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Adenovirus-mediated ubiquitination alters protein–RNA binding and aids viral RNA processing

Nature Microbiology volume 5, pages 1217–1231 (2020)Cite this article

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Abstract

Viruses promote infection by hijacking the ubiquitin machinery of the host to counteract or redirect cellular processes. Adenovirus encodes two early proteins, E1B55K and E4orf6, that together co-opt a cellular ubiquitin ligase complex to overcome host defences and promote virus production. Adenovirus mutants lacking E1B55K or E4orf6 display defects in viral RNA processing and protein production, but previously identified substrates of the redirected ligase do not explain these phenotypes. Here, we used a quantitative proteomics approach to identify substrates of E1B55K/E4orf6-mediated ubiquitination that facilitate RNA processing. While all currently known cellular substrates of E1B55K and E4orf6 are degraded by the proteasome, we uncovered RNA-binding proteins as high-confidence substrates that are not decreased in overall abundance. We focused on two RNA-binding proteins, RALY and hnRNP-C, which we confirm are ubiquitinated without degradation. Knockdown of RALY and hnRNP-C increased levels of viral RNA splicing, protein abundance and progeny production during infection with E1B55K-deleted virus. Furthermore, infection with E1B55K-deleted virus resulted in an increased interaction of hnRNP-C with viral RNA and attenuation of viral RNA processing. These data suggest that viral-mediated ubiquitination of RALY and hnRNP-C relieves a restriction on viral RNA processing and reveal an unexpected role for non-degradative ubiquitination in the manipulation of cellular processes during virus infection.

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Fig. 1: E1B55K deletion or inhibition of cullin-mediated ubiquitination decreases Ad late RNA splicing and overall RNA processing.
Fig. 2: Unbiased proteomics reveals RBPs among putative non-degraded substrates of the Ad E1B55K–E4orf6 complex.
Fig. 3: RALY and hnRNP-C are non-degraded substrates of Ad E1B55K/E4orf6-mediated ubiquitination.
Fig. 4: Knockdown of RALY and hnRNP-C rescues the RNA processing defect caused by the absence of a functional E1B55K–E4orf6 complex.
Fig. 5: Interactions of hnRNP-C with viral late RNA increases in the absence of a functional E1B55K–E4orf6 complex.

Data availability

All MS data for this study have been deposited in the CHORUS database under project ID 1648, experiment IDs 3476 (K-ɛ-GG), 3477 (K-ɛ-GG-matched whole-cell proteome) and 3478 (RBR-ID of hnRNP-C). The eCLIP-seq data have been deposited in the NCBI’s Gene Expression Omnibus (GEO) and are accessible through GEO series accession number GSE145411. The eCLIP-Seq data have been deposited in the GEO97 and are accessible through GEO series accession number GSE145411. Additional supporting data are available from the corresponding author upon request. Source data are provided with this paper.

Code availability

The proteomics data were analysed using standard methods. The implementation of the analysis was performed using R software. The scripts are available from the corresponding author upon request or can be accessed via GitHub at https://github.com/JosephDybas/AdenovirusProteomics.

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Acknowledgements

We thank members of the Weitzman Lab for insightful discussions and input. We thank P. Choi for help with eCLIP-seq, and G. Yeo and E. Van Nostrand for their input regarding analysis of corresponding data. We are grateful to A. Berk, P. Branton, R. Greenberg, G. Ketner, A. Levine, K. Lynch, D. Ornelles, J. Wilson and H. Wodrich for gifts of reagents. We thank S. Schaffer for technical advice, and D. Avgousti, L. Busino, P. Choi, K. Lynch and J. Weitzman for careful reading of the manuscript. We thank the UPenn Cell and Developmental Biology Microscopy Core for imaging assistance. This research was supported by NIAID grant nos. R01-AI145266 (to M.D.W.), R01-AI121321 (to M.D.W.) and R01-AI118891 (to B.A.G.), and NCI grant no. R01-CA97093 (to M.D.W.). Additional support came from the NCI T32 Training Grant in Tumor Virology T32-CA115299 (to A.M.P.) and Individual National Research Service Awards F32-AI147587 (to J.M.D.) and F32-AI138432 (to A.M.P.) from the National Institutes of Health.

