Unexpected complexity in the interference activity of a cloned influenza defective interfering RNA

Figures

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  Fig. 1. a. Schematic diagram of influenza segment 1244 DI RNA (1/244), segment 2265 DI RNA (2/265), segment 3262 DI RNA (3/262), and the Seg 1-GFP RNA expressed from plasmids. The names derive from the 244, 265 and 262 nt remaining at the 3′ end of the cognate vRNAs respectively.The total number of nucleotides comprising each RNA is shown on the right. The nucleotide positions of the breakpoints that result in the deleted genome RNAs used in this study (positive-sense, 5′ to 3′) are indicated. Solid arrows indicate the primers used in the primer extension assays for cRNA and mRNA analyses and dashed arrows indicate primers used for vRNA analyses. For the construction of Seg 1-GFP RNA see Methods. b. Sequence of 1/244 DI RNA in cRNA sense indicating the open reading frame, and the predicted protein sequence in single letter amino acid code. The 35 residue PB1 binding domain of PB2 is indicated by the dark grey box, and the 22 residue mitochondrial interaction domain of PB2 is indicated by the light grey box. The boxed amino acid sequence downstream from the central deletion that gave rise to 1/244 DI RNA do not arise from the PB2 ORF. The three G → C mutations at nucleotide positions 30,…

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  Fig. 2. Detection of RNAs synthesised by 1/244 DI RNA and 1/244 KO DI RNA by Northern blot and primer extension and protection of mice from influenza. a. Northern blot of RNA extracted from cells 48 h after infection with 1/244 DI virus and helper virus to detect positive-sense influenza RNA transcribed from genome segment 1 and the 1/244 RNA itself. Lane 1: total RNA; lane 2: non-polyadenylated RNA; lane 3 polyadenylated mRNA. The positions of size markers (nt) are indicated. b. Viral RNAs synthesised by 1/244 DI RNA and 1/244 AUG KO DI RNA and detected by primer extension. Samples were taken 48 h after transfection with plasmids expressing Seg 1-GFP, PB1, PB2, PA and NP proteins and 1/244 and 1/244 AUG KO DI RNAs. Lane 1: a 10 nt size ladder; lane 2: RNA made in the presence of 1/244 DI RNA; lane 3: RNA made in the presence of 1/244 AUG KO DI RNA. The positions of vRNA and mRNA are indicated. 5S ribosomal RNA was measured as a loading control. c. Mice were inoculated intranasally with A/WSN alone (10 LD50, 1000 f.f.u.), A/WSN + 1/244 AUG KO DI virus, A/WSN + 1/244 DI virus, A/WSN + inactivated 1/244 AUG KO DI virus, A/WSN + inactivated 1/244 DI virus, or saline alone. Each DI virus…

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  Fig. 3. Effect of increasing amounts of DI and full-length genome segment RNAs on expression of GFP from a segment 1 reporter gene construct. 293T cells were transfected with the segment 1-GFP plasmid, plasmids expressing PB1, PB2, PA and NP proteins, and increasing amounts of an additional PolI plasmid expressing a DI RNA (1/244, 1/244 KO, 2/265 or 3/262) or a full-length vRNA (segment 1, 4 or 6). Cells were examined for fluorescence 2 days after transfection. a. Pairs of cell monolayer images taken by phase-contrast (left) and epifluorescence microscopy (right). The amount of each plasmid expressing the various RNAs used as putative inhibitors is shown on the left. Control cells (top) were transfected with an empty vector (1 μg). b. Quantitation of fluorescence in cells generated in the presence of transfected plasmids expressing 1/244 DI, 1/244 KO DI, 3/262 DI, full-length segment 1 and full-length segment 6 RNAs, as indicated in the key. Columns show the mean of 3 (1/244 RNA, segment 1 and segment 6) or 2 (1/244 KO DI RNA and 3/262) independent experiments, and bars are standard errors of the mean. c Quantitation of fluorescence in cells generated in the presence of transfected…

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  Fig. 4. Effect of DI RNA on the accumulation and encapsidation of viral RNAs. 293T cells were transfected with increasing amounts of the 1/244 DI PolI plasmid (0, 0.1, 0.5 and 1.0 μg) and a constant amount of the plasmids needed for the expression of infectious A/WSN virus. After co-cultivation with MDCK cells RNA was extracted from cells and from virus particles purified from culture fluids. a. RNA extracted from cell lysates (top panel) and virus particles from supernatants (lower panel) at 1, 2 and 3 days post co-cultivation was analysed with probes specific for segment 1 RNA, segment 7 RNA, and 1/244 DI RNA. The sizes of RNA markers are shown on the left and the identity of the RNAs on the right. b. Cell lysate RNA and virion RNA extracted on day 3 were analysed with probes specific for segment 2 RNA and segment 7 RNA. c. A/WSN infectivity in cell supernatants measured by microplaque assay. The infectivities on 1 (■), 2 (▲) and 3 (●) days after co-cultivation are shown. Data are the mean of 2 independent experiments with the bar representing the range. d. The ratios of levels of segment 1 RNA to segment 7 in virions on days 2 (■) and 3 (●) and of segment 2 RNA to segment 7 in virions…

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  Fig. 5. Analysis of influenza segment 1-directed RNA synthesis by primer extension in the presence of influenza 1/244 DI RNA, segment1 RNA, or segment 6 RNA. 293T cells were transfected with plasmids as described in Fig. 3. Primer extension analysis of viral RNA directed by segment 1-GFP in the absence or presence of increasing amounts of plasmid encoding 1/244 DI RNA (panel a), genome segment 1 vRNA (c) or genome segment 6 (e). 5S rRNA detected from the same RNA preparations were used as an internal control. The primer extension products are identified on the left of each panel. Quantitation of viral RNA from three independent experiments by phosphorimaging analysis is shown in panels (b), (d), and (f). The values of band intensities were normalised against the relevant 5S rRNA and are expressed as a percentage of the maximum value for each RNA analysed. Basal levels of vRNA generated from the target plasmid were subtracted from the total as decribed in the text. The error bars represent the standard error of the mean of at least 3 replicates. vRNA (■), mRNA (▲) and cRNA (▼)

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  Fig. 6. The effect of influenza 1/244 DI RNA on the level of RNA transcribed from influenza genome segments 2, 3 and 6. 293T cells were transfected with plasmids as described in Fig. 3. Analysis of RNA derived from segment 2 is shown in panel (a), segment 3 in panel (c) and segment 6 in panel (d) in the presence of increasing amounts of 1/244 DI RNA. Quantitation of viral RNA from two independent experiments is shown for segment 2 (b) and segment 3 (d) and three independent experiments for segment 6 (f). The error bars represent the range of data for three experiments. vRNA (■), mRNA (▲) and cRNA (▼)

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  Fig. 7. The effect of 1/244 DI RNA on its own levels of transcribed RNA in the presence or absence of seg 1-GFP. Human 293T cells were transfected with pPolI-244 and expression plasmids as described in Fig. 3. Primer extension analysis of levels of RNA transcribed from 1/244 DI RNA in the presence (a) or absence (c) of 1.0 μg segment 1-GFP. The faint band indicated by (*) is the extension product from the pcDNA PB2 expression plasmid used in the transfection. b shows the quantitation of viral RNA levels from four independent experiments with the standard error of the mean, and (d) shows the range for two replicates. vRNA (■), positive-sense RNA (cRNA + mRNA) (▲). The effect of omitting the plasmid expressing the influenza PB2 protein on virus RNA synthesis is shown in panel (e)