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

Ashe A, Sarkies P, Le Pen J, Tanguy M, Miska EA - J. Virol. (2015)

Bottom Line: 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.We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research United Kingdom Gurdon Institute, University of Cambridge, Cambridge, United Kingdom Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia ashe@sydney.edu.au eric.miska@gurdon.cam.ac.uk.

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sid-1 and sid-2 are not required for viral resistance. (A) qRT-PCR showing the relative levels of Orsay virus 4 days after infection in N2 and in drh-1, sid-1, and sid-2 mutants. drh-1 mutant animals show significantly higher levels of Orsay virus RNA than N2 animals do (P < 0.05, t test), but there is no significant difference between either sid-1 or sid-2 mutant and N2 animals. Data were normalized to gapdh and then N2. (B) Shown are the 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from panel C (sid-1) and Fig. 1 (N2) normalized to library size and the level in the P0 generation. emb, embryo. (C) 5′ independent small RNA sequencing of P0 and F1 sid-1 mutant animals after Orsay virus exposure. P0 animals were assayed as a mixed-stage population of predominantly adults, and F1 animals were synchronized and assayed at three different ages as indicated. Data are shown as sense or antisense and ordered according to the size of the RNA molecule. The values on the y axis are reads per million. The 5′ nucleotide is indicated by color as follows: red, A; green, C; blue, G; pink, U.
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Figure 5: sid-1 and sid-2 are not required for viral resistance. (A) qRT-PCR showing the relative levels of Orsay virus 4 days after infection in N2 and in drh-1, sid-1, and sid-2 mutants. drh-1 mutant animals show significantly higher levels of Orsay virus RNA than N2 animals do (P < 0.05, t test), but there is no significant difference between either sid-1 or sid-2 mutant and N2 animals. Data were normalized to gapdh and then N2. (B) Shown are the 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from panel C (sid-1) and Fig. 1 (N2) normalized to library size and the level in the P0 generation. emb, embryo. (C) 5′ independent small RNA sequencing of P0 and F1 sid-1 mutant animals after Orsay virus exposure. P0 animals were assayed as a mixed-stage population of predominantly adults, and F1 animals were synchronized and assayed at three different ages as indicated. Data are shown as sense or antisense and ordered according to the size of the RNA molecule. The values on the y axis are reads per million. The 5′ nucleotide is indicated by color as follows: red, A; green, C; blue, G; pink, U.

Mentions: Given the limited ability of the antiviral RNAi response generated against the Orsay virus to spread between cells, we hypothesized that systemic RNAi may not be important in defending against viral infection. Supporting this view, Guo et al. recently reported results suggesting that, indeed, Orsay virus accumulated to similar levels in N2 and a sid-1 mutant (35). We confirmed this result and additionally showed that sid-2 is dispensable for viral defense (Fig. 5A). In order to further study the role of sid-1 in viral defense, we assayed the siRNAs present in Orsay-infected animals and their offspring by small RNA sequencing as described above for N2 animals (Fig. 5B and C). We could detect no difference in small RNAs between the sid-1 mutant strain and N2, further confirming that sid-1 transport of mobile siRNAs is not required for their generation in the context of Orsay virus infection.


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

Ashe A, Sarkies P, Le Pen J, Tanguy M, Miska EA - J. Virol. (2015)

sid-1 and sid-2 are not required for viral resistance. (A) qRT-PCR showing the relative levels of Orsay virus 4 days after infection in N2 and in drh-1, sid-1, and sid-2 mutants. drh-1 mutant animals show significantly higher levels of Orsay virus RNA than N2 animals do (P < 0.05, t test), but there is no significant difference between either sid-1 or sid-2 mutant and N2 animals. Data were normalized to gapdh and then N2. (B) Shown are the 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from panel C (sid-1) and Fig. 1 (N2) normalized to library size and the level in the P0 generation. emb, embryo. (C) 5′ independent small RNA sequencing of P0 and F1 sid-1 mutant animals after Orsay virus exposure. P0 animals were assayed as a mixed-stage population of predominantly adults, and F1 animals were synchronized and assayed at three different ages as indicated. Data are shown as sense or antisense and ordered according to the size of the RNA molecule. The values on the y axis are reads per million. The 5′ nucleotide is indicated by color as follows: red, A; green, C; blue, G; pink, U.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4645334&req=5

Figure 5: sid-1 and sid-2 are not required for viral resistance. (A) qRT-PCR showing the relative levels of Orsay virus 4 days after infection in N2 and in drh-1, sid-1, and sid-2 mutants. drh-1 mutant animals show significantly higher levels of Orsay virus RNA than N2 animals do (P < 0.05, t test), but there is no significant difference between either sid-1 or sid-2 mutant and N2 animals. Data were normalized to gapdh and then N2. (B) Shown are the 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from panel C (sid-1) and Fig. 1 (N2) normalized to library size and the level in the P0 generation. emb, embryo. (C) 5′ independent small RNA sequencing of P0 and F1 sid-1 mutant animals after Orsay virus exposure. P0 animals were assayed as a mixed-stage population of predominantly adults, and F1 animals were synchronized and assayed at three different ages as indicated. Data are shown as sense or antisense and ordered according to the size of the RNA molecule. The values on the y axis are reads per million. The 5′ nucleotide is indicated by color as follows: red, A; green, C; blue, G; pink, U.
Mentions: Given the limited ability of the antiviral RNAi response generated against the Orsay virus to spread between cells, we hypothesized that systemic RNAi may not be important in defending against viral infection. Supporting this view, Guo et al. recently reported results suggesting that, indeed, Orsay virus accumulated to similar levels in N2 and a sid-1 mutant (35). We confirmed this result and additionally showed that sid-2 is dispensable for viral defense (Fig. 5A). In order to further study the role of sid-1 in viral defense, we assayed the siRNAs present in Orsay-infected animals and their offspring by small RNA sequencing as described above for N2 animals (Fig. 5B and C). We could detect no difference in small RNAs between the sid-1 mutant strain and N2, further confirming that sid-1 transport of mobile siRNAs is not required for their generation in the context of Orsay virus infection.

Bottom Line: 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.We conclude that systemic or transgenerational RNAi does not play a role in the defense against natural Orsay virus infection.

View Article: PubMed Central - PubMed

Affiliation: Wellcome Trust Cancer Research United Kingdom Gurdon Institute, University of Cambridge, Cambridge, United Kingdom Department of Biochemistry, University of Cambridge, Cambridge, United Kingdom School of Molecular Bioscience, University of Sydney, Sydney, NSW, Australia ashe@sydney.edu.au eric.miska@gurdon.cam.ac.uk.

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Related in: MedlinePlus