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Cited by 1interferon and immune responsesRNA Interference and Gene DeliveryPlant Virus Research Studies

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Characterizing the role of RSD-6 in the biogenesis of virus-derived small interfering RNAs and the modulation of viral pathogenesis

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Dahal, Krishna, Xia, Mingli, Lu, Jinfeng, Yan, Teng, Lu, Rui·2026·Journal of Virology
DOI 10.1128/jvi.01516-25PMC PMC12424992

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Abstract

Small interfering RNAs (siRNAs) produced through the processing of viral double-stranded RNAs mediate potent antiviral RNA interference (RNAi) in eukaryotes. In Caenorhabditis elegans , such an antiviral defense is further amplified through the production of secondary siRNAs, yet the mechanisms by which secondary virus-derived siRNAs (vsiRNAs) confer protection remain poorly understood. Here, we characterize the role of rsd-6 , which encodes a Tudor domain protein and plays an important role in antiviral RNAi, in vsiRNA biogenesis and the modulation of viral pathogenesis. Using CRISPR Cas9-generated rsd-6 null mutants, we show that both primary and secondary vsiRNAs accumulate normally in the absence of RSD-6, indicating that it functions downstream of secondary vsiRNA biogenesis. We further showed that secondary vsiRNAs generated in an rrf-1 -independent manner remained detected in the absence of RSD-6 and that viral replication is further enhanced in rrf-1;rsd-6 double mutants compared to rrf-1 single mutants, suggesting a role of rsd-6 in mediating antiviral guided by all secondary vsiRNAs. Consistently, rsd-6 mutants exhibited more severe pathogenesis upon Orsay virus infection compared to rrf-1 mutants, underscoring its role as a major determinant of viral disease outcome. Domain characterization established that the N-terminal tandem domains of RSD-6 are required for antiviral activity, while the C-terminal Tudor domains are dispensable. Functional conservation was confirmed in C. briggsae , where silencing of the rsd-6 homolog enhanced viral replication. Together, our findings identify RSD-6 as a key effector acting downstream of secondary vsiRNA production and highlight its conserved role in modulating viral replication and pathogenesis across Caenorhabditis species. IMPORTANCE: In C. elegans , the RNAi-mediated antiviral defense relies on the production of secondary virus-derived siRNAs (vsiRNAs) to achieve an amplified antiviral effect. However, the mechanism by which these secondary vsiRNAs confer protection remains poorly understood. This is primarily due to the limited number of identified key effector genes. To address this knowledge gap, we profiled vsiRNA biogenesis in loss-of-function mutants and discovered that rsd-6 is dispensable for the production of secondary vsiRNAs, suggesting a role of rsd-6 in mediating antiviral defense downstream of secondary vsiRNA biogenesis. Worm survival assay further confirmed that rsd-6 is a critical modulator of viral pathogenesis and its antiviral function is conserved across Caenorhabditis species. The RSD-6 protein features three N-terminal tandem domains of unknown function and two tandem Tudor domains at its C-terminus. Our domain analyses demonstrated that the N-terminal tandem domains, but not the C-terminal Tudor domains, are essential for antiviral function. The identification of rsd-6 as a key effector gene acting downstream of vsiRNA biogenesis provides a solid foundation for elucidating the mechanism of antiviral RNAi amplification.

