An Evolutionarily Conserved Pathway Essential for Orsay Virus Infection of Caenorhabditis elegans

ABSTRACT Many fundamental biological discoveries have been made in Caenorhabditis elegans. The discovery of Orsay virus has enabled studies of host-virus interactions in this model organism. To identify host factors critical for Orsay virus infection, we designed a forward genetic screen that utilizes a virally induced green fluorescent protein (GFP) reporter. Following chemical mutagenesis, two Viro (virus induced reporter off) mutants that failed to express GFP were mapped to sid-3, a nonreceptor tyrosine kinase, and B0280.13 (renamed viro-2), an ortholog of human Wiskott-Aldrich syndrome protein (WASP). Both mutants yielded Orsay virus RNA levels comparable to that of the residual input virus, suggesting that they are not permissive for Orsay virus replication. In addition, we demonstrated that both genes affect an early prereplication stage of Orsay virus infection. Furthermore, it is known that the human ortholog of SID-3, activated CDC42-associated kinase (ACK1/TNK2), is capable of phosphorylating human WASP, suggesting that VIRO-2 may be a substrate for SID-3 in C. elegans. A targeted RNA interference (RNAi) knockdown screen further identified the C. elegans gene nck-1, which has a human ortholog that interacts with TNK2 and WASP, as required for Orsay virus infection. Thus, genetic screening in C. elegans identified critical roles in virus infection for evolutionarily conserved genes in a known human pathway. IMPORTANCE Orsay virus is the only known virus capable of naturally infecting the model organism Caenorhabditis elegans, which shares many evolutionarily conserved genes with humans. We exploited the robust genetic tractability of C. elegans to identify three host genes, sid-3, viro-2, and nck-1, which are essential for Orsay virus infection. Mutant animals that lack these three genes are highly defective in viral replication. Strikingly, the human orthologs of these three genes, activated CDC42-associated kinase (TNK2), Wiskott-Aldrich syndrome protein (WASP), and noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) are part of a known signaling pathway in mammals. These results suggest that TNK2, WASP, and NCK1 may play important roles in mammalian virus infection. IMPORTANCE Orsay virus is the only known virus capable of naturally infecting the model organism Caenorhabditis elegans, which shares many evolutionarily conserved genes with humans. We exploited the robust genetic tractability of C. elegans to identify three host genes, sid-3, viro-2, and nck-1, which are essential for Orsay virus infection. Mutant animals that lack these three genes are highly defective in viral replication. Strikingly, the human orthologs of these three genes, activated CDC42-associated kinase (TNK2), Wiskott-Aldrich syndrome protein (WASP), and noncatalytic region of tyrosine kinase adaptor protein 1 (NCK1) are part of a known signaling pathway in mammals. These results suggest that TNK2, WASP, and NCK1 may play important roles in mammalian virus infection.

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Punctuated loci on chromosome IV determine natural variation in Orsay virus susceptibility of Caenorhabditis elegans strains Bristol N2 and Hawaiian CB4856

Journal of Virology ◽

10.1128/jvi.02430-20 ◽

2021 ◽

Author(s):

Mark G. Sterken ◽

Lisa van Sluijs ◽

Yiru A. Wang ◽

Wannisa Ritmahan ◽

Mitra L. Gultom ◽

...

Keyword(s):

