An Alternative STAT Signaling Pathway Acts in Viral Immunity in Caenorhabditis elegans

Abstract

Across metazoans, innate immunity is vital in defending organisms against viral infection. In mammals, antiviral innate immunity is orchestrated by interferon signaling, activating the STAT transcription factors downstream of the JAK kinases to induce expression of antiviral effector genes. In the nematode Caenorhabditis elegans, which lacks the interferon system, the major antiviral response so far described is RNA interference (RNAi), but whether additional gene expression responses are employed is not known. Here we show that, despite the absence of both interferon and JAK, the C. elegans STAT homolog STA-1 orchestrates antiviral immunity. Intriguingly, mutants lacking STA-1 are less permissive to antiviral infection. Using gene expression analysis and chromatin immunoprecipitation, we show that, in contrast to the mammalian pathway, STA-1 acts mostly as a transcriptional repressor. Thus, STA-1 might act to suppress a constitutive antiviral response in the absence of infection. Additionally, using a reverse genetic screen, we identify the kinase SID-3 as a new component of the response to infection, which, along with STA-1, participates in the transcriptional regulatory network of the immune response. Our work uncovers novel physiological roles for two factors in viral infection: a SID protein acting independently of RNAi and a STAT protein acting in C. elegans antiviral immunity. Together, these results illustrate the complex evolutionary trajectory displayed by innate immune signaling pathways across metazoan organisms.IMPORTANCE Since innate immunity was discovered, a diversity of pathways has arisen as powerful first-line defense mechanisms to fight viral infection. RNA interference, reported mostly in invertebrates and plants, as well as the mammalian interferon response and JAK/STAT pathway are key in RNA virus innate immunity. We studied infection by the Orsay virus in Caenorhabditis elegans, where RNAi is known to be a potent antiviral defense. We show that, in addition to its RNAi pathway, C. elegans utilizes an alternative STAT pathway to control the levels of viral infection. We identify the transcription factor STA-1 and the kinase SID-3 as two components of this response. Our study defines C. elegans as a new example of the diversity of antiviral strategies.

Keywords: Caenorhabditis elegans; Orsay virus; RNA virus; STAT signaling; innate immunity.

FIG 1

Identification of a STAT transcription factor signature in immune response against the Orsay virus (OrV) in C. elegans. (A) Schematic representation of known antiviral pathways in C. elegans. (B) MEME motif enrichment in genes regulated upon infection and conservation of the de novo-identified motif by Tom-tom. ID, identifier. (C) Schematic representation of conserved domains between the human STAT-1 and C. elegans STATs. The red bars depict the predicted phosphorylation sites (20). aa, amino acids. (D) Phylogenetic analysis of STAT transcription factors. Nematodes are indicated in blue, and vertebrates are indicated in red. Trichinella spiralis, Brugia malayi, Caenorhabditis briggsae, Drosophila melanogaster, Homo sapiens, and Strongylocentrotus purpuratus STATs are shown on the tree. The tree is based on full-length sequences. Alignment was performed with Muscle. Branch support values are from bootstrap using 1,000 iterations. Only values lower than 0.8 are depicted. The bar shows a branch length of 0.7 nucleotide substitutions per position.

FIG 2

STA-1 is a key transcription factor in the immune response upon Orsay virus (OrV) infection. (A, C, and D) Viral loads in different strains measured by RT-qPCR on the Orsay virus RNA1 genome after 3 days of infection. The wild-type (WT) control N2 strain, rde-1(ne219) strain, and strains with sta-1(ok587) and sta-2(ok1860) mutations were infected. Values that were significantly different (P < 0.01) from the value for the wild-type control strain (unless otherwise indicated) by a two-tailed Mann-Whitney U test are indicated with an asterisk. Values that were not significantly different (ns) are indicated. n = 6 for panels A and C, and n = 4 for panel D. (B) Expression of a single-copy transgene coding for a GFP::STA-1 fusion protein under the control of a sur-5 promoter in adult animals. DIC, differential interference contrast.