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Figures

Figure 1. Generation and characterization of an rsd-6 null allele using genome editing. A. Schematic representation of the wild-type and mutant alleles of the rsd-6 gene. Exons are shown as solid boxes; introns and untranslated regions are represented by lines. The predicted protein sizes from conceptual translation are indicated for each allele. B. Upper panel: Northern blot detection of FR1gfp genomic and subgenomic RNAs in wild-type N2 and rsd-6 mutant (kd622)) strains. A labeled cDNA probe derived from the GFP coding sequence was used.in this northern blotting assay. Lower panel: Quantification of FR1gfp subgenomic RNA3 levels from the northern blot using ImageJ. C. RT-qPCR quantification of FR1gfp genomic RNA1 accumulation in wild-type and rsd-6(kd622) mutants. A primer pair specific for FR1gfp RNA1 was used. Amplification of the endogenous act-1 gene served as an internal control. ****, P < 0.00001.***, p < 0.0001. *, P < 0.01. D. Upper panel: Schematic of the rsd-6 transgene under control of a heat-inducible promoter (HIP). UTR: 3′ untranslated region of unc-54. Lower panel: Overexpression of wildtype rsd-6 rescues antiviral RNAi in rsd-6(kd622) mutants (with red fluorescence in head). The image was recorded 24 hours post heat induction.
Figure 1. Generation and characterization of an rsd-6 null allele using genome editing. A. Schematic representation of the wild-type and mutant alleles of the rsd-6 gene. Exons are shown as solid boxes; introns and untranslated regions are represented by lines. The predicted protein sizes from conceptual translation are indicated for each allele. B. Upper panel: Northern blot detection of FR1gfp genomic and subgenomic RNAs in wild-type N2 and rsd-6 mutant (kd622)) strains. A labeled cDNA probe derived from the GFP coding sequence was used.in this northern blotting assay. Lower panel: Quantification of FR1gfp subgenomic RNA3 levels from the northern blot using ImageJ. C. RT-qPCR quantification of FR1gfp genomic RNA1 accumulation in wild-type and rsd-6(kd622) mutants. A primer pair specific for FR1gfp RNA1 was used. Amplification of the endogenous act-1 gene served as an internal control. ****, P < 0.00001.***, p < 0.0001. *, P < 0.01. D. Upper panel: Schematic of the rsd-6 transgene under control of a heat-inducible promoter (HIP). UTR: 3′ untranslated region of unc-54. Lower panel: Overexpression of wildtype rsd-6 rescues antiviral RNAi in rsd-6(kd622) mutants (with red fluorescence in head). The image was recorded 24 hours post heat induction.CC BY-NC-ND · PubMed Central
Figure 2. Quantification of siRNAs derived from Orsay virus genomic RNA1 in different genetic backgrounds. A. Left panel: Northern blot analysis of Orsay virus genomic RNA1 in the indicated C. elegans strains. An alkaline phosphatase-labeled cDNA probe specific to Orsay virus RNA1 was used. Ribosomal RNA stained with methylene blue served as a loading control. Right panel: Quantification of Orsay virus RNA1 levels from the northern blot using ImageJ. B. Size distribution and 5′ nucleotide composition of Orsay virus RNA1-derived siRNAs in the same worm strains shown in panel A. C. Size distribution and 5′ nucleotide composition of Orsay virus RNA1-derived siRNAs following treatment with Terminator phosphatase to clone secondary vsiRNAs with 5′ tri-phosphorylate during sequencing library construction. Worm strains analyzed are the same as in panel A. D. Left panel: Quantification of 22–24 nt Orsay virus RNA1-derived primary siRNAs in the same worm strains as in panel A. Right panel: Quantification of secondary siRNAs derived from Orsay virus RNA1 in the same strains. Read counts were normalized to one million total miRNAs.