Genetic Variation ◽

Caenorhabditis Elegans ◽

Virus Infection ◽

Genetic Architecture ◽

Recombinant Inbred Lines ◽

Model Organism ◽

C Elegans ◽

Viral Susceptibility ◽

Orsay Virus ◽

Insight Into

Host-pathogen interactions play a major role in evolutionary selection and shape natural genetic variation. The genetically distinct Caenorhabditis elegans strains, Bristol N2 and Hawaiian CB4856, are differentially susceptible to the Orsay virus (OrV). Here we report the dissection of the genetic architecture of susceptibility to OrV infection. We compare OrV infection in the relatively resistant wild-type CB4856 strain to the more susceptible canonical N2 strain. To gain insight into the genetic architecture of viral susceptibility, 52 fully sequenced recombinant inbred lines (CB4856 x N2 RILs) were exposed to OrV. This led to the identification of two loci on chromosome IV associated with OrV resistance. To verify the two loci and gain additional insight into the genetic architecture controlling virus infection, introgression lines (ILs) that together cover chromosome IV, were exposed to OrV. Of the 27 ILs used, 17 had an CB4856 introgression in an N2 background and 10 had an N2 introgression in a CB4856 background. Infection of the ILs confirmed and fine-mapped the locus underlying variation in OrV susceptibility and we found that a single nucleotide polymorphism in cul-6 may contribute to the difference in OrV susceptibility between N2 and CB4856. An allele swap experiment showed the strain CB4856 became as susceptible as the N2 strain by having an N2 cul-6 allele, although having the CB4856 cul-6 allele did not increase resistance in N2. Additionally, we found that multiple strains with non-overlapping introgressions showed a distinct infection phenotype from the parental strain, indicating that there are punctuated locations on chromosome IV determining OrV susceptibility. Thus, our findings reveal the genetic complexity of OrV susceptibility in C. elegans and suggest that viral susceptibility is governed by multiple genes. Importance Genetic variation determines the viral susceptibility of hosts. Yet, pinpointing which genetic variants determine viral susceptibility remains challenging. Here, we have exploited the genetic tractability of the model organism C. elegans to dissect the genetic architecture of Orsay virus infection. Our results provide novel insight into natural determinants of Orsay virus infection.

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Heat Stress Reduces the Susceptibility of Caenorhabditis elegans to Orsay Virus Infection

Genes ◽

10.3390/genes12081161 ◽

2021 ◽

Vol 12 (8) ◽

pp. 1161

Author(s):

Yuqing Huang ◽

Mark G. Sterken ◽

Koen van Zwet ◽

Lisa van Sluijs ◽

Gorben P. Pijlman ◽

...

Keyword(s):

Heat Stress ◽

Caenorhabditis Elegans ◽

Virus Infection ◽

Intracellular Pathogen ◽

Potential Candidate ◽

Molecular Responses ◽

C Elegans ◽

Pathogen Response ◽

Orsay Virus ◽

Natural Virus

The nematode Caenorhabditis elegans has been a versatile model for understanding the molecular responses to abiotic stress and pathogens. In particular, the response to heat stress and virus infection has been studied in detail. The Orsay virus (OrV) is a natural virus of C. elegans and infection leads to intracellular infection and proteostatic stress, which activates the intracellular pathogen response (IPR). IPR related gene expression is regulated by the genes pals-22 and pals-25, which also control thermotolerance and immunity against other natural pathogens. So far, we have a limited understanding of the molecular responses upon the combined exposure to heat stress and virus infection. We test the hypothesis that the response of C. elegans to OrV infection and heat stress are co-regulated and may affect each other. We conducted a combined heat-stress-virus infection assay and found that after applying heat stress, the susceptibility of C. elegans to OrV was decreased. This difference was found across different wild types of C. elegans. Transcriptome analysis revealed a list of potential candidate genes associated with heat stress and OrV infection. Subsequent mutant screens suggest that pals-22 provides a link between viral response and heat stress, leading to enhanced OrV tolerance of C. elegans after heat stress.

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Caenorhabditis elegans as an Emerging Model for Virus-Host Interactions

Journal of Virology ◽

10.1128/jvi.00509-17 ◽

2017 ◽

Vol 91 (23) ◽

Cited By ~ 10

Author(s):

Don B. Gammon

Keyword(s):

Caenorhabditis Elegans ◽

Virus Infection ◽

Fungal Pathogens ◽

Model Organism ◽

Artificial Infection ◽

Viral Pathogen ◽

Content Type ◽

Host Interactions ◽

C Elegans ◽

Infection Models

ABSTRACT Since 1999, Caenorhabditis elegans has been extensively used to study microbe-host interactions due to its simple culture, genetic tractability, and susceptibility to numerous bacterial and fungal pathogens. In contrast, virus studies have been hampered by a lack of convenient virus infection models in nematodes. The recent discovery of a natural viral pathogen of C. elegans and development of diverse artificial infection models are providing new opportunities to explore virus-host interplay in this powerful model organism.

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Balancing selection of the Intracellular Pathogen Response in natural Caenorhabditis elegans populations

10.1101/579151 ◽

2019 ◽

Author(s):

Lisa van Sluijs ◽

Kobus J. Bosman ◽

Frederik Pankok ◽

Tatiana Blokhina ◽

Joost A. G. Riksen ◽

...

