Fig 1.
(A) Schematic presentation of the EBOV genome. The terminal 3’-leader and 5’-trailer regions (p, promoters) are relevant to viral RNA synthesis and encapsidation. The genome harbors 7 protein genes whose mRNA expression levels (schematically shown below the genome, terminal dots indicating the 5’-cap) decrease from the first (NP) to the last (L) mRNA. Potential secondary structures involving the 7 transcription start signals (TSS, in light blue) are depicted above the genome. White boxes indicate the coding regions for EBOV proteins (NP, VP35, VP40, GP, VP30, VP24, L) and light gray boxes 5’- and 3’-UTRs. Dark gray areas mark the position of the potential secondary structures depicted above the genome. (B) Close-up of the genomic 3’-leader promoter (top) and the complementary antigenomic sequence (bottom). The shown secondary structures form in the free RNAs as confirmed by structure probing [13,26,44]. Promoter elements (PE) 1 and 2 of the bipartite leader promoter are marked in green, with pink U residues denoting UN5 hexamers in the region between nt 51 to 128. The TSS (in cyan) and and a spacer sequence (orange residues) form the NP hairpin. Hexamer phasing in the leader promoter, manifesting as the need for a multiple of 6 nt between position 51 and 80, is crucial for EBOV replication and transcription initiation at the 3’-leader promoter [13,18,40].
Fig 2.
RNA-Seq analysis of (A) small RNAs (<200 nt) or (B) poly(A) RNAs derived from EBOV-infected HuH7 cells. (A) Length of EBOV leader transcripts in small RNA libraries. The vast majority of reads had their 5’-end at position 2, thus a 55-meric leaderRNA had its 3’-end at the TSS (position 56); leaderRNA reads with lengths between 15 and 100 nt were were defined as viral leader transcripts and used as read pool for leaderRNA length/3’-end analysis. Error bars represent standard errors of the mean (SEM) calculated for each transcript length based on three biological replicates (S3 Table). Dashed vertical lines demarcate arbitrary length windows, with percentages indicated; the antigenomic leader and part of the 5’-UTR of the NP mRNA are shown schematically below the graph. (B) Analysis of NP mRNA reads with 5’-ends between antigenome position 54 and 70 (% of reads at each position). The dashed vertical line marks the annotated EBOV transcription start site (TSS). Error bars indicate SEM based on three biological replicates (S3 Table). The sketch at the bottom shows the genomic 3’-leader with the expected transcription start site at nt 56 (light blue arrow). The RNA-Seq analysis is consistent with position 56 being the major TSS, but left the possibility open that some transcripts may be initiated at position 57 and 58 in EBOV-infected cells.
Fig 3.
Northern Blot and qRT-PCR analysis of total RNA derived from EBOV-infected HuH7 cells.
(A) After electrophoresis on a native 20% PAA gel, leaderRNAs were detected by Northern blotting using a complementary, digoxigenin-labeled in vitro-transcribed RNA probe (nt 80–1 of the genomic RNA; schematically illustrated on the left). A T7-transcribed reference RNA (+) 2–78, corresponding to nt 2–78 of the antigenome, served as positive control and size marker; T7 transcripts (+) 56–158 and (-) 154–1 (representing the corresponding antigenome or genome sequence; see Fig 1B) were included as negative controls. Total RNA from Mock (non-infected) cells was used as additional negative control. Leader transcripts of ~ 60–70 nt were detected 15 min and 30 min post exposure. (B) Quantification of the NP mRNA:leaderRNA and leaderRNA:cRNA ratios for 3 biological replicates with 2 technical replicates each based on CT values determined according to strategy 1 depicted in S2A Fig. The column graph illustrates the resulting mean ratio ± SEM for NP mRNA/leaderRNA (8.75 ± 1.02) and leaderRNA/cRNA (4.19 ± 0.34). An exemplary calculation for one of the replicates is shown in S3 Fig. (C) Molar NP mRNA/leaderRNA ratio based on CT values obtained according to RT-PCR strategy 4 (S2D Fig) and including standard curves, either based on the synthetic 73- or 65-meric leaderRNA mimic as standard.
Fig 4.