Figure 2. Quantification of siRNAs derived from Orsay virus genomic RNA1 in different genetic backgrounds. A. Left panel: Northern blot analysis of Orsay virus genomic RNA1 in the indicated C. elegans strains. An alkaline phosphatase-labeled cDNA probe specific to Orsay virus RNA1 was used. Ribosomal RNA stained with methylene blue served as a loading control. Right panel: Quantification of Orsay virus RNA1 levels from the northern blot using ImageJ. B. Size distribution and 5′ nucleotide composition of Orsay virus RNA1-derived siRNAs in the same worm strains shown in panel A. C. Size distribution and 5′ nucleotide composition of Orsay virus RNA1-derived siRNAs following treatment with Terminator phosphatase to clone secondary vsiRNAs with 5′ tri-phosphorylate during sequencing library construction. Worm strains analyzed are the same as in panel A. D. Left panel: Quantification of 22–24 nt Orsay virus RNA1-derived primary siRNAs in the same worm strains as in panel A. Right panel: Quantification of secondary siRNAs derived from Orsay virus RNA1 in the same strains. Read counts were normalized to one million total miRNAs.CC BY-NC-ND · PubMed Central
Figure 3. RSD-6 contributes to the function of virus-derived secondary siRNAs. A, Upper panel, northern blot detection of OrV genomic RNA1 in the indicated C. elegans strains. Experimental conditions were as described in Figure 2A. Lower panel, quantification of OrV RNA1 levels from the upper panel, analyzed using ImageJ. B, Size distribution and 5′ nucleotide composition of OrV RNA1-derived vsiRNAs in the indicated double mutants. Data represent direct sequencing of small RNAs (primary siRNAs). C, Size distribution and 5′ nucleotide composition of OrV RNA1-derived vsiRNAs in the same double mutants as in (A), but with Terminator phosphatase-treated small RNA samples to detect secondary siRNAs. D, Left panel, quantification of 22–24 nt OrV RNA1-derived primary vsiRNAs in the indicated strains. Read counts were normalized to one million total miRNA reads. Right panel, quantification of OrV RNA1-derived secondary vsiRNAs in the indicated strains. Secondary siRNA reads were similarly normalized to one million total miRNAs.
Figure 3. RSD-6 contributes to the function of virus-derived secondary siRNAs. A, Upper panel, northern blot detection of OrV genomic RNA1 in the indicated C. elegans strains. Experimental conditions were as described in Figure 2A. Lower panel, quantification of OrV RNA1 levels from the upper panel, analyzed using ImageJ. B, Size distribution and 5′ nucleotide composition of OrV RNA1-derived vsiRNAs in the indicated double mutants. Data represent direct sequencing of small RNAs (primary siRNAs). C, Size distribution and 5′ nucleotide composition of OrV RNA1-derived vsiRNAs in the same double mutants as in (A), but with Terminator phosphatase-treated small RNA samples to detect secondary siRNAs. D, Left panel, quantification of 22–24 nt OrV RNA1-derived primary vsiRNAs in the indicated strains. Read counts were normalized to one million total miRNA reads. Right panel, quantification of OrV RNA1-derived secondary vsiRNAs in the indicated strains. Secondary siRNA reads were similarly normalized to one million total miRNAs.CC BY-NC-ND · PubMed Central
Figure 4. Functional domain characterization of RSD-6. (A) Schematic domain structure and predicted molecular weight (kDa) of RSD-6 and its derivatives. SN: staphylococcal nuclease domain; Tudor: Tudor domain; cbrRSD-6: C. briggsae RSD-6 ortholog. (B) Western blot analysis of HA-tagged RSD-6 and its derivatives as indicated. No HI, no heat induction; * indicates non-induced samples). (C) Quantitative analysis of FR1gfp replicon RNA1 accumulation by RT-qPCR in rsd-6(kd622) mutants expressing either wild-type RSD-6 or its derivatives. All strains contained both the FR1gfp replicon transgene and rsd-6(kd622) null allele. Statistical significance: ***, p < 0.0001, ****, p < 0.00001.
Figure 4. Functional domain characterization of RSD-6. (A) Schematic domain structure and predicted molecular weight (kDa) of RSD-6 and its derivatives. SN: staphylococcal nuclease domain; Tudor: Tudor domain; cbrRSD-6: C. briggsae RSD-6 ortholog. (B) Western blot analysis of HA-tagged RSD-6 and its derivatives as indicated. No HI, no heat induction; * indicates non-induced samples). (C) Quantitative analysis of FR1gfp replicon RNA1 accumulation by RT-qPCR in rsd-6(kd622) mutants expressing either wild-type RSD-6 or its derivatives. All strains contained both the FR1gfp replicon transgene and rsd-6(kd622) null allele. Statistical significance: ***, p < 0.0001, ****, p < 0.00001.CC BY-NC-ND · PubMed Central