Keyword(s):

Genetic Variation ◽

Caenorhabditis Elegans ◽

Balancing Selection ◽

Model Organism ◽

Evolutionary Strategies ◽

Intracellular Pathogen ◽

C Elegans ◽

Pathogen Response ◽

Orsay Virus ◽

Selection Of

AbstractBackgroundGenetic variation in host populations may lead to differential viral susceptibilities. Here, we investigate the role of natural genetic variation present for an antiviral pathway, the Intracellular Pathogen Response (IPR), underlying susceptibility to Orsay virus in the model organism Caenorhabditis elegans. The IPR involves transcriptional activity of 80 genes including the pals-genes. The pals-genes form an expanded gene family which hints they could be shaped by an evolutionary selective pressure. Here we examine the genetic variation in the pals-family for traces of selection and explore the molecular and phenotypic effects of having distinct pals-gene alleles.ResultsGenetic analysis of 330 world-wide C. elegans strains reveals that genetic diversity within the IPR-related pals-genes can be categorized in a few haplotypes worldwide. Importantly, two key-IPR regulators, pals-22 and pals-25, are in a genomic region carrying signatures of balancing selection. Therefore, distinct pals-22/pals-25 alleles have been maintained in C. elegans populations over time, which suggests different evolutionary strategies exist in IPR regulation. We investigated the IPR by infecting two C. elegans strains that represent distinct pals-22/pals-25 haplotypes, N2 and CB4856, with Orsay virus to determine their susceptibility and transcriptional response to infection. Our data suggests that regulatory genetic variation underlies constant high activity of IPR genes in CB4856 which could determine the host transcriptional defense. We found that CB4856 shows initially lower viral susceptibility than N2. High basal IPR expression levels might help counteract viral infection directly, whereas N2-like strains that need to activate the IPR genes first may have a slower response. Nevertheless, most wild strains harbor N2-like alleles for the pals-genes.ConclusionsOur work provides evidence for balancing genetic selection of immunity genes in C. elegans and illustrated how this may shape the transcriptional defense against pathogens. The transcriptional and genetic data presented in this study therefore provide a novel perspective on the functional diversity that can develop within a main antiviral response in natural host populations.

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Natural Viruses of Caenorhabditis Nematodes

Annual Review of Genetics ◽

10.1146/annurev-genet-112618-043756 ◽

2019 ◽

Vol 53 (1) ◽

pp. 313-326 ◽

Cited By ~ 4

Author(s):

Marie-Anne Félix ◽

David Wang

Keyword(s):

Model Organism ◽

Laboratory Model ◽

Intestinal Cells ◽

Caenorhabditis Briggsae ◽

C Elegans ◽

The Past ◽

Antiviral Genes ◽

Evolutionarily Conserved ◽

Positive Sense ◽

Orsay Virus

Caenorhabditis elegans has long been a laboratory model organism with no known natural pathogens. In the past ten years, however, natural viruses have been isolated from wild-caught C. elegans (Orsay virus) and its relative Caenorhabditis briggsae (Santeuil virus, Le Blanc virus, and Melnik virus). All are RNA positive-sense viruses related to Nodaviridae; they infect intestinal cells and are horizontally transmitted. The Orsay virus capsid structure has been determined and the virus can be reconstituted by transgenesis of the host. Recent use of the Orsay virus has enabled researchers to identify evolutionarily conserved proviral and antiviral genes that function in nematodes and mammals. These pathways include endocytosis through SID-3 and WASP; a uridylyltransferase that destabilizes viral RNAs by uridylation of their 3′ end; ubiquitin protein modifications and turnover; and the RNA interference pathway, which recognizes and degrades viral RNA.

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The Dietary Restriction-Like Genedrl-1, Which Encodes a Putative Serine/Threonine Kinase, Is Essential for Orsay Virus Infection inCaenorhabditis elegans

Journal of Virology ◽

10.1128/jvi.01400-18 ◽

2018 ◽

Vol 93 (3) ◽

Author(s):

Luis Enrique Sandoval ◽

Hongbing Jiang ◽

David Wang

Keyword(s):

Caenorhabditis Elegans ◽

Virus Infection ◽

Dietary Restriction ◽

Model Organism ◽

Serine Threonine Kinase ◽

Content Type ◽

Host Interactions ◽

Threonine Kinase ◽

Orsay Virus ◽

Natural Virus

ABSTRACTOrsay virus is the only known natural virus pathogen ofCaenorhabditis 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 ofdrl-1led 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 ofC. elegansas a model to identify novel virus-host interactions essential for virus infection.IMPORTANCEThe recent discovery of Orsay virus, the only known natural virus ofCaenorhabditis 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.