(A) RNA-Seq analysis of leaderRNA length/3’ termination sites in small RNA (<200 nt) libraries derived from cells transfected with the wt MG and coexpressing VP30. The shown mean values (± SEM) are based on 3 to 5 biological replicates each. For more details, see legend to Fig 2A and S3 Table. (B) Quantification of viral mRNA to leaderRNA ratio in MG-transfected HEK293 cells by a two-step qRT-PCR. The qRT-PCR setup is schematically depicted in S2B Fig (strategy 2). Relative levels (2-ΔCT values; normalized to Firefly luciferase mRNA) of mRNA+cRNA, leRNA+cRNA, and cRNA in the presence (+) or absence (-) of the viral polymerase L are shown (mean 2-ΔCT = ~314 for mRNA+cRNA; mean 2-ΔCT = ~4.6 for leaderRNA+cRNA, and mean 2-ΔCT = ~2 for cRNA alone). The horizontal line marks the cRNA level. Mean values (± SEM) were derived from 5 independent experiments (exp.) with 2 (1 of 5 exp.) or 3 (4 of 5 exp.) technical replicates each. (C) Molar Rluc mRNA/leaderRNA ratio based on CT values obtained according to RT-PCR strategy 4 (S2D Fig) and including standard curves, either based on the synthetic 73- or 65-meric leaderRNA mimic as standard. (D) LeaderRNA 5’-ends (start sites) in RNA-Seq libraries derived from EBOV-infected HuH7 cells (upper bar), wt MG-transfected HEK293 cells (central bar), or a mock (non-infected/non-transfected) HEK293 control spiked with a synthetic 65-meric leaderRNA (lower bar; see S2 Table for sequence). The color code is specified on the right. The number of biological replicates (n) is indicated on the right (for details, see S3 Table).
Fig 5.
Analysis of replication-competent (RC) and replication-deficient (RD) MGs with mutated genome 3’-ends.
(A) Genome 3’-end of the wt MG and three mutant derivatives that either lack the 3’-terminal G residue (Δ1), the 3’-terminal two residues (Δ2) or carry an extra 3’-G residue (+G). (B-C) Corresponding reporter gene assays of lysates from cells transfected with MG variants illustrated in panel A, either as part of the (B) RC MG or (C) RD MG backbone. Mean activity values (± SEM) of the native 3’-leader MG (dark gray bars) and mutant MGs (light blue bars) were derived from 3 independent experiments with 3 technical replicates each; data for the wt MG were set to 100%. As negative control, the plasmid encoding the L gene was omitted during transfection (–L; black bars). (D-F) Corresponding two-step qRT-PCR of RC MG samples using the same cells as in panel B (for qRT-PCR setup, see S2C Fig); color code as in panel B and C. Mean 2-ΔΔCT values (± SEM) of viral mRNA (D), cRNA (E) and vRNA (F) derived from 3 independent experiments with 3 technical replicates each. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 (unpaired Welch’s t test).
Fig 6.
Testing for alternative gene end (GE) signals in the genomic 3’-leader promoter using a bicistronic (bici) MG system reflecting the EBOV NP-VP35 gene border.
(A) Bici MGs encode a Renilla (light blue box) and Firefly (light red box) luciferase (for further details on color coding, see Fig 1). In construct bici NP-NP, the native VP35 5’-UTR hairpin at the second cistron was exchanged with the NP 5’-UTR hairpin. Then, the MG variant bici NPGEmut-NP was constructed, in which the native NP GE sequence (GEmut, light green box) was mutated (marked by red vertical lines, mutations shown in the box at the bottom). For this construct, one would expect Firefly luciferase expression only if the NP HP harbored an alternative GE signal. In the third test construct (bici NPGEmut-leNP), a derivative of bici NPGEmut-NP, the entire 3’-terminal 78 nt of the 3’-leader promoter were inserted into the intergenic region. In construct bici NPGEmut-VP40, the NP HP at the second cistron was replaced with the VP40 5’-UTR HP that introduces a functional second GE signal (GE2) to replace the inactivated GE signal at the end of the NP gene; this variant served as positive control. (B-C) Corresponding Renilla (B) and Firefly (C) luciferase reporter gene assays were used as an indirect readout for viral transcription of mRNA 1 encoding Renilla and mRNA 2 encoding Firefly luciferase. Activity values of the wt NP-NP MG were set to 100%.–L, negative control in which the plasmid encoding L was omitted during transfection. Mean values ± SEM were derived from 3 independent experiments with at least 2 technical replicates each. *p < 0.05; ****p < 0.0001; n.s., not significant (unpaired Welch’s t test).
Fig 7.
leaderRNA synthesis in the presence or absence of VP30.
(A-C) Two-step strand-specific qRT-PCR (see S2B Fig) quantification of (A) mRNA+cRNA, (B) cRNA or (C) leaderRNA+cRNA using the 2-ΔΔCT method as described [18]. HEK293 cells were transfected with the RC MG encoding the native 3’-leader, either with or without coexpression of VP30. Transfections without the plasmid encoding L served as negative controls. Mean 2-ΔΔCT values (± SEM) were derived from 5 independent experiments (exp.) with 3 (4 of 5 exp.) or 2 (1 of 5 exp.) technical replicates. (D) Mean 2-ΔCT values (± SEM) of leaderRNA+cRNA versus cRNA, corresponding to samples analyzed in panel C and B, respectively. 2-ΔCT values were determined according to strategy 2 (S1 Text, S2B Fig). *p < 0.05; ***p < 0.001; ****p < 0.0001 (unpaired Welch’s t test).
Fig 8.
RNA-Seq analysis of relative leader transcript and Renilla mRNA levels in RC MG systems, with (+) and without (-) cotransfection of the plasmid encoding VP30.
(A) Illustration of the MG encoding the wt NP HP and mutant derivatives either encoding the NheI HP [44] destabilized particularly on the mRNA level or the Δ5’ spacer variant [40] devoid of any hairpin structure on the genomic and mRNA level. Minimum free energies (MFE; ΔG) of the secondary structures were predicted by RNAfold using the default parameters [80]. Black underlined residues in the NheI HP mark mutated residues. For more contextual details, see Fig 1B. (B) Fraction of leaderRNA reads (in %) in small RNA (< 200 nt) libraries derived from cells transfected with the MGs illustrated in panel A; the corresponding fraction (0.11 ± 0.06) is also shown for EBOV-infected cells. For the definition of leaderRNAs, see legend to Fig 2A; error bars are standard errors. The difference between the relative read numbers of leaderRNAs with and without VP30 cotransfection is significant if all MGs are pooled together (***p = 0.00025, Welch’s t test). The individual difference for the NheI NP construct is significant as well (**p = 0.0022, Welch’s t test) but not for the other MGs (p = 0.13 for wt NP, p = 0.054 for Δ5’ spacer, Welch’s t test). (C) Fraction of Rluc mRNA reads (in %) in corresponding poly(A) RNA-enriched libraries. For details (panel B and C) on sample and library preparation, Illumina Sequencing, sequencing analyses and biological replicates, see the S1 Text and S3 Table. (D, E) Analysis of Rluc mRNA reads with 5’-ends between antigenome position 54 and 70 (% of reads at each position) in poly(A) RNA-enriched libraries derived from cells transfected with the Δ5’ spacer MG in (D) the presence (+) or (E) absence (-) of VP30. Percent values were normalized to the sum of all mRNA reads with 5’-ends mapping to positions 54 to 70, based on two biological replicates (± SEM) in panel D and three biological replicates (± SEM) in panel E (see S3 Table for details). The data indicate position 56 as the major transcription initiation site.
Fig 9.
Comparative RNA-Seq analysis of leaderRNA lengths in (A) EBOV-infected HuH7 cells or HEK293 cells transfected with (B) MG NP HP (wt), (C) MG NheI HP or (D) MG Δ5’ spacer. Panel A is identical to Fig 2A and included for comparison; the mean vales (± SEM) in panels B to D are based on four (each wt MG and MG NheI) or five (MG Δ5’ spacer) biological replicates. For more details, see legend to Fig 2A and S3 Table.
Fig 10.
qRT-PCR analysis of leaderRNA synthesis from MG templates that deviate from hexamer phasing.
(A) Illustration of RC MG variants, either encoding the wt NP HP, a derivative thereof with a single nt deletion, construct NP-1 (stem), or a variant in which the NP HP was replaced with the corresponding hairpin of the VP35 gene; in the latter two constructs, the distance between nt 51 and 80 is not a multiple of 6 (29 nt). For detailed information on color code see legend to Fig 1B; ΔA, deletion of a single A residue. (B-D) qRT-PCR analysis (performed as described in Fig 7 and S2B Fig, strategy 2) of MG variants illustrated in panel A; (B) mRNA+cRNA level, (C) leRNA+cRNA level, (D) cRNA level. Mean 2-ΔΔCT values derive from 3 independent experiments with 3 technical replicates each.
Fig 11.
Analysis of innate immunity induction in HEK293 cells upon transfection with chemically synthesized, 5’-triphosphorylated EBOV leaderRNA mimics (65-mer, 73-mer), using mRNA levels for IFN-β and CXCL10 as readout; mRNA levels were determined by qRT-PCR.
Vesicular Stomatitis Virus (VSV) genomic RNA isolated as described [81] and a validated RIG-I-activating duplex RNA (3P-G/AS G24; [49]) were used as positive controls and bulk tRNA from Saccharomyces cerevisiae as negative control. In the mock control, transfection was performed with H2O instead of RNA. The graph with mean values and standard deviations is based on six independent experiments each. Statistical significance was evaluated using the Wilcoxon test *, p < 0.05; ns, not significant. For sequence and structure of the 65-mer, 73-mer and duplex 3P-G/AS G24, see S2 Table; for methodological details, see S1 Text.