Figure 5. RSD-6 modulates viral pathogenesis in C. elegans. (A) Survival assay of mock-infected C. elegans strains. All worms were maintained on OP50 E. coli food. (B) Survival assay of Orsay virus-infected C. elegans strains as indicated. Animals were challenged with Orsay virus at the L1 stage, and survival rates were monitored every 24 hours post-infection. Statistical significance: *, *p* < 0.02; **, *p* < 0.002; ***, *p* < 0.0002; ****, *p* < 0.00002.
Figure 5. RSD-6 modulates viral pathogenesis in C. elegans. (A) Survival assay of mock-infected C. elegans strains. All worms were maintained on OP50 E. coli food. (B) Survival assay of Orsay virus-infected C. elegans strains as indicated. Animals were challenged with Orsay virus at the L1 stage, and survival rates were monitored every 24 hours post-infection. Statistical significance: *, *p* < 0.02; **, *p* < 0.002; ***, *p* < 0.0002; ****, *p* < 0.00002.CC BY-NC-ND · PubMed Central
Figure 6. The rsd-6 homolog contributes to antiviral immunity in C. briggsae. (A) Northern blot detection of Santeuil virus RNA2 in C. briggsae strains after RNAi knockdown of indicated genes. Total RNA (5 μg per lane) from Santeuil virus-infected animals was probed with cDNA derived from the viral RNA2 segment. JU1018, C. briggsae strain with C. elegans sid-2 transgene. JU1264, original Santeuil virus host. L4440, empty vector; eGFP, dsRNA targeting eGFP. rRNA, methylene blue-stained ribosomal RNA serving as the loading control. svRNA2, Santeuil virus RNA2. B. Quantification of Santeuil virus RNA2 levels from (A) using ImageJ. (C) RT-qPCR quantification of Santeuil virus RNA2 in JU1018 worms fed with dsRNA targeting indicated genes. Data represent mean ± SD of three independent experiments. Statistical analysis: *, p < 0.05; *****, p < 0.00005; ns, not significant.
Figure 6. The rsd-6 homolog contributes to antiviral immunity in C. briggsae. (A) Northern blot detection of Santeuil virus RNA2 in C. briggsae strains after RNAi knockdown of indicated genes. Total RNA (5 μg per lane) from Santeuil virus-infected animals was probed with cDNA derived from the viral RNA2 segment. JU1018, C. briggsae strain with C. elegans sid-2 transgene. JU1264, original Santeuil virus host. L4440, empty vector; eGFP, dsRNA targeting eGFP. rRNA, methylene blue-stained ribosomal RNA serving as the loading control. svRNA2, Santeuil virus RNA2. B. Quantification of Santeuil virus RNA2 levels from (A) using ImageJ. (C) RT-qPCR quantification of Santeuil virus RNA2 in JU1018 worms fed with dsRNA targeting indicated genes. Data represent mean ± SD of three independent experiments. Statistical analysis: *, p < 0.05; *****, p < 0.00005; ns, not significant.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Dahal, K., Xia, M., Lu, J., Yan, T., & Lu, R.
Affiliations
  • Louisiana State University (US)
  • Department of Neurology, The Taub Institute for Research on Alzheimer's Disease and the Aging Brain, Gertrude H. Sergievsky Center, College of Physicians and Surgeons, Columbia University, The New York Presbyterian Hospital
Corresponding author
Lu, Rui
ORCID
Lu, Rui
Venue
Journal of Virology
Publisher
American Society for Microbiology
Published
2026-01-20
Volume
100
Issue
1
Pages
e0151625-e0151625
ISSN
  • 0022-538X (ISSN-L)
  • 1098-5514
DOI
10.1128/jvi.01516-25
PMC
PMC12424992
OpenAlex
W4417304987
Cited by
1
References
46
Citations by year
2026: 1
FWCI
0.37
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Virology (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institutes of Health (1R01GM119012-01A1, 1R03AI171860-01A1)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › interferon and immune responses
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Agricultural and Biological Sciences › Plant Science › Plant Virus Research Studies
Keywords
Tudor domain protein, virus-derived small interfering RNAs, antiviral RNA interference, Caenorhabditis elegans, Caenorhabditis briggsae, Orsay virus, viral pathogenesis, viral replication

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