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The Caenorhabditis elegans ABL-1 Tyrosine Kinase Is Required for Shigella flexneri Pathogenesis

Applied and Environmental Microbiology ◽

10.1128/aem.00558-06 ◽

2006 ◽

Vol 72 (7) ◽

pp. 5043-5051 ◽

Cited By ~ 20

Author(s):

Elizabeth A. Burton ◽

Ann Marie Pendergast ◽

Alejandro Aballay

Keyword(s):

Caenorhabditis Elegans ◽

Tyrosine Kinase ◽

Diarrheal Disease ◽

Shigella Flexneri ◽

Model Organism ◽

Host Susceptibility ◽

Virulence Plasmid ◽

Virulence Determinants ◽

C Elegans

ABSTRACT Shigellosis is a diarrheal disease caused by the gram-negative bacterium Shigella flexneri. Following ingestion of the bacterium, S. flexneri interferes with innate immunity, establishes an infection within the human colon, and initiates an inflammatory response that results in destruction of the tissue lining the gut. Examination of host cell factors required for S. flexneri pathogenesis in vivo has proven difficult due to limited host susceptibility. Here we report the development of a pathogenesis system that involves the use of Caenorhabditis elegans as a model organism to study S. flexneri virulence determinants and host molecules required for pathogenesis. We show that S. flexneri-mediated killing of C. elegans correlates with bacterial accumulation in the intestinal tract of the animal. The S. flexneri virulence plasmid, which encodes a type III secretory system as well as various virulence determinants crucial for pathogenesis in mammalian systems, was found to be required for maximal C. elegans killing. Additionally, we demonstrate that ABL-1, the C. elegans homolog of the mammalian c-Abl nonreceptor tyrosine kinase ABL1, is required for S. flexneri pathogenesis in nematodes. These data demonstrate the feasibility of using C. elegans to study S. flexneri pathogenesis in vivo and provide insight into host factors that contribute to S. flexneri pathogenesis.

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Antiviral RNA Interference against Orsay Virus Is neither Systemic nor Transgenerational in Caenorhabditis elegans

Journal of Virology ◽

10.1128/jvi.03664-14 ◽

2015 ◽

Vol 89 (23) ◽

pp. 12035-12046 ◽

Cited By ~ 26

Author(s):

Alyson Ashe ◽

Peter Sarkies ◽

Jérémie Le Pen ◽

Mélanie Tanguy ◽

Eric A. Miska

Keyword(s):

Caenorhabditis Elegans ◽

Virus Infection ◽

Viral Infection ◽

Rna Virus ◽

Rnai Pathway ◽

Content Type ◽

C Elegans ◽

Systemic Rnai ◽

Endogenous Role ◽

Orsay Virus

ABSTRACTAntiviral RNA-mediated silencing (RNA interference [RNAi]) acts as a powerful innate immunity defense in plants, invertebrates, and mammals. InCaenorhabditis elegans, RNAi is systemic; i.e., RNAi silencing signals can move between cells and tissues. Furthermore, RNAi effects can be inherited transgenerationally and may last for many generations. Neither the biological relevance of systemic RNAi nor transgenerational RNAi is currently understood. Here we examined the role of both pathways in the protection ofC. elegansfrom viral infection. We studied the Orsay virus, a positive-strand RNA virus related toNodaviridaeand the first and only virus known to infectC. elegans. Immunity to Orsay virus infection requires the RNAi pathway. Surprisingly, we found that genes required for systemic or transgenerational RNAi did not have a role in antiviral defense. Furthermore, we found that Orsay virus infection did not elicit a systemic RNAi response even when a target for RNAi was provided by using transgenes. Finally, we show that viral siRNAs, the effectors of RNAi, are not inherited to a level that provides any significant resistance to viral infection in the next generation. We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection. Furthermore, our data suggest that there is a qualitative difference between experimental RNAi and antiviral RNAi. Our data are consistent with a model of systemic and transgenerational RNAi that requires a nuclear or germ line component that is lacking in almost all RNA virus infections.IMPORTANCESince its discovery inCaenorhabditis elegans, RNAi has proven a valuable scientific tool in many organisms. InC. elegans, exogenous RNAi spreads throughout the organism and can be passed between generations; however, there has been controversy as to the endogenous role(s) that the RNAi pathway plays. One endogenous role for which spreading both within the infected organism and between generations would be advantageous is a role in viral defense. In plants, antiviral RNAi is systemic and the spread of RNAi between cells provides protection against subsequent viral infection. Here we investigated this by using the only naturally occurring virus known to infectC. elegans, Orsay virus, and surprisingly found that, in contrast to the exogenous RNAi pathway, the antiviral RNAi response targeted against this virus does not spread systemically throughout the organism and cannot be passed between generations. These results suggest that there are differences between the two pathways that remain to be discovered.

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Behavioral Effects of Volatile Anesthetics in Caenorhabditis elegans

Anesthesiology ◽

10.1097/00000542-199610000-00027 ◽

1996 ◽

Vol 85 (4) ◽

pp. 901-912 ◽

Cited By ~ 38

Author(s):

Michael C. Crowder ◽

Laynie D. Shebester ◽

Tim Schedl

Keyword(s):

Nervous System ◽

Caenorhabditis Elegans ◽

Time Course ◽

Model Organism ◽

Volatile Anesthetic ◽

Volatile Anesthetics ◽

Effective Concentration ◽

Forward Genetics ◽

C Elegans ◽

Median Effective Concentration

Background The nematode Caenorhabditis elegans offers many advantages as a model organism for studying volatile anesthetic actions. It has a simple, well-understood nervous system; it allows the researcher to do forward genetics; and its genome will soon be completely sequenced. C. elegans is immobilized by volatile anesthetics only at high concentrations and with an unusually slow time course. Here other behavioral dysfunctions are considered as anesthetic endpoints in C. elegans. Methods The potency of halothane for disrupting eight different behaviors was determined by logistic regression of concentration and response data. Other volatile anesthetics were also tested for some behaviors. Established protocols were used for behavioral endpoints that, except for pharyngeal pumping, were set as complete disruption of the behavior. Time courses were measured for rapid behaviors. Recovery from exposure to 1 or 4 vol% halothane was determined for mating, chemotaxis, and gross movement. All experiments were performed at 20 to 22 degrees C. Results The median effective concentration values for halothane inhibition of mating (0.30 vol%-0.21 mM), chemotaxis (0.34 vol%-0.24 mM), and coordinated movement (0.32 vol% - 0.23 mM) were similar to the human minimum alveolar concentration (MAC; 0.21 mM). In contrast, halothane produced immobility with a median effective concentration of 3.65 vol% (2.6 mM). Other behaviors had intermediate sensitivities. Halothane's effects reached steady-state in 10 min for all behaviors tested except immobility, which required 2 h. Recovery was complete after exposure to 1 vol% halothane but was significantly reduced after exposure to immobilizing concentrations. Conclusions Volatile anesthetics selectively disrupt C. elegans behavior. The potency, time course, and recovery characteristics of halothane's effects on three behaviors are similar to its anesthetic properties in vertebrates. The affected nervous system molecules may express structural motifs similar to those on vertebrate anesthetic targets.

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Stochastic left–right neuronal asymmetry in Caenorhabditis elegans

Philosophical Transactions of the Royal Society B Biological Sciences ◽

10.1098/rstb.2015.0407 ◽

2016 ◽

Vol 371 (1710) ◽

pp. 20150407 ◽

Cited By ~ 16

Author(s):

Amel Alqadah ◽

Yi-Wen Hsieh ◽

Rui Xiong ◽

Chiou-Fen Chuang

Keyword(s):

Caenorhabditis Elegans ◽

Molecular Mechanisms ◽

Neurological Diseases ◽

Calcium Signalling ◽

Model Organism ◽

Brain Asymmetry ◽

C Elegans ◽

Left And Right ◽

Left Right Asymmetry ◽

Neuronal Asymmetry

Left–right asymmetry in the nervous system is observed across species. Defects in left–right cerebral asymmetry are linked to several neurological diseases, but the molecular mechanisms underlying brain asymmetry in vertebrates are still not very well understood. The Caenorhabditis elegans left and right amphid wing ‘C’ (AWC) olfactory neurons communicate through intercellular calcium signalling in a transient embryonic gap junction neural network to specify two asymmetric subtypes, AWC OFF (default) and AWC ON (induced), in a stochastic manner. Here, we highlight the molecular mechanisms that establish and maintain stochastic AWC asymmetry. As the components of the AWC asymmetry pathway are highly conserved, insights from the model organism C. elegans may provide a window onto how brain asymmetry develops in humans. This article is part of the themed issue ‘Provocative questions in left–right asymmetry’.

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