Doctor of Philosophy (PhD)
RNA interference (RNAi) is a wide-spread gene silencing mechanism that control diverse biological functions and triggered by small interfering RNAs (siRNAs) processed from the viral genome or its replication intermediates. Mechanistic studies of antiviral RNAi in Caenorhabditis elegans has led to the identification of several critical factors involved in the process. As a result, whether antiviral RNAi requires additional novel genes remains to be an open question.
Viruses are intracellular parasites that rely on host products for reproduction. Disrupting their interaction with host factors can significantly compromise their replication and keep them under control. Thus, identification of host genes involved in viral genome replication will facilitate the development of antiviral drugs.
In this dissertation, I present how I designed and conducted genome-wide genetic screens to look for novel worm genes required for antiviral immunity and viral genome replication.
To identify novel factors required for antiviral RNAi but not for classical RNAi, a reporter worm strain containing four transgenes corresponding to known antiviral RNAi gene was developed for a biased genetic screen. It was expected that any loss-of-function alleles derived from these four known genes will be automatically rejected during the screen. Altogether 25 candidate alleles were identified and assigned to 2 known antiviral RNAi genes and 11 novel genes. Specifically, rsd-6 was confirmed as one of the candidate genes through mapping-by-sequencing strategy and 2 candidate genes as key requirement of antiviral RNAi but dispensable for classical RNAi. I believe that these 2 candidate genes are novel antiviral RNAi genes since drh-1 is so far the only one that falls into this category and has been excluded during the screen.
To look for worm genes required for Orsay virus genome replication I used a triple mutant that carries the FR1gfp replicon transgene as reporter for loss of viral genome replication. The transgene-mediated viral genome replication also ensures that no false positive mutants will be picked up because of failure in virus genome replication initiation. Altogether 16 candidate alleles were identified and assigned to 12 novel genes. Most importantly, I found that most of these 12 candidate genes also play essential role in directing the genome replication of Orsay virus, which naturally infects C. elegans. To my knowledge, this is the first work that has successfully led to the identification of critical worm factors required for viral genome replication.
Meng, Fei, "Identification of Novel Host Genes Required for Antiviral Immunity and Viral Genome Replication in C. Elegans" (2020). LSU Doctoral Dissertations. 5411.