Abstract

Over an evolutionary timeline, viruses have evolved ways to exploit biochemical pathways of the host for their propagation, whereas host organisms developed mechanisms to recognize virus infection and cope with them. Consequently, a better understanding of the different host factors involved in virus replication could provide insights into potential biological pathways or molecules for therapeutic targeting. Furthermore, detailed dissection of the mechanisms of virus sensing could reveal fundamentally important aspects of virus sensing that are evolutionarily conserved to mammals and offer insights into potentially underexplored areas of research. The C. elegans experimental model offers a useful platform for studying evolutionarily conserved host genes, as C. elegans and mammals share many important genes and biological mechanisms as exemplified by the discoveries made in RNA biology, developmental biology, neuroscience, and genetics. Combined with the Orsay virus, a natural pathogen to C. elegans, the C. elegans–Orsay virus experimental system offers a novel platform for interrogating host-pathogen interactions in unique ways compared to traditional tissue culture or animal models. For example, the small size of nematodes permits culturing in large numbers. Unlike mammalian animal models such as mice, C. elegans allows for forward mutagenesis screens at a whole-organism level. In my thesis, I established a forward chemical mutagenesis screen aimed at isolating host factors involved in the replication phase of the Orsay virus life cycle. This was achieved by leveraging a transgenic C. elegans model in which the virus lifecycle can be initiated from a stably integrated transgene. By performing random chemical mutagenesis, I isolated three mutant lines that harbored independent missense mutations in a previously uncharacterized gene, Y55F3BL.4 (renamed viro-9), and validated this result by trans-complementing the wild type viro-9. viro-9 contains a Pfam-annotated SRR1 domain with a poorly defined function. Since the function of the viro-9 is not known in C. elegans, I performed physiological assays to define any gross morphological defects in the null allele mutants and found that ablation of the viro-9 locus moderately reduced the life span and brood size. To gain insights into the potential functional conservation of the gene, I asked whether CBG23913, the Caenorhabditis briggsae (C. briggsae) ortholog of viro-9, can rescue the virus infection defect in viro-9 mutant C. elegans animals. The C. briggsae CBG23913 was sufficient for restoring virus replication, suggesting that the function of the SRR1 domain is conserved across 80–110 million years. Additionally, I compared the SRR1 domain-containing sequences across different eukaryotic kingdoms and identified highly conserved residues that were altered in the C. elegans mutant lines isolated during the chemical mutagenesis. Although the alteration of the chemically mutagenized sites did not dramatically affect the predicted VIRO-9 structure, it could be speculated that these residues may play important roles for protein-protein interactions. Besides studying pro-viral C. elegans host factors, I addressed the fundamental question of how virus replication is sensed in C. elegans upon infection. Although many experimental caveats remain to be resolved, I found that the expression of the replication-defective Orsay virus RNA1 segment in the genomic and complementary orientations, mimicking the double-stranded RNA (dsRNA), was sufficient for activating an antiviral response in C. elegans. This highlights that the mechanism of virus sensing in C. elegans is potentially similar to dsRNA recognition in mammalian cells, which depends on RIG-I-like receptor (RLR) family of proteins. In summary, I was able to address the theme of host-virus interactions from different angles. I have expanded our knowledge of post-entry C. elegans host factors by identifying viro-9 as a pro-viral host gene with an evolutionarily conserved function. Additionally, I identified potential substrates that may be involved in virus recognition in C. elegans, which may share similarities with those previously described in other organisms. Overall, these findings could open research avenues on biochemical pathways that could potentially be targeted as part of a therapeutic intervention.

Committee Chair

David Wang

Committee Members

Carolina Lopez; Megan Baldridge; Michael Nonet; Siyuan Ding; Tim Schedl

Degree

Doctor of Philosophy (PhD)

Author's Department

Biology & Biomedical Sciences (Molecular Genetics & Genomics)

Author's School

Graduate School of Arts and Sciences

Document Type

Dissertation

Date of Award

5-22-2025

Language

English (en)

Author's ORCID

https://orcid.org/0009-0002-3256-7127

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