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Authors and Affiliations

  1. Division of Protective Immunity and Division of Cancer Pathobiology, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Christin Herrmann, Joseph M. Dybas, Jennifer C. Liddle, Alexander M. Price, Caitlin E. Purman, Matthew Charman, Eui Tae Kim & Matthew D. Weitzman

  2. Cell and Molecular Biology Graduate Group, University of Pennsylvania, Philadelphia, PA, USA

    Christin Herrmann

  3. Department of Biomedical and Health Informatics, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA

    Joseph M. Dybas & Katharina E. Hayer

  4. Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Joseph M. Dybas, Jennifer C. Liddle, Alexander M. Price, Caitlin E. Purman, Matthew Charman, Eui Tae Kim & Matthew D. Weitzman

  5. Epigenetics Institute, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Richard Lauman, Benjamin A. Garcia & Matthew D. Weitzman

  6. Graduate Group in Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Richard Lauman

  7. Department of Biochemistry and Biophysics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA

    Benjamin A. Garcia

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Contributions

M.D.W., C.H., J.M.D. and J.C.L. conceived the project. C.H. performed the experiments and received assistance from J.C.L., A.M.P. and E.T.K. C.H. prepared the figures with input from the other authors. J.M.D. performed the bioinformatics and proteomics data analyses. J.C.L. and R.L. performed the MS experiments. A.M.P. performed RNA FISH. A.M.P., C.E.P. and C.H. performed RT–qPCR, CLIP–qPCR and eCLIP-seq sample preparation. K.E.H. performed the bioinformatics analysis of the eCLIP-seq data. M.C. performed microscopy. C.H., J.C.L. and E.T.K. performed the IP assays. C.H., J.M.D., J.C.L. and M.D.W. wrote the manuscript with input from the other authors. M.D.W. and B.A.G. supervised the research.

Corresponding author

Correspondence to Matthew D. Weitzman.

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Extended data

Extended Data Fig. 1 E1B55K deletion or inhibition of Cullin-mediated ubiquitination does not decrease late RNA transcription, decay, or early RNA processing.

a, Quantification of results shown in Fig. 1b. b, Analysis of nascent transcription and mRNA half-life by labeling RNA with 4-thiouridine (4sU) for 30 min at 23.5 hpi in HeLa cells infected with WT or ΔE1B Ad5 at m.o.i. = 10. Nascent 4sU-labeled RNA was purified for RT-qPCR to determine relative transcription rates of two early (E1A and E4) and one late (MLP) viral transcripts. mRNA half-life was approximated using the ratio of nascent and total input RNA levels normalized to GAPDH. c, Analysis of decay of viral early (E1A) and late (MLP) RNA species by Actinomycin D pulse at 24 hpi in HeLa cells infected with WT or ΔE1B Ad5 at m.o.i. 10 by normalization to input levels. d, Schematic illustrating primer design to differentiate spliced and unspliced viral transcripts. e-g, HeLa cells infected with WT or ΔE1B Ad5 (MOI = 10) in the presence of DMSO or NEDDi (neddylation inhibitor MLN4924) added at 8 hours post-infection (hpi). Cells were harvested for RNA analysis at 24 hpi. e, Bar graph representing spliced RNA levels of viral early transcripts E1A by RT-qPCR, f, Bar graph representing splicing efficiency as the ratio of spliced to unspliced transcripts of E1A relative to the WT DMSO control by RT-qPCR. g, Representative RNA FISH visualizing the localization of fiber (green) and GAPDH (magenta) transcripts in relation to nuclear DNA stained with DAPI (blue). Nuclei are labeled with the classification of each cell according to the pattern of fiber used for Fig. 1d. Scale bar=10 μm. All data are representative of two (g) or three (others) biologically independent experiments. All graphs show mean+SD. Statistical significance was calculated using a paired (a) or unpaired (others), two-tailed Student’s t-test, * p < 0.05, ** p < 0.01.

