Genetic and Functional Characterization of Novel Host Factors Regulating Virus Infection in Caenorhabditis elegans
Date of Award
Doctor of Philosophy (PhD)
Viruses infect the majority of eukaryotic life on the planet and remain a global threat to human health. These pathogens are constrained to intracellular life cycles, as they exploit and rely on host factors and machinery throughout their entire reproduction process. While many of these viral life cycle factors have been reported and studied, our knowledge in the identity and function of these factors remains incomplete and a challenge in fundamental virology and the development of antiviral therapeutics. Caenorhabditis elegans offers an innovative approach for discovering novel host factors required for virus infection in a multicellular and simple model organism. Although C. elegans has been extensively used as a model to study bacterial and fungal infections, viral studies have been highly restricted due to the lack of a naturally infecting C. elegans virus. Therefore, the discovery of Orsay virus as the first natural viral pathogen of C. elegans presents an unprecedented opportunity to advance our understanding of vital virus interactions with its host. As many C. elegans genes are evolutionarily conserved in higher organisms, pro-viral factors in C. elegans may have a conserved function in humans for pathogenic viruses. Thus, the study of the C. elegans-Orsay virus model may ultimately lead to the development of antiviral therapeutics. In this dissertation, I exploit the power of this nascent C. elegans-Orsay virus system to identify and characterize two novel host factors that Orsay virus relies upon for infection, one of which was also found to play an evolutionarily conserved pro-viral role in human cells. First, I identified the C. elegans protein dietary restriction-like 1(DRL-1) as a novel host factor required for Orsay virus infection. Through a chemical mutagenesis screen, based on a virus-inducible green fluorescent protein (GFP) transcriptional reporter, I isolated a C. elegans mutant lacking a functional drl-1 gene. This drl-1 mutant demonstrated a greater than 10,000-fold reduction in Orsay virus RNA levels, which could be rescued by ectopic expression of wild-type DRL-1. Additionally, DRL-1 was dispensable for Orsay RNA replication from an endogenous transgene replicon, suggesting that DRL-1 affects a pre-replication stage of the Orsay life cycle. As drl-1 encodes a putative serine-threonine kinase that shares similarity with human mitogen activated protein kinases (MAPK), it implicates for the first time in C. elegans, a role for a MAPK pathway in viral infection. Second, as the chemical mutagenesis aforementioned was not saturated, I expanded this forward genetic screen, and identified the C. elegans protein huntingtin-interacting protein related 1 (HIPR-1) as another novel host factor required for Orsay virus infection. HIPR-1 belongs to the Huntingtin-interacting protein family, whose members are known to be key factors in clathrin-mediated endocytosis through inositol phospholipid, clathrin light and heavy chain, and F-actin binding. Ablation of HIPR-1 in C. elegans also resulted in a significant defect in viral RNA replication, a defect which could be bypassed through an endogenous transgene replicon system, suggesting a lack of HIPR-1 also affects an early, pre-replication step of the virus life cycle stage. To characterize the functional nature of HIPR-1, I performed a domain analysis and demonstrated that neither the clathrin light chain binding domain nor the clathrin heavy chain binding motif were needed for virus infection, whereas the inositol phospholipid binding and F-actin binding domains were obligatory. In human cell culture, deletion of the human HIPR-1 orthologs, HIP1 and HIP1R, led to decreased infection by Coxsackie B3 virus, demonstrating a conservation of its pro-viral function through evolution. To further establish functional conservation, I determined a chimeric HIPR-1 protein harboring the corresponding human HIP1 inositol phospholipid binding domain was able to rescue Orsay infection in C. elegans. Overall, these results provide novel insights into the mechanism of HIP proteins in virus infection, which appears to be clathrin binding independent.Taken together, I have contributed to the base knowledge of the early C. elegans virology field by expanding our narrow knowledge of cellular factors impacting viral infection in C. elegans. This increase will better enable the community to explore the nature of the virus lifecyle and host-virus interactions in either C. elegans or mammals, or both.
Chair and Committee
Jacco Boon, Michael Diamond, Michael Nonet, Stephen Pak,
Sandoval, Luis Enrique, "Genetic and Functional Characterization of Novel Host Factors Regulating Virus Infection in Caenorhabditis elegans" (2020). Arts & Sciences Electronic Theses and Dissertations. 2341.