A deletion polymorphism in the Caenorhabditis elegans RIG-I homolog disables viral RNA dicing and antiviral immunity

Thank you for sending your work entitled “A deletion polymorphism in the C. elegans RIG-I homolog disables viral RNA dicing and antiviral immunity” for consideration at eLife. Your article has been favorably evaluated by a Senior editor (Detlef Weigel) and 2 reviewers.

The Senior editor has assembled the following comments to help you prepare a revised submission.

Ashe and colleagues use an appealing mix of quantitative and molecular genetics to identify and characterize a natural polymorphism in the C. elegans DRH-1 gene that affects production of viral-derived primary siRNAs. The gene emerged from an analysis of natural variation in sensitivity to the Orsay virus, and by a combination of GWAS, introgression mapping, and whole-genome sequencing, the authors managed to pin down a small deletion as a major-effect locus. Using mostly if not exclusively genetics, the authors provide evidence that DRH-1 is likely to specifically recruit the Dicer protein DCR-1 to viral dsRNA substrates. This clearly is a new finding, especially in an organism with a single Dicer. While we find the work very interesting, a minimal set of additional experiments would help support the inferences of the genetic analyses by answering the following questions: 1) Does viral infection affect the expression and subcellular location of DRH-1 protein, as assayed with a GFP fusion?

2) Is there evidence that EXO RNAi is enhanced in drh-1 mutants because absence of DRH-1-bound Dicer would increase Dicer pools? That DRH-1 is not involved in endosiRNA production is consistent with a specific role for SRH-3 in bringing Dicer onto endosiRNA substrates. If the answer to the initial question is yes, this could support the scenario for the possible benefit of the drh-1 deletion in a significant fraction of the wild accessions because of enhanced environmental RNAi (which may confer unknown fitness advantages).

3) A critical experiment for supporting the proposed model would be whether the naturally found truncated form of DRH-1 indeed fails to interact with DCR-1 and RDE-4. We understand that antibodies are not available to test this. Thus, mechanistic inferences about DRH-1 action should be toned down in the revision.

1) Does viral infection affect the expression and subcellular location of DRH-1 protein, as assayed with a GFP fusion?

With regard to changes in drh-1 mRNA levels we have recently published a study analysing gene expression changes upon Orsay virus infection (Sarkies et al., Genome Research 2013). We observed no statistically significant changes in drh-1 transcript levels upon infection in wild-type (N2) animals. Thus drh-1 is unlikely to be transcriptionally regulated upon viral infection.

With regards to the subcellular localization of DRH-1 protein, we agree with the reviewers that the cell biology of viral infection in C. elegans is of great interest, including changes in the subcellular localization of DRH-1 before and after viral infection. However, we do not agree that this analysis relates directly to the manuscript that we have submitted: any change or lack of change in subcellular localization would neither strengthen nor contradict our model. Furthermore, we believe that a thorough analysis of DRH-1 behaviour upon infection would have to include analysis of the subcellular dynamics of the Orsay virus in order to be meaningful. Since we do not fully understand this yet, a combined analysis should be the focus of an independent future study. Nevertheless, we have generated a transgenic line expressing a DRH-1-GFP fusion protein. We found that these transgenic animals localize GFP diffusely in the cytoplasm of intestinal and other cells (Author response image 1 below, transgene driven by a “ubiquitous” promoter, sur-5). We observed that the cytoplasmic localization of GFP does not change in response to infection with the Orsay virus. However, in the absence of a suitable antibody that works in immunofluorescence, we are unable to assess the localization of the endogenous protein, thus we cannot rule out that changes in DRH-1 subcellular localization occur upon infection. We should therefore be cautious about the interpretation of such a negative result and we would prefer not to include these data in the manuscript. We have therefore added a paragraph to the Discussion referencing the gene expression paper, and we have referred to the results of the DRH-1GFP fusion protein analysis as data not shown.

Author response image 1

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2) Is there evidence that EXO RNAi is enhanced in drh-1 mutants because absence of DRH-1-bound Dicer would increase Dicer pools? That DRH-1 is not involved in endosiRNA production is consistent with a specific role for DRH-3 in bringing Dicer onto endosiRNA substrates. If the answer to the initial question is yes, this could support the scenario for the possible benefit of the drh-1 deletion in a significant fraction of the wild accessions because of enhanced environmental RNAi (which may confer unknown fitness advantages).

The reviewers have raised an interesting point. We therefore performed RNAi experiments using a dilution series of unc-22 RNAi (see Materials and methods) and found that exo-RNAi is not enhanced in drh-1 mutants, while it is enhanced in eri-1 mutants. We conclude that there is no evidence that DCR-1 interaction with RDE-4 and DRH-1 limits its availability for exo-RNAi. We have included this data as Figure 4–figure supplement 4. We also added a discussion point to the Discussion.

3) A critical experiment for supporting the proposed model would be whether the naturally found truncated form of DRH-1 indeed fails to interact with DCR-1 and RDE-4. We understand that antibodies are not available to test this. Thus, mechanistic inferences about DRH-1 action should be toned down in the revision.

The truncated DRH-1 (nDf250) might fail to interact with RDE-4/DRH-1, the Orsay RNA, or both. By analogy to RIG-I we suggest that DRH-1 (niDf250) fails to recognize the Orsay RNA. However, we agree with the reviewers: we have toned down our mechanistic inferences by suggesting that DRH-1 may be necessary for either recognition of viral RNA or correct Dicer processing “assisting DCR-1 processing of the double-stranded viral RNA.”