to NP residue F346, I identified two other adjacent aromatic residues in NP (Y385 & F479) capable of selectively regulating NA gene segment expression, suggesting a larger role for this domain in gene-segment specific regulation. These findings demonstrate a novel role for interactions between the gene segment UTRs and NP in regulating IAV gene segment expression. RNA structures are proposed to play an important role in regulating IAV gene segment expression and packaging; however, their study has previously been limited to in vitro or in silico approaches that predicted structures present in naked IAV RNAs which were likely not representative of the structural conformations that would be present in the RNAs in the context of vRNPs in infected cells and virions. Using the selective 2′-hydroxyl acylation analyzed by primer extension and mutational profiling (SHAPE-MaP) protocol, I was able to generate a comprehensive map of the RNA structural profile of the eight gene segments in the context of vRNPs isolated from virions purified from the A/Puerto Rico/8/1934 (PR8) and PR8 NP:F346S viruses. Each segment exhibited its own unique RNA structural profile. Initially, I used this data to determine whether the NP:F346S substitution affects the NP binding distribution along the NA segment and leads to the differential formation of RNA structures important for NP-dependent regulation. However, the RNA structural profiles of the NA segment were highly similar in the presence of NP:WT and NP:F346S, and introducing silent substitutions to disrupt predicted RNA structures in specific regions of the segment had no effect on the NP:F346S phenotype. Using this data set, I aim to perform future experiments to probe the roles that the observed RNA structures in the eight segments and interactions between them may play in regulating gene segment expression, packaging, and the production of aberrant RNA replication species (DIs, mvRNAs etc…). Only a small fraction of particles in IAV populations are fully infectious with the remaining portion consisting primarily of semi-infectious particles (SIPs) which fail to express one or more viral proteins. As SIPs lack one or more segments, they require co-infection with other IAV particles in order to obtain the complete complement of segments required for a productive infection. IAV strains can vary significantly in terms of the fraction of their population that exists as SIPs, suggesting that there is a genetic determinant underlying SIP production. However, the exact identity of the gene segment(s) that determine the SIP production phenotype of different IAV strains remains unknown. Using high SIP producing (A/Fort Monmouth/1/1947 (H1N1)) and low SIP producing (A/California/04/2009 (H1N1)) strains of IAV, I generated reassortant viruses containing a distinct set of gene segments from each parental strain and quantified the ratio of total particles to infectious particles for each reassortant using the hemagglutination assay and plaque assay respectively. I found that the SIP production phenotype was linked to the genetic background of the viral replicase complex (vRNP). These findings are suggestive of a potential role for gene segment replication efficiency and/or packaging in determining the fraction of IAV particles that are fully or semi-infectious. My finding that the NP:F346S substitution can selectively regulate the expression of individual gene segments via interactions with the UTRs demonstrates a novel mechanism of intersegment gene expression regulation in IAV by which a segment’s RNA sequence can influence its inherit susceptibility to regulation by the same viral protein. In addition, my finding that specific RNA structures within the NA segment influence its expression levels enhances our understanding of the role that RNA structures play in IAV biology. Finally, my finding that the vRNP complex genotype influences an IAV strain’s SIP production phenotype suggests that the baseline efficiency of gene segment replication regulates SIP formation frequency by either modulating the efficiency by which IAV can successfully complete all steps of its replication cycle and evade the host immune response or modulating the fraction of gene segments that get incorporated into viral particles. Altogether, these studies have important consequences for further developing our understanding of the factors that drive the replication and evolution of IAV.

Graduation Semester

2022-08

Type of Resource

Thesis

Copyright and License Information

Copyright 2022 Meghan Diefenbacher

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