Article Figures & Data
- FIG 1
The positive-sense DVG strand is associated with the strong antiviral activity induced during infection. (A) MEFs were infected with SeV HD or SeV LD (MOI = 1.5 TCID50/cell) or left untreated (N/T) and challenged 6 h later with IAV (MOI of 1.5). Cells were harvested 12 h after challenge, and the expression of IAV NP mRNA was examined by qRT-PCR. The data are the percentage of IAV NP mRNA relative to that of N/T cells. (B) Expression of antiviral genes examined by RT-qPCR. Results are expressed as the mean ± the standard error of the mean of three independent experiments. (C) Representation of genomic and copy-back DVG RNAs produced during SeV infection. (D) IFNB1 mRNA expression and ratio of positive/negative-sense DVG RNA copy numbers determined by RT-qPCR from A549 cells infected with SeV HD at an MOI of 1.5 TCID50/cell. Experiments were independently repeated at least three times. Each assay was performed in triplicate. A representative graph is shown. (E) Representative staining of positive-sense DVG (green), positive-sense SeV genome or mRNA (red), IRF3 (purple), and nuclei (4′,6-diamidino-2-phenylindole [DAPI], blue) on LLC-MK2 cells at 6 h after SeV HD infection or on mock-treated controls. Magnification, ×100. (F) Quantification of IRF3 nuclear translocation within all of the cells, DVG-containing cells, or DVG-negative cells. Results are expressed as the mean ± the standard error of the mean of three independent experiments. ***, P < 0.001; ns, nonsignificant (one-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for glyceraldehyde 3-phosphate dehydrogenase (GAPDH).
- FIG 2
Identification of a candidate motif essential for type I IFN induction by positive-sense DVG RNA. (A) Representation deletion of mutant DVGs. The length of the deletion (dashed line) and the total final length of each molecule are indicated. The mutant DVG with both complementary sequences (gray) deleted is DVG-IS (internal sequence). (B) Expression of IFNB1 and IFIT1 mRNA measured by RT-qPCR from LLC-MK2 cells transfected for 6 h with 4.15 pmol of the ivtDVG indicated. The data are the mean ± the standard error of the mean of all of the experiments (total n = 5 to 11/group). (C) Folding prediction of highly stimulatory and poorly stimulatory mutant DVGs with the RNAfold ViennaRNA software. A temperature of 37°C was chosen to determine structures based on minimal free energy. The candidate motif (DVG70-114) is circled. (D) Relative position of the candidate stimulatory DVG70-114 motif and its sequence in the genome of DVG-546. The AU content of DVG70-114, compared with that of DVG-546 and the full-length genome, is shown in the inset. (E) Folding predictions for DVG-268 and of the mutants derived from it. The structures of intact DVG-268 and of the DVG70-114 motif deleted (Δ70-114) molecules are shown. The position of DVG70-114 is highlighted. Point mutations within the DVG70-114 motif are indicated in the red square. (F) LLC-MK2 cells were transfected with 4.15 pmol of the ivtDVG indicated, IFNB1 and IFIT1 mRNA expression was analyzed by RT-qPCR at 6 h posttransfection. The data are the mean ± the standard error of the mean of all of the experiments (total n = 3/group). (G) Murine TC-1 cells were transfected with 4.15 pmol of DVG-268 or DVG-268Δ70-114, and IFN-β protein and mRNA levels were measured by ELISA and RT-qPCR, respectively. The data are the mean ± the standard error of the mean of three independent experiments. Each experiment was performed in duplicate. Bars correspond to the standard error of the mean. *, P <0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (two-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH.
- FIG 3
Recombinant SeV iDVGs with an intact DVG70-114 motif preserve their stimulatory activity in the context of infection. (A) Schematic of the recombinant SeV DVG generation system. BSR-T7 cells were infected with partially inactivated SeV 52 and transfected with a plasmid encoding either DVG-324 or DVG-354. Cells and supernatants were collected 48 h later and inoculated into 10-day-old embryonated chicken eggs. SeV containing rDVGs was collected from the allantoic fluid. T7 pro, T7 promoter sequence; Riboz., ribozyme; T7 term, T7 polymerase terminator sequence. (B) LL-CMK2 cells were infected with virus rDVG-324 or rDVG-354 at an MOI of 5 TCID50/cell and analyzed at 6 h postinfection by RT-qPCR for the expression of IFNB1 and IFIT1 mRNA, SeV NP mRNA, and DVGs. The relative copy number of DVG RNA was quantified by RT-qPCR with the DVG comp primers (see Table S1 in the supplemental material). The data are the average of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (one-way ANOVA with Bonferroni's post hoc test and two-tailed t test in DVG RNA quantification). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. (C and D) LL-CMK2 cells were infected with SeV LD at an MOI of 1.5 TCID50/cell and transfected 24 h later with 4.15 pmol of DVG-268 or DVG-268Δ70-114 RNA or left untreated (NT). Expression of SeV NP and SeV (P/V) (C) and antiviral genes (D) was measured at 6 h posttransfection. The data are the average of three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (one-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH.
