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.

Keywords: Caenorhabditis elegans; Orsay virus; TNK2; WASP; sid-3; viro-2.

FIG 1

A forward genetic screen identified mutant strains defective in GFP expression and Orsay virus replication. (A) GFP reporter expression patterns of Viro mutants after Orsay virus or N. parisii infection. BF, bright-field microscopy. Bars, 200 μm. (B) Orsay virus RNA levels quantified by real-time qRT-PCR. Values are means plus standard deviations (error bars) for three replicate wells. Values that were significantly different (P < 0.0005) are indicated by a bar and asterisks (****). Values that were not significantly different (P > 0.05) (NS) are indicated.

FIG 2

Impact of ectopic expression of SID-3 in sid-3(vir9) mutant on Orsay virus replication and GFP expression. (A) Neighbor-joining phylogenetic tree of SID-3 orthologs from humans (Homo sapiens) and multiple model organisms. The model organisms include Gallus gallus, Danio rerio, Xenopus tropicalis, Drosophila melanogaster, Saccharomyces pombe, and Mus musculus. The bar indicates the amino acid substitution rate (0.1 amino acid substitution per position). (B) Schematic representation of protein domain organization of C. elegans SID-3 and human TNK2. (C) Imaging of Orsay virus-infected sid-3(vir9) mutant that overexpressed wild-type SID-3 or the K139A kinase catalytic domain mutant. BF, bright-field microscopy. Bars, 200 μm. (D) Real-time qRT-PCR of Orsay virus-infected sid-3(vir9) mutant that overexpressed wild-type SID-3 or the K139A kinase catalytic domain mutant. **, P < 0.01; NS, not significant (P > 0.05).

FIG 3

RNAi knockdown of candidate causal genes in drh-1 mutant strain. Real-time qRT-PCR of Orsay virus RNA levels after feeding RNAi knockdown of individual genes. ***, P < 0.005; NS, not significant (P > 0.05).

FIG 4

Infection of strains carrying independent mutant alleles of sid-3 and viro-2. (A) GFP expression patterns of reporter strains that carry independent mutant alleles of sid-3 and viro-2. BF, bright-field microscopy. Bars, 200 μm. (B) Quantification of Orsay virus RNA levels by real-time qRT-PCR. *****, P < 0.00005.

FIG 5

Impact of ectopic expression of VIRO-2 in the viro-2(vir10) mutant on Orsay virus replication and GFP expression. (A) Neighbor-joining phylogenetic tree of VIRO-2 orthologs from human and multiple model organisms. The bar indicates the amino acid substitution rate (0.1 amino acid substitution per position). (B) Schematic representation of protein domain organization for C. elegans VIRO-2 and human N-WASP. (C) GFP expression after ectopic VIRO-2 expression in Viro-2. BF, bright-field microscopy. Bars, 200 μm. (D) Real-time qRT-PCR of Orsay virus-infected viro-2(vir10) mutant that overexpressed VIRO-2. **, P < 0.01.

FIG 6

Impact of sid-3 and viro-2 on Orsay virus RNA1 transgene-initiated replication. (A) Imaging of heat-induced strains carrying either wild-type or mutant Orsay virus RNA1 in the jyIs8;rde-1 background. BF, bright-field microscopy. Bars, 200 μm. (B) Imaging of heat-induced strains carrying either wild-type or mutant Orsay virus RNA1 in the sid-3(vir9) background. Bars, 200 μm. (C) Quantification of Orsay RNA1 replication from heat-induced transgenic C. elegans. To control for possible variability in the copy number of the extrachromosomal arrays, two independent lines for each plasmid construct were tested. NS, not significant (P > 0.05).

FIG 7

SID-3 and VIRO-2 colocalize in transgenic strains. (A and B) Confocal microscopy of a representative animal of the rde-1(ne219) strain (sid-3::YFP and viro-2::mCherry fusion proteins driven by the universal sur-5 gene promoter) 48 h after Orsay virus infection. BF, bright-field microscopy. Bars, 10 μm. The zoomed-in area shown in the micrograph in panel B is indicated by the magenta box in panel A. The graph in panel B shows fluorescence intensity (in AU [arbitrary unit]) on the y axis and distance (in nm) on the x axis. The graph shows fluorescence intensity profiling of the position marked by the magenta arrow. The intestinal apical side is indicated in the graph by a black bar.

FIG 8

RNAi knockdown of candidate genes. Real-time qRT-PCR of Orsay virus RNA levels after feeding RNAi knockdown of individual genes. *, P < 0.05; NS, not significant (P > 0.05).