Source data

Extended Data Fig. 2 Quantification for number of peptides and proteins identified in di-glycine remnant profiling and whole cell proteome data sets.

a-c, HeLa cells transduced with rAd E1B55K/E4orf6 (m.o.i. = 10). a, Immunoblot of time course of E1B55K/E4of6 expression showing degradation kinetics of known substrates. hpt=hours post-transduction. Representative of 3 biologically independent experiments. b-c, Peptide and protein counts for three biologically independent replicates (dark, medium, and light blue bars). b, Total peptides and corresponding proteins identified following K-ɛ-GG antibody enrichment combined with mass spectrometry analysis at 0, 6, 8, and 10 hours post E1B55K/E4orf6 expression. c, Total proteins identified by whole cell proteomics analysis at time 0 and 10 h of E1B55K/E4orf6 expression.

Source data

Extended Data Fig. 3 Di-glycine remnant profiling and whole cell proteome data for RNA-binding proteins enriched within predicted E1B55K/E4orf6 substrates.

a-b, Gene ontology analysis identified RNA-binding proteins enriched in proteins that exhibited an increase in normalized protein-based ubiquitin abundance of log2 fold change >1 following 10 h expression of E1B55K/E4orf6. a, Enriched RNA-binding proteins ubiquitination log2 fold changes (left) and whole cell protein abundance log2 fold changes (right) comparing 0 and 10 h of E1B55K/E4orf6 expression. b, Heat map showing relative ubiquitination of indicated lysine residues at 0, 6, 8, and 10 h of E1B55K/E4orf6 expression within enriched RNA-binding proteins. Colors correspond to the average z-score of the ubiquitination. All data are representative of three biologically independent experiments.

Extended Data Fig. 4 Network analysis of predicted E1B55K/E4orf6 substrates identifies a ‘RALY/hnRNP-C module’ enriched for RNA-binding proteins.

a, The Reactome-FI application in Cytoscape was utilized to generate a protein-protein interaction network for proteins increased in ubiquitination following E1B55K/E4orf6 transduction. Nodes represent proteins and edges represent Reactome-based protein-protein interactions. Node size corresponds to relative protein-based ubiquitination log2 fold change and node color corresponds to whole cell proteome log2 fold change following 10 h expression of E1B55K/E4orf6. Reactome-FI interaction module analysis was performed to identify clusters of highly interacting proteins. b, Gene ontology analysis for molecular function, as generated by ReactomeFI (FDR is Benjamini-Hochberg method), identified enrichment of RNA-binding and Poly(A) RNA-binding proteins within the RALY/hnRNP-C network module. c, RALY/hnRNP-C network module protein ubiquitin log2 fold changes (left) and whole cell protein abundance log2 fold changes (right) comparing 0 and 10 h of E1B55K/E4orf6 expression. d, Heat map showing relative ubiquitin abundance of indicated lysine residues at 0, 6, 8, and 10 h of E1B55K/E4orf6 expression from proteins within the RALY/hnRNP-C network module. Colors correspond to the average z-score of ubiquitination. All data are representative of three biologically independent experiments.

Extended Data Fig. 5

MS2 evidence for ubiquitination site localization in RALY (a) and hnRNP-C (b–f) peptides. Spectra were obtained from LC-MS/MS analyses using collision-induced dissociation (CID) at 35%, and identified in MaxQuant 1.6.0.16. All modified residues can be confidently identified by confirming ions, except for hnRNP-C K198 (d), which lacks ions to distinguish between K197 and K198. Best evidence spectra were selected for annotation of b-ion (blue) and y-ion (red) series and their masses for singly- and doubly-charged fragments. All data are representative of three biologically independent experiments.

Extended Data Fig. 6 RALY and hnRNP-C are not decreased upon transduction in multiple cell lines.

a, Quantification of results shown in Fig. 3c. b, HEK293 cells transfected with the indicated constructs for 24 h, followed by denaturing IP with HA antibody, and immunoblot analysis of hnRNP-C2-Flag. c, Quantification of results shown in Fig. 3d. d, Immunoblot analysis of protein levels in HeLa cells over a time course of transduction with recombinant Ad vectors expressing only E1B55K and E4orf6 (m.o.i. = 10). e, Immunoblot analysis of protein levels in HeLa, U2OS, A549 and HEK293 cells. HeLa, U2OS and A459 cells were transduced with recombinant Ad vectors expressing only E1B55K and E4orf6 for 24 h. HEK293 cells, which contain an endogenous copy of E1B55K, were mock transfected or transfected with plasmids expressing E4orf6 or GFP. f, Bar graphs of RALY and hnRNP-C RNA levels over a time course of infection with Ad5 WT (m.o.i. = 10) relative to mock as determined by RT-qPCR. g, Quantification of results shown in Fig. 3i. All data are representative of three (four for RALY in g) biologically independent experiments. All graphs show mean+SD. Statistical significance was calculated using a paired, two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005.

