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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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No evidence of inheritance of viRNAs after Orsay virus infection. (A) The 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from Fig. 1 normalized to library size and the level in the P0 generation. (B) Percentages of P0 and F1 animals displaying the dpy- or unc-encoded phenotype following exposure to RNAi. Error bars show the standard deviation of three (P0) or four (F1) biological replicates. **, P < 0.005; NS, not significant (t test). (C) The experimental design for the data shown in panel D. (D) qRT-PCR data for the relative levels of Orsay virus 4 days after exposure in animals whose parents were either infected with Orsay virus (+V+V) or uninfected (−V+V) (Orsay virus infection of parents was confirmed by qRT-PCR). Data are normalized to gapdh levels and the level of infection of the +V parents. Error bars show the standard error of the mean. (E) Graph showing a comparison of Orsay virus infection levels between liquid- and agar-based infection protocols in both the drh-1 mutant and N2 strains with three different concentrations of virus. Each condition was performed with five biological replicates, and error bars represent the standard error of the mean.
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Figure 2: No evidence of inheritance of viRNAs after Orsay virus infection. (A) The 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from Fig. 1 normalized to library size and the level in the P0 generation. (B) Percentages of P0 and F1 animals displaying the dpy- or unc-encoded phenotype following exposure to RNAi. Error bars show the standard deviation of three (P0) or four (F1) biological replicates. **, P < 0.005; NS, not significant (t test). (C) The experimental design for the data shown in panel D. (D) qRT-PCR data for the relative levels of Orsay virus 4 days after exposure in animals whose parents were either infected with Orsay virus (+V+V) or uninfected (−V+V) (Orsay virus infection of parents was confirmed by qRT-PCR). Data are normalized to gapdh levels and the level of infection of the +V parents. Error bars show the standard error of the mean. (E) Graph showing a comparison of Orsay virus infection levels between liquid- and agar-based infection protocols in both the drh-1 mutant and N2 strains with three different concentrations of virus. Each condition was performed with five biological replicates, and error bars represent the standard error of the mean.

Mentions: To better understand the antiviral RNAi response, we used high-throughput sequencing to assess the small RNAs present in biological duplicates of N2 animals infected with Orsay virus (N2 P0) and compared them with their uninfected offspring (N2 F1) (Fig. 1B to E). As shown previously (30), the N2 P0 sample (Fig. 1B) shows Dicer products mapping both sense and antisense to the viral genome and abundant 22G RNAs mapping antisense to the viral RNA. There are considerably less viral interfering RNAs (viRNAs) in F1 animals than in their parents and they decrease over time (Fig. 1C to E and 2A).


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)

No evidence of inheritance of viRNAs after Orsay virus infection. (A) The 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from Fig. 1 normalized to library size and the level in the P0 generation. (B) Percentages of P0 and F1 animals displaying the dpy- or unc-encoded phenotype following exposure to RNAi. Error bars show the standard deviation of three (P0) or four (F1) biological replicates. **, P < 0.005; NS, not significant (t test). (C) The experimental design for the data shown in panel D. (D) qRT-PCR data for the relative levels of Orsay virus 4 days after exposure in animals whose parents were either infected with Orsay virus (+V+V) or uninfected (−V+V) (Orsay virus infection of parents was confirmed by qRT-PCR). Data are normalized to gapdh levels and the level of infection of the +V parents. Error bars show the standard error of the mean. (E) Graph showing a comparison of Orsay virus infection levels between liquid- and agar-based infection protocols in both the drh-1 mutant and N2 strains with three different concentrations of virus. Each condition was performed with five biological replicates, and error bars represent the standard error of the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: No evidence of inheritance of viRNAs after Orsay virus infection. (A) The 23-nt sense (left) Dicer products and 22G antisense (right) secondary RNAs from Fig. 1 normalized to library size and the level in the P0 generation. (B) Percentages of P0 and F1 animals displaying the dpy- or unc-encoded phenotype following exposure to RNAi. Error bars show the standard deviation of three (P0) or four (F1) biological replicates. **, P < 0.005; NS, not significant (t test). (C) The experimental design for the data shown in panel D. (D) qRT-PCR data for the relative levels of Orsay virus 4 days after exposure in animals whose parents were either infected with Orsay virus (+V+V) or uninfected (−V+V) (Orsay virus infection of parents was confirmed by qRT-PCR). Data are normalized to gapdh levels and the level of infection of the +V parents. Error bars show the standard error of the mean. (E) Graph showing a comparison of Orsay virus infection levels between liquid- and agar-based infection protocols in both the drh-1 mutant and N2 strains with three different concentrations of virus. Each condition was performed with five biological replicates, and error bars represent the standard error of the mean.
Mentions: To better understand the antiviral RNAi response, we used high-throughput sequencing to assess the small RNAs present in biological duplicates of N2 animals infected with Orsay virus (N2 P0) and compared them with their uninfected offspring (N2 F1) (Fig. 1B to E). As shown previously (30), the N2 P0 sample (Fig. 1B) shows Dicer products mapping both sense and antisense to the viral genome and abundant 22G RNAs mapping antisense to the viral RNA. There are considerably less viral interfering RNAs (viRNAs) in F1 animals than in their parents and they decrease over time (Fig. 1C to E and 2A).

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