FIG 3

STA-1 acts at the promoter of virus response genes and represses their expression. (A) Schematic representation of the RNA-seq experiment performed with strain N2 and sta-1(ok587) animals. (B) Representation of the number of genes showing differential expression (DE) upon infection in strain N2 (N2 inf), infection in sta-1 mutant animals, or infection in sta-1 mutants compared to N2, as measured by RNA-seq. (C) Gene expression measured by RNA-seq in a sta-1 mutant animals normalized to the N2 control. All genes or a subset of genes that show upregulation upon infection in N2 animals are depicted. The box shows the interquartile range, and the whiskers extend to the greatest value ≤1.5 times the interquartile range from the box. The significance of the enrichment is represented as the P value of the Wilcoxon unpaired test. (D) ChIP-sequencing after immunoprecipitation of the GFP::STA-1 transgene. (Top) The ChIP signal is plotted 1 kb upstream and 1 kb downstream of all genes anchored at TSS as in references and . The heatmap was generated using k-means clustering (three clusters, C1 [cluster 1], C2, and C3]) of all transcription start sites (TSS) using the GFP::STA-1 signal for clustering. (E) Motif identification and conservation by Centrimo (54) for the 1-kb sequence centered around the summits of the peaks in cluster 2 (C2) in panel D. The asterisk indicates that the IRF1 motif identified by Tom-tom matches the reverse complement of the motif found. (F) Example of a upregulated sta-1 gene showing binding of STA-1 by ChIP-seq in two independent biological replicates (rep1 [replicate 1 ] and rep2). The putative STA-1 binding motif is indicated. (G) ChIP peak enrichment in different sets of genes. The three sets of genes were genome-wide genes (all genes), genes upregulated in sta-1 versus strain N2 (sta-1 up), and genes differentially expressed upon infection and upregulated in sta-1 (sta-1 up and infection).

FIG 4

STA-1 is required for normal life span. (A) Survival plot showing the relationship between the survival and age of the wild-type N2 and sta-1 mutant animals. (B) Descriptive statistics of the life span experimental data in panel A. The associated log rank test Bonferroni P value is 2.7e−6. std. error, standard error; 95% C.I., 95% confidence interval.

FIG 5

The tyrosine kinase SID-3 enables efficient viral replication. (A) Activation of the JAK/STAT signaling pathway in RNA virus infection in humans. Upon recognition of the viral RNA by a sensor like RIG-I, type 1 interferon (alpha/beta interferon) is released in the environment, recognized by the IFN receptor, and activates the JAK tyrosine kinase to trigger nuclear translocation of activated STAT transcription factors. IFNa/b, alpha/beta interferon; ISG, interferon-stimulated genes; P, phosphate. (B) Overview of the RNAi screen. (C) RNAi treatment leading to increased resistance to OrV infection. The viral RNA accumulation after RNAi treatment is represented by the Z-score of the ΔΔC_T values (relative to empty vector [eV]). The red broken lines represent the 99% confidence interval of the control RNAi (eV), calculated as plus or minus 2.7 standard deviations. The red bars represent the median of the biological replicates for the genes tested or for controls. The plus signs represent outliers. The inset at the top of the graph depicts all tested clones.

FIG 6

SID-3 and STA-1 regulate a common set of genes. (A) Schematic representation of the different sid-3 alleles available. The sid-3 ok973 allele induces a deletion of 1,330 bp and insertion of 12 nucleotides, deleting exons 11 and 12 and part of exon 13, after the tyrosine kinase domain. The sid-3 tm342 allele is a null allele, where an early 821-bp-long deletion (exon 1 to exon 3) and insertion of 7 nucleotides lead to a premature stop codon. chr X, chromosome X. (B and C) Viral load after 3 days of infection, measured by RT-qPCR on the Orsay virus RNA1 genome. *, P < 0.01 by two-tailed Mann-Whitney test. n = 6 for each strain. (D) RNA-seq analysis showing an enrichment for infection genes in sid-3(ok973) upregulated genes. (E) Overlap between genes showing differential expression by RNA-seq upon infection or upregulated in sta-1 or upregulated in sid-3. (F) Overlap between STA-1-bound genes, as shown by ChIP-seq and sid-3 upregulated genes. (G and H) Viral load in the indicated strains, measured by RT-qPCR on the Orsay virus RNA1 genome after 3 days of infection. The sid-3 allele used is sid-3(ok973). In panel G, *, P < 0.01 by two-tailed Mann-Whitney test and n = 6 for each strain. In panel H, *, P < 0.05 by two-tailed Mann-Whitney test and n = 3 for each strain except N2 and n = 4 for strain N2.

FIG 7

A STA-1 pathway controls the response to viral infection in C. elegans, a model.