Source data

Extended Data Fig. 7 RALY and hnRNP-C single knockdowns rescue late protein, RNA and splice efficiency during infection with Ad ΔE1B.

a-d, HeLa cells transfected with control (siCTRL) or RALY and hnRNP-C (siRALY/hnRNP-C) siRNA 24 h prior to infection with Ad5 WT or ΔE1B (m.o.i. = 10), harvested at respective time points. a, Quantification of results shown in Fig. 4a. b, Extended immunoblot analysis of viral and cellular protein levels. c, Bar graph representing spliced RNA levels of viral early transcript E1A measured by RT-qPCR. d, Bar graph representing splicing efficiency (ratio of spliced to unspliced transcripts) of E1A measured by RT-qPCR. e-g, HeLa cells transfected with control siRNA (siCTRL), siRNA for RALY (siRALY), siRNA for hnRNP-C (sihnRNP-C) or siRNA for both RALY and hnRNP-C (siRALY/hnRNP-C) 24 h prior to infection with Ad5 WT or ΔE1B (m.o.i. = 10) and harvested at 24 hpi. e, Immunoblot analysis of viral and cellular protein levels. f, Bar graph representing spliced RNA levels of E1A, MLP and fiber measured by RT-qPCR. g, Bar graph representing splicing efficiency of E1A, MLP and fiber measured by RT-qPCR. h-j. HeLa cells transfected with control (siCTRL) or RALY and hnRNP-C (siRALY/hnRNP-C) siRNA 24 h prior to infection with Ad5 WT (m.o.i. = 10), treated with either DMSO or NEDDi at 8 hpi and processed at 24 hpi. h, Bar graph representing spliced RNA levels of E1A measured by RT-qPCR. i, Bar graph representing splicing efficiency as defined as the ratio of spliced to unspliced transcripts of E1A measured by RT-qPCR. j, Quantification of results shown in Fig. 4i. All data are representative of three biologically independent experiments. All graphs show mean+SD. Statistical significance was calculated using paired (a and j) or unpaired (others), two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005.

Source data

Extended Data Fig. 8 No dramatic difference in protein localization and protein-complex formation of RALY and hnRNP-C between Ad WT and ΔE1B infection.

a, Representative images of immunofluorescence comparing the localization of RALY and hnRNP-C (both green) in mock, Ad WT and ΔE1B infection of HeLa cells (m.o.i. = 10, 24 hpi). Viral replication centers are stained by DBP or USP7 (both magenta) and nuclear DNA by DAPI (grey). Scale bar=10 μm. b, Immunoblot analysis of RALY and hnRNP-C protein complexes formed upon mock, Ad WT and ΔE1B infection of HeLa cells (m.o.i. = 10) and treatment with indicated concentrations of disuccinimidyl suberate (DSS) for 30 min at 24 hpi. c, Schematic for targeted hnRNP-C RNA-binding region identification (RBR-ID) and interactome. d, Data for RALY from hnRNP-C RBR-ID experiment comparing mock (grey), Ad5 WT (black), and ΔE1B (red) at 24 hpi (m.o.i. = 10). Shown are smoothed residue-level RBR-ID score plotted along the primary sequence (left) and smoothed residue-level fold change between crosslinked and control conditions with a significance threshold of p < 0.05 (right). RALY domain structure with ubiquitination site is shown above graphs. RNA-binding regions are highlighted in blue. e, Correlation matrix for hnRNP-C interactome between replicates of mock, Ad5 WT, and Ad5 ΔE1B. Color gradient is based on the Pearson correlation coefficient. f, Comparison of z-scores for top 20 proteins identified in hnRNP-C interactome during WT Ad5 infection (m.o.i. = 10, 24 hpi). Mock=grey, Ad5 WT = black, Ad5 ΔE1B = red. All data are representative of three biologically independent experiments.

