An antiviral role for the RNA interference machinery in Caenorhabditis elegans - PubMed (original) (raw)

An antiviral role for the RNA interference machinery in Caenorhabditis elegans

Daniel H Schott et al. Proc Natl Acad Sci U S A. 2005.

Abstract

RNA interference (RNAi) is a sequence-specific gene-silencing mechanism triggered by exogenous dsRNA. In plants an RNAi-like mechanism defends against viruses, but the hypothesis that animals possess a similar natural antiviral mechanism related to RNAi remains relatively untested. To test whether genes needed for RNAi defend animal cells against virus infection, we infected wild-type and RNAi-defective cells of the nematode C. elegans with vesicular stomatitis virus engineered to encode a GFP fusion protein. We show that upon infection, cells lacking components of the RNAi apparatus produce more GFP and infective particles than wild-type cells. Furthermore, we show that mutant cells with enhanced RNAi produce less GFP. Our observation that multiple genes required for RNAi are also required for resistance to vesicular stomatitis virus suggests that the RNAi machinery functions in resistance to viruses in nature.

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Figures

Fig. 1.

Fig. 1.

VSV infection of wild-type and rde-1 mutant C. elegans cells. (A) Schematic of the genome structure of the recombinant VSV used in this study, encoding a GFP–P fusion protein. (B) C. elegans cells 7 days after infection at moi = 0.003 (15). Green/yellow, GFP fluorescence; blue, Hoechst 33342 staining for DNA. (Scale bar, 100 μm.) (C) Time course of virus titers capable of infecting mammalian cells, in medium after infection of wild type and rde-1(ne219) cells at moi = 3. Error bars represent SEM; n = 3 cell-culture wells. Titer in medium over rde-1(ne219) cells at day 7 is different from titer over wild-type cells at day 7 (P = 0.03 in two-tailed t test). (D) C. elegans cells 7 days after infection at moi = 3. Green, GFP fluorescence; blue, Hoechst 33342 staining for DNA. (Scale bar, 50 μm.)

Fig. 2.

Fig. 2.

VSV replication in infected wild-type and RNAi-defective C. elegans cells. (A) GFP levels in RNAi-defective mutants relative to wild-type 7 days after infection at moi = 3. Values are ratios of GFP fluorescence to Hoechst 33342 fluorescence, relative to wild type. Error bars represent the 95% confidence interval in t distribution based on four to eight worm embryonic cell isolations. Different from wild type in two-tailed t tests: rde-1(ne219), P = 7 × 10–4; rde-3(ne298), P = 0.03; rde-4(ne301), P = 0.02; rrf-1(pk1417), P = 2 × 10–4; sid-1(qt9), P = 0.07. Different from sid-1(qt9) in two-tailed t tests: rde-1(ne219), P = 9 × 10–4; rde-3(ne298), P = 0.04; rde-4(ne301), P = 0.02; rrf-1(pk1417), P = 1 × 10–5. (B) Quantities of VSV (–)-strand RNA in cells 7 days after infection at moi = 3, relative to virus used to infect cells. (C) Quantities of VSV (–)-strand RNA in rde-1(ne219) cells 1 h and 52 h after infection at moi = 3, relative to virus used to infect cells. (D) Virus titers capable of infecting mammalian cells, in medium 7 days after infection of wild-type and mutant cells at moi = 3. Error bars represent SEM; n = 4 cell culture wells. Different from wild type in two-tailed t tests: rde-1(ne219), P = 1 × 10–3; rde-3(ne298), P = 4 × 10–3; rde-4(ne301), P = 7 × 10–6; rrf-1(pk1417), P = 2 × 10–3.

Fig. 3.

Fig. 3.

GFP levels in dsRNA-treated wild-type and sid-1(qt9) cell cultures relative to wild type (wt) treated with irrelevant (dpy-10) dsRNA 7 days after infection at moi = 3. Values are ratios of GFP fluorescence to Hoechst 33342 fluorescence, relative to wild type. Error bars represent the 95% confidence interval in t distribution based on samples within one experiment, but data are representative of at least two experiments. Different from dpy-10 dsRNA-treated wild type in two-tailed t tests: rde-1 dsRNA-treated wild type, P = 2 × 10–12; dcr-1 dsRNA-treated wild type, P = 2 × 10–7; C04F12.1 dsRNA-treated wild type, P = 7 × 10–12. Different from corresponding sid-1(qt9) cells in two-tailed t tests: rde-1 dsRNA-treated wild type, P = 7 × 10–13; dcr-1 dsRNA-treated wild type, P = 1 × 10–8; C04F12.1 dsRNA-treated wild type, P = 7 × 10–10.

Fig. 4.

Fig. 4.

GFP levels in RNAi enhanced mutants relative to wild type 7 days after infection at moi = 6. Values are ratios of GFP fluorescence to Hoechst 33342 fluorescence, relative to wild type. Error bars represent the 95% confidence interval in t distribution based on samples within one experiment, but data are representative of at least two experiments. Different from wild type in two-tailed t tests: rrf-3(pk1426), P = 6 × 10–4; eri-1(mg366), P = 3 × 10–4; lin-15B(n744), P = 2 × 10–8; eri-1(mg366);lin-15B(n744), P = 2 × 10–8.

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