The Dietary Restriction-Like Gene drl-1, Which Encodes a Putative Serine/Threonine Kinase, Is Essential for Orsay Virus Infection in Caenorhabditis elegans

Abstract

Orsay virus is the only known natural virus pathogen of Caenorhabditis elegans, and its discovery has enabled virus-host interaction studies in this model organism. Host genes required for viral infection remain understudied. We previously established a forward genetic screen based on a virus-inducible green fluorescent protein transcriptional reporter to identify novel host factors essential for virus infection. Here, we report the essential role in Orsay virus infection of the dietary restriction-like (drl-1) gene, which encodes a serine/threonine kinase similar to the mammalian MEKK3 kinase. Ablation of drl-1 led to a >10,000-fold reduction in Orsay virus RNA levels, which could be rescued by ectopic expression of DRL-1. DRL-1 was dispensable for Orsay replication from an endogenous transgene replicon, suggesting that DRL-1 affects a prereplication stage of the Orsay life cycle. Thus, this study demonstrates the power of C. elegans as a model to identify novel virus-host interactions essential for virus infection.IMPORTANCE The recent discovery of Orsay virus, the only known natural virus of Caenorhabditis elegans, provides a unique opportunity to study virus-host interactions that mediate infection in a genetically tractable multicellular model organism. As viruses remain a global threat to human health, better insights into cellular components that enable virus infection and replication can ultimately lead to the development of new targets for antiviral therapeutics.

Keywords: Caenorhabditis elegans; Orsay virus; drl-1.

FIG 1

Chemical mutagenesis screen identified a mutant strain with a defect in GFP expression and viral RNA replication. (A) GFP reporter expression of Viro-4 mutants infected with Orsay virus or N. parisii at 3 days postinfection. (B) Orsay virus RNA2 levels quantified by real-time qRT-PCR at 3 days postinfection. Values are means plus standard deviations for three replicate wells. ****, P < 0.0005.

FIG 2

RNAi knockdown of candidate causal genes in the drh-1 mutant strain. Shown are results of real-time qRT-PCR of Orsay virus RNA2 levels 2 days after Orsay virus infection of animals fed RNAi against independent genes. Values are means plus standard deviations (error bars) for three independent biological experiments, each with three replicate wells. Statistically significant differences were determined by one-way analysis of variance (ANOVA) [F(3,32) = 14.47; P < 0.0001], with statistical difference identified between empty vector and drl-1 (P = 0.0001 by Dunnett’s multiple-comparison test). ****, P < 0.0005; NS, not significant (P > 0.05).

FIG 3

Orsay RNA replication and GFP expression patterns in Viro-4 mutants carrying a fosmid containing the endogenous drl-1 locus or overexpressing DRL-1. (A) GFP response at 3 days postinfection of Viro-4 mutants expressing the drl-1 locus or ectopically expressing DRL-1 infected with Orsay virus. The Pmyo2::YFP plasmid was coinjected as a transgenic marker. (B) Orsay virus RNA2 levels at 3 days postinfection of Viro-4 mutants expressing the drl-1 locus or overexpressing DRL-1 infected with Orsay virus. ***, P < 0.005; ****, P < 0.0005.

FIG 4

Impact of drl-1 on Orsay virus RNA replication induced from a transgene. (A) GFP expression of strains carrying either wild-type or polymerase-dead mutant Orsay virus RNA1 under a heat-inducible promoter in the drl-1(vir11) background 3 days after heat shock. The Pmyo2::YFP plasmid was coinjected as a transgenic marker. (B) Quantification of Orsay virus RNA1 replication 3 days after heat shock by real-time qRT-PCR. *, P < 0.05.