Source data

Extended Data Fig. 9 In the absence of E1B55K, hnRNP-C and RALY interact more with viral late RNA.

a, Control immunoblot for hnRNP-C CLIP-qPCR in Fig. 5b. Higher molecular weight complexes represent hnRNP-C crosslinked to RNA. * indicates antibody heavy chain. b, Schematic of Ad5 genome and viral transcription units. Amplicons for early (E1A, DBP, E4) and late (MLP, Penton, Hexon, 100 K, Fiber) transcription units indicated. c, HeLa cells infected with WT Ad5 or ΔE1B (m.o.i. = 10), UV-crosslinked and harvested at 24 hpi, subjected to hnRNP-C CLIP with 10% input as compared to Fig. 5b and RT-qPCR of indicated transcripts. d, HeLa cells infected with WT Ad5 or ΔE1B (m.o.i. = 10), without UV-crosslinking, and harvested at 24 hpi, subjected to hnRNP-C CLIP and RT-qPCR of indicated transcripts. e, HeLa cells infected with WT Ad5 or ΔE1B (m.o.i. = 10), UV-crosslinked and harvested at 24 hpi, subjected to IgG CLIP and RT-qPCR of indicated transcripts. f, HeLa cells induced for RALY-Flag expression with doxycycline for 3 days total, infected with WT Ad5 or ΔE1B (m.o.i. = 10), UV-crosslinked and harvested at 24 hpi, subjected to Flag CLIP and RT-qPCR of indicated transcripts. For all CLIP-qPCR experiments: GAPDH = negative control, hnRNP-C = positive control. g, Immunoblot for hnRNP-C CLIP-qPCR in Fig. 5c. Higher molecular weight complexes represent hnRNP-C crosslinked to RNA. * indicates antibody heavy chain. h, Number of mapped hnRNP-C eCLIP-Seq reads for the indicated conditions. i, Percentage of mapped reads that uniquely mapped to Ad5 genome for different hnRNP-C eCLIP-Seq conditions. j, PCA plot for hnRNP-C peaks mapped to host transcripts comparing mock (grey), Ad5 WT (black), and Ad5 ΔE1B (red). k, Top 6 hnRNP-C binding motifs in WT and ΔE1B infection, WT infection only, and ΔE1B infection only. n = 1043, 162 peaks, and 267 peaks, respectively. Enriched motifs were identified with HOMER Software Package using hypergeometric enrichment calculations and adjustments for multiple comparisons. All data are representative of two (h-k) or three (a-g) independent experiments. Graphs in c-f show mean+SD. Statistical significance was calculated using unpaired, two-tailed Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.005.

Source data

Extended Data Fig. 10 Non-degradative ubiquitination of RNA-binding proteins promotes efficient adenoviral RNA processing.

During wild-type (WT) Ad5 infection the E1B55K/E4orf6 complex induces ubiquitination of RNA-binding proteins RALY and hnRNP-C to facilitate efficient viral late RNA processing. Ubiquitination regulates interaction of these host proteins with viral RNA to facilitate viral infection. In the absence of the E1B55K/E4orf6 complex ubiquitin ligase activity, the RBPs bind more to viral late mRNAs and limit RNA processing and protein production. RNP = ribonucleoprotein.

Supplementary information

Supplementary Information

Supplementary Sections 1 and 2: literature review of identified RBPs and primer sequences.

Reporting Summary

Supplementary Tables

Supplementary Table 1: Complete di-glycine remnant and WCP data. Normalized and log2-transformed proteomics data for K-ɛ-GG peptide-based quantification (tab 1), transformation to K-ɛ-GG protein-based quantification (tab 2) and whole-cell proteome quantification. Data were generated from three biologically independent experiments. Fold changes were calculated in log2 and P values were calculated using unpaired, two-sided Student’s t-tests. Supplementary Table 2: Di-glycine remnant and WCP data for predicted E1B55K/E4orf6 substrates. Normalized and log2-transformed proteomics data for K-ɛ-GG protein-based (tab 1) and peptide-based (tab 2) quantifications for predicted E1B55K/E4orf6 substrates. Data were generated from three biologically independent experiments. Significance tests for whole-cell proteome abundance changes were calculated using unpaired, two-sided Student’s t-tests. K-ɛ-GG abundance changes were normalized to whole-cell proteome abundