Principles of RNA recruitment to viral ribonucleoprotein condensates in a segmented dsRNA virus
- Max Planck Institute of Biochemistry, Germany
- Department of Biochemistry, University of Cambridge, United Kingdom
- Molecular Immunology Laboratory, International Centre for Genetic Engineering and Biotechnology, Italy
- Department of Physics and Center for Nanoscience, Ludwig Maximilian University, Germany
Figures
Figure 1 with 2 supplements
RNA ccumulation during Rotavirus infection.
(a) Experimental design for detecting EGFP-tagged viroplasms during early infection (3–5 hpi) stages. (b) Schematics of the FISH probe design for detecting all 11 RV transcripts. A pooled set of 3–4 …
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Figure 1—source data 1
Pearson’s correlation coefficients (PCCs) of colocalising NSP5-EGFP-tagged viral factories and RV transcripts.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig1-data1-v2.xlsx
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Figure 1—source data 2
Signal intensity distribution histograms for GAPDH transcripts and RV transcript foci.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig1-data2-v2.zip
Figure 1—figure supplement 1
smFISH of GAPDH mRNA and RV transcripts.
GAPDH mRNA red, (a) and RV transcripts yellow, (b) imaged using two distinct sets of FISH probes targeting GAPDH mRNA (Quasar 670 dye-labelled probes) and RV transcripts (Quasar 570 dye-labelled …
Figure 1—figure supplement 2
Viroplasms accumulate RV transcripts but not polyadenylated mRNAs.
FISH visualisation of polyadenylated transcripts (oligo (dT)30-ATTO647N probes, see Methods, red signal) and RV transcripts (Seg4, yellow) at 4 hpi. NSP5-EGFP-labelled viroplasms contain RV …
Figure 2 with 2 supplements
Segment-specific transcript accumulation during Rotavirus infection.
(a) smFISH of RV transcripts at early infection time points (2–6 hpi) using segment-specific probes. Two sets of 48 gene-specific probes were designed for each Seg3 (magenta, Quasar570) and Seg4 …
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Figure 2—source data 1
Colocalisation of Seg3 and Seg4 transcripts.
Pearson’s correlation coefficients (PCCs) of colocalising Seg3 and Seg4 transcripts.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data1-v2.csv
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Figure 2—source data 2
Seg4/EGFP and Seg3 RNA signal intensities in RV-infected cells at 4 HPI.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data2-v2.zip
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Figure 2—source data 3
Raw signal intensity distributions (counts) of diffraction-limited smFISH-detected spots were derived for Seg3 (Quasar 670 dye-labelled probes) and Seg4 (Quasar 570 dye-labelled probes) transcripts at 2 and 4 hpi.
Each spot was detected after applying a constrained deconvolution algorithm (see Methods).
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data3-v2.xlsx
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Figure 2—source data 4
Integrated signal intensities of Seg3, Seg4 transcripts and NSP5-EGFP-tagged viral condensates at different infection time points.
Integrated signals were calculated for single cells separately, N=24 (2 hpi), N=21 (4 hpi), N=30 (6 hpi).
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data4-v2.zip
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Figure 2—source data 5
Pearson’s correlation coefficients (PCCs) calculated for a single cell.
N=37 (2 hpi), N=62 (4 hpi), N=51 (6 hpi).
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data5-v2.xlsx
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Figure 2—source data 6
Seg4/EGFP and Seg3 RNA signal intensities in RV-infected cells at 6 hpi.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data6-v2.txt
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Figure 2—source data 7
Integrated intensities for Seg4 RNA and Seg3 RNA for shNSP2 and WT MA104 cells.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data7-v2.xlsx
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Figure 2—source data 8
Western blot of NSP2 from RV-infected cells (WT MA104 and NSP2-specific shRNA-expressing MA104 cells).
MA104 cells stably expressing shRNA targetting NSP2 transcripts were infected at MOI = 1, and cell lysates were analysed for NSP2 expression by western blotting at 8 hpi.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig2-data8-v2.zip
Figure 2—figure supplement 1
Analysis of Seg3 and Seg4 transcript accumulation and localisation in RV-infected cells.
(a) Signal intensity distributions of Seg3 and Seg4 transcript foci at 2 hpi and 4 hpi. Raw signal intensity distributions (counts) of smFISH-detected spots were derived for Seg3 and Seg4 …
Figure 2—figure supplement 2
NSP2 knockdown in MA104 cells results in the apparent loss of RV RNA clustering.
(a) Wild-type MA104 cells infected with RV (MOI = 1) at 6 hpi, with a schematic showing Seg3 and Seg4 RNA clustering. (b) MA104 cells expressing shRNAs that target NSP2 (Methods) were infected with …
Figure 3
DNA-PAINT analysis of the RV RNA oligomerisation.