- FIG 4
3′-to-5′ complementarity and additional proximal secondary structures are not necessary for IFN induction by DVG-derived RNA. (A) Illustration of DVG-324 and the related 3′ complementary sequence deletion mutant (DVG-324NC). The relative position of the immunostimulatory motif DVG70-114 is highlighted with a red square. The length of the deletion (dashed line) is indicated. (B) Expression of IFNB1 and IFIT1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of the ivtDVG indicated. (C) Folding predictions for DVG-324NCΔ70-114, DVG-324NCΔ5-51, and parental DVG-324NC. Motif DVG70-114 is indicated by a red oval. Motif DVG5-51 is indicated by a blue oval. (D) Expression of IFNB1 mRNA by RT-qPCR in LLC-MK2 cells transfected for 6 h with 4.15 pmol of the ivtDVG indicated. (E) Illustration of the DVG5-51 motif and its relative position in the DVG-324NC construct. The sequence of DVG5-51 is shown below the construct. (F) Folding prediction for DVG-324NC motif1+ compared to that for DVG-324NC. (G) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of the ivtDVG illustrated in panel H. All transfection experiments were independently repeated at least three times. The data are the mean ± the standard error of the mean of all of the experiments (total n = 3 to 5/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, nonsignificant (one-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH.
- FIG 5
The DVG70-114 motif transfers strong immunostimulatory activity to inert RNA molecules. (A) Folding predictions for HCV X region, X region DVG70-114, and X region DVG5-51. Motif DVG70-114 is in a red balloon, and motif DVG5-51 is in a blue balloon. (B) Expression of IFNB1 and IFIT1 mRNA measured by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of gel-purified poly(U/UC), X region, X region DVG70-114, or X region DVG5-51 ivtDVGs. The data are the mean ± the standard error of the mean of all of the experiments (total n = 3 to 5/group). *, P < 0.05; **, P < 0.01; ns, nonsignificant (one-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH.
- FIG 6
The DVG70-114 motif strongly stimulates RLR signaling. (A) Expression of IFNB1 mRNA determined by RT-qPCR from wild-type (WT), Mavs−/−, and Ddx58−/− (RIGI−/−) MEFs transfected for 6 h with 4.15 pmol of ivtDVG or poly (I:C). (B) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with ivtDVGs left untreated or treated with AP. (C) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of DVG-268 not treated (NT) or treated with RNase A, V1, A/V1 or mock transfected. (D and E) EMSA of DVG-268 and DVG-268Δ70-114 RNA with increasing doses of RIG-I deltaCARD in the absence (D) or presence (E) of 1 µM ATP. All of the experiments in panel A to E were independently repeated at least three times. Each RT-qPCR assay was performed in triplicate. The data are the average values of all of the experiments (total n ≥3/group). *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 (two-way [A] or one-way [B and C] ANOVA with Bonferroni's post hoc test). RT-qPCR data are expressed as the copy number relative to that of the housekeeping gene for GAPDH and/or ACTB (for LL-CMK2 cells) and Rps11 (for MEFs). Representative gel pictures are shown in panels D and E.
Supplemental Material
Figure S1
Production of positive-sense DVG during DVG replication closely correlates with expression of IFNB1 mRNA. (A) Scheme of PCR amplification strategy utilized to detect and differentiate positive- and negative-sense strands of DVG-546. RT of the positive- and negative-sense DVGs was performed with the primers indicated. RT-qPCR of each sense-specific RT product was performed with a combination of the DVG 1 and DVG J primers to produce identical PCR products at equivalent efficiencies. While the DVG 1 primer will serve to reverse transcribe the positive-sense strand of both DVG-546 and the SeV HD genome, the DVG J primer spans the junction site between the inverse trailer and the internal DVG sequence, which is unique to the DVG-546 sequence and is not present in the SeV HD genome. (B) Positive- and negative-sense DVG qPCR efficiency test of TC-1 mouse lung epithelial cells infected with SeV HD at an MOI of 1.5 TCID50/cell. Total RNA (1 µg) isolated 12 h after infection was reverse transcribed for either positive- or negative-sense DVG. Ten-fold serial dilutions were analyzed for RT-qPCR, and cycle threshold (CT) values for all of the dilutions were graphed relative to the log10 of the number of picograms of initial RNA. The linear region of the standard curve is shown and was used to determine the R2 value and PCR efficiency, which was calculated as 10−1/slope − 1. (C) Copy numbers of positive- and negative-sense DVGs over the course of A549 infection with SeV HD at an MOI of 1.5 TCID50/cell. (D) IFNB1 mRNA expression and ratio of positive/negative-sense DVG RNA determined by RT-qPCR from LLC-MK2 cells infected with SeV HD at an MOI of 1.5 TCID50/cell. All of the experiments shown in panels B to D were independently repeated three times, and each assay was performed in triplicate. A representative data set is shown. Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. Download Figure S1, TIF file, 1.7 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S2
ivtDVGs with an intact stimulatory motif stimulate IFN responses in both mouse and human cells, and the responses are sustained for up to 24 h posttransfection. (A) Expression of IFNB1 mRNA determined by RT-qPCR of TC-1 cells transfected for 6 h with 4.15 pmol of the ivtDVGs indicated. (B) Expression of IFNB1 mRNA by RT-qPCR of A549 cells transfected for 6 h with 4.15 pmol of the ivtDVGs indicated. Each RT-qPCR assay was performed in triplicate. The data are the average values of all of the experiments (total n = ≥3/group). *, P <0.05; **, P < 0.001; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Bonferroni's post hoc test). RT-qPCR data are expressed as the copy number relative to that of the housekeeping gene for GAPDH for LL-CMK2 cells and Rps11 for TC-1 cells. (C) Expression of IFNB1 and IFIT1 mRNA at 4, 8, 12, and 24 h posttransfection of LL-CMK2 with 4.15 pmol of the ivtDVG RNA indicated. One representative experiment out of three is shown. Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. Download Figure S2, TIF file, 1.6 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S3
Quality control of ivtDVG RNAs. (A) Electrophoretic analysis of ivtDVGs was performed with an Agilent 2100 Bioanalyzer. (B) Endotoxin levels on ivtDVGs used in this study measured by Limulus assay. (C) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of gel-purified ivtDVG. The experiment was independently repeated three times, and each assay was performed in triplicate. A representative qPCR is shown. RT-qPCR data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. (D) The amount of ivtDVGs present upon each transfection was quantified at 6 h posttransfection by RT-qPCR with a common set of primers directed to the 5′-complementary end of the DVGs (DVG comp). Two representative transfections of LL-CMK2 cells are shown (EXPs 1 and 2). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. (E) Immunofluorescence staining of LL-CMK2 cells infected with SeV LD (MOI of 1.5 TCID50/cell) at 24 hpi with SeV NP (red) and nuclei (DAPI, blue). Magnification, ×40. Download Figure S3, TIF file, 1.5 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S4
Verification of DVG70-114 motif activity in DVG-546. (A) Electrophoretic analysis for DVG-268 ivtDVGs with an Agilent 2100 Bioanalyzer. (B) Electrophoretic analysis for DVG-546 ivtDVGs with an Agilent 2100 Bioanalyzer. (C) Folding prediction for DVG-546 with the motif deleted (DVG-546Δ70-114) and parental DVG-546. The DVG70-114 motif is indicated by a red oval. (D) Expression of IFNB1 mRNA by RT-qPCR in LLC-MK2 cells transfected for 6 h with 4.15 pmol of the ivtDVG indicated. (E) Folding prediction for DVG-546 motif1+ and parental DVG-546. The DVG70-114 motif is indicated by a red oval. The mutated DVG70-114 motif in DVG-546 motif1+ is indicated by an orange oval. (F) Expression of IFNB1 mRNA by RT-qPCR of LLC-MK2 cells transfected for 6 h with 4.15 pmol of ivtDVG illustrated in panel E. All transfection experiments were independently repeated at least three times. The data are the mean ± the standard error of the mean of all of the experiments (total n = 3 to 5/group). **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. Download Figure S4, TIF file, 2.7 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S1
Primers used in mutagenesis and mutant DVG verification. Table S1, DOCX file, 0.1 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S5
DVG-derived ivtDVG RNAs induce levels of antiviral responses equivalent to those induced by known viral RIG-I ligands. (A) EMSA of 6 pmol of HCV poly(U/UC), X region, and DVG-268 in the presence of ATP and increasing doses (0 to 20 pmol) of full-length RIG-I. The product was resolved on a 2% agarose gel. (B and C) Expression of IFNB1 and IFIT1 mRNA measured by RT-qPCR from LLC-MK2 cells transfected for 6 h with 4.15 pmol of ivtDVGs or equivalent amounts of HCV poly(U/UC), X region, or poly(I ⋅ C) (high molecular weight). The data are the mean ± the standard error of the mean of all of the experiments (total n = 3/group). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 (one-way ANOVA with Bonferroni's post hoc test). Data are expressed as the copy number relative to that of the housekeeping gene for GAPDH. Download Figure S5, TIF file, 2.6 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Figure S6
DVG70-114 improves the binding of DVGs to RIG-I and MDA5. (A) EMSA of DVG-268 and DVG-200, which lack immunostimulatory motif DVG70-114, in the presence of ATP and increasing doses of RIG-I deltaCARD. (B) EMSA of DVG-268 and DVG-268Δ70-114 RNA in the presence of increasing doses of MDA5 deltaCARD. Download Figure S6, TIF file, 0.7 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Table S2
Primers used in qPCR assays. Table S2, DOCX file, 0.1 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Text S1
Additional materials and methods used in this study. Download Text S1, DOCX file, 0.1 MB.
Copyright © 2015 Xu et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-ShareAlike 3.0 Unported license, which permits unrestricted noncommercial use, distribution, and reproduction in any medium, provided the original author and source are credited.
Additional Files
Supplementary Data
Supplementary Data
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