(a) Schematics of DNA-PAINT imaging of RV transcripts. RNA hybridisation is used to install unique DNA ‘docking’ P1 strands for Seg3 RNA targets. These P1 sequences bind to complementary …
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Figure 3—source data 1
The apparent kon value for RNA structures detected at 2, 4, and 6 hpi.
(e) Unimodal distribution of kon values calculated for single Seg3 transcripts detected at 2 hpi. The resulting average kon value calculated from a Gaussian fit is 10–5 (Ms)–1, corresponding to a single Seg3 transcript. (f) Estimated numbers of Seg3 transcripts (mean ± SD) at 4 hpi and 6 hpi are 24±12 (x̃=21) and 53±36 (x̃=42), respectively. N=245 (2 hpi), N=515 (4 hpi), N=1181 (6 hpi). Scale bars, 5 µm (b), 500 nm (c).
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig3-data1-v2.xlsx
Figure 4 with 1 supplement
Universal DNA exchange-smFISH approach for single-cell imaging of the RV transcriptome.
(a) Schematics of the Universal DNA Exchange-smFISH approach (UDEx-FISH). FISH probes targeting different RNA segments are extended with 12 nucleotide-long orthogonal DNA sequences A1-An (DNA …
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Figure 4—source data 1
The integrated signal intensity after each successive round of imager dissociation and re-association.
Each image plane for each channel was recorded with the same image acquisition parameters.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig4-data1-v2.xlsx
Figure 4—figure supplement 1
Validation of the UDEx-FISH approach in RV-infected MA104 cells.
(a) Washing and labelling efficiencies of smFISH probes with orthogonal DNA handles designed for Segments 1–9. After each wash step, no residual RNA signal was detected for each segment tested in …
Figure 5 with 1 supplement
Single-cell analysis of the RV transcriptome.
(a) In situ analysis of the RV transcriptome at 6 hpi. Scale bar, 20 µm. (b) Zoomed-in images of the highlighted regions containing viroplasms and 11 distinct RNA targets. Scale bar: 5 µm. (b) The …
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Figure 5—source data 1
Relative fractions of individual RV transcripts in the RV transcriptome at 6 hpi (Seg1-Seg11).
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig5-data1-v2.txt
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Figure 5—source data 2
Single-cell analysis of the relative fractions of each segment-specific transcript (N=15 cells) estimated from the UDEx-FISH images data.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig5-data2-v2.zip
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Figure 5—source data 3
Relative abundance of individual transcripts in viroplasms (N=21) estimated by UDEx-FISH.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig5-data3-v2.zip
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Figure 5—source data 4
Quantitative comparison of the RV transcriptome estimated by UDEx-FISH and RNA-Seq at 6 hpi.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig5-data4-v2.zip
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Figure 5—source data 5
Integrated signal intensities for individual viroplasm-localized RV transcripts used for correlation analysis of the RV RNA stoichiometry in viroplasms.
- https://cdn.elifesciences.org/articles/68670/elife-68670-fig5-data5-v2.xlsx
Figure 5—figure supplement 1
Quantification of transcripts by UDEx-FISH.
(a) UDEx-FISH and RNA-Seq quantitation of RV transcripts at 6 hpi (MA104 cells, as described in Methods). Relative fractions of RV transcripts quantified by RNA-Seq (e.g. mean values shown in Figure …
Figure 6
3’ UTR is required for RV transcript localisation to viroplasms.
(a) Schematics of the EGFP transcript construct containing the EGFP-coding sequence flanked by the 5’UTR and 3’UTR sequences derived from Seg1 RNA (5’UTR-EGFP-3’UTR) or only 3’UTR sequence (denoted …
Figure 7
3’ UTRs and the viral polymerase are required for RV transcript localisation.
(a) Polyadenylated RV mRNAs coding EGFP do not partition to viroplasms. Top - experimental design of the smFISH probes to target RV mRNAs coding EGFP in cells infected with the wild-type rotavirus. …
Figure 8
A proposed model of viral transcript partitioning into viroplasms.
RV transcripts (for clarity, only 3 out of 11 distinct transcripts are shown) associate with viral RNA-binding proteins NSP2 (cyan doughnut-like octamers), as well as the RV RNA-dependent RNA …
Author response image 1
Author response image 2
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Chlorocebus aethiops) | MA104 Clone 1 | ATCC | ATCC CRL-2378.1; RRID:CVCL_3845 | Stable cell line |
| Cell line (Chlorocebus aethiops) | MA104-NSP5-EGFP | Cells obtained from Dr. O. Burrone and G. Papa, ICGEB, Trieste, Italy | https://doi.org/10.1128/JVI.01110-19 | Stable cell line |
| Cell line (Chlorocebus aethiops) | MA104-shRNA-NSP2 | This manuscript | MA104-shRNA-NSP2 | Made by transfection with pPB[shRNA]-EGFP:T2A:Puro-U6 plasmid |
| Cell line (Chlorocebus aethiops) | MA104-NSP2-mCherry | Geiger et al., 2021 | https://doi.org/10.15252/embj.2021107711 | Stable cell line |
| Strain, strain background (Group A rotavirus) | Bovine rotavirus group A, strain RF [G6P6(1)] | Dr Ulrich Desselberger, University of Cambridge, UK | https://doi.org/10.1371/journal.pone.007432 | |
| Strain, strain background (Group A rotavirus) | Simian rotavirus group A, strain SA11/tsC mutant | Dr Sarah McDonald Esstman, Wake Forest University, USA | https://doi.org/10.1016/j.virusres.2021.198488 | |
| Sequence-based reagents | FISH probes | Integrated DNA Technologies | RNA FISH DNA probes | Sequences listed in Supplementary file 1 |
| Antibody | Goat polyclonal, HRP-conjugated, anti-guinea pig | ThermoFisher | A18775, RRID:AB_2535552 | 1:10,000 dilution used for Western Blotting |
| Antibody | Anti-NSP2 (polyclonal, anti-guinea pig) | Dr. O. Burrone, ICGEB, Trieste, Italy; Papa et al., 2019 | https://doi.org/10.1128/JVI.01110-19 | 1:1,000 dilution used for Western Blotting |
| Sequence-based reagents | FISH Probes, fluorescently labelled Seg3RF-Q670 Seg4RF-Q570 GAPDH-Q670 Comb-Q570 For_UTR Rev_UTR Rev_dUTR | LGC Biosearch Technologies | Seg3RF-Q670 Seg4RF-Q570 GAPDH-Q670 Comb-Q570 | Sequences provided in the Supplementary file 1 |
| Other | Stellaris RNA FISH Hybridization Buffer | LGC Biosearch Technologies | SMF-HB1-10 | |
| Recombinant DNA reagent | pPB[shRNA]-EGFP:T2A:Puro-U6 | VectorBuilder https://en.vectorbuilder.com | pPB[shRNA]-EGFP:T2A:Puro-U6 | Sequence provided in Supplementary file 1 |
| Recombinant DNA reagent | pCMV-HyPBase | Dr. O.Burrone, ICGEB, Trieste, Italy; Papa et al., 2019 | https://doi.org/10.1128/JVI.01110-19 | |
| Recombinant DNA reagent | pT7-NSP3-EGFP | Dr. O.Burrone, ICGEB, Trieste, Italy; Papa et al., 2019 | https://doi.org/10.1128/JVI.01110-19 | |
| Commercial assay or kit | NEBNEXT rRNA Depletion kit | New England Biolabs | E6350S | Mouse/Rat/Human species |
| Commercial assay or kit | Faustovirus Capping Enzyme | New England Biolabs | M2081S | |
| Commercial assay or kit | mRNA Cap 2´-O-methyltransferase | New England Biolabs | M0366 | |
| Other | Lipofectamine 3000 | Invitrogen | L3000001 | |
| Commercial assay or kit | MycoSPY | Biontex Laboratories, Germany | M030-050 | PCR detection of Mycoplasma sp. |
| Commercial assay or kit | NEBNEXT Ultra II FS DNA Library Prep kit | New England Biolabs | E7805S | |
| Commercial assay or kit | RNA extraction kit, RNEasy | Qiagen | 74034 | |
| Other | SuperScript II reverse transcriptase | Invitrogen | 18064014 | |
| Commercial assay or kit | HighScribe T7 kit | New England Biolabs | E2040S | |
| Other | DAPI stain | Invitrogen | D1306 | |
| Other | Atto647N-Oligo(dT30) | Integrated DNA Technologies | https://doi.org/10.1016/j.molcel.2017.10.015 | |
| Software, algorithm | OriginPro | OriginLab | RRID:SCR_014212 | |
| Software, algorithm | Icy | (http://icy.bioimageanalysis.org/) | RRID:SCR_010587 | Colocalisation analysis |
| Software, algorithm | Picasso | Developed in- house (Kowalewski, 2023) https://github.com/jungmannlab/picasso | Schnitzbauer et al., Nat. Protoc. 12, 1198–1228 (2017). | |
| Software, algorithm | Stellaris Designer | LGC Biosearch Technologies | https://www.biosearchtech.com/stellaris-designer | RNA FISH probe designer |
| Software, algorithm | Salmon | https://github.com/COMBINE-lab/salmon (COMBINE lab, 2022) | Patro, R. et al., Nat. Methods 14, 417–419 (2017). |
Additional files
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Supplementary file 1
GenBank IDs and sequences of the DNA oligonucleotides used in the study.
- https://cdn.elifesciences.org/articles/68670/elife-68670-supp1-v2.xlsx
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Supplementary file 2
RNA-Seq transcriptome analysis of RV-infected MA104 cells at 6 hpi (RVA strain RF).
- https://cdn.elifesciences.org/articles/68670/elife-68670-supp2-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/68670/elife-68670-transrepform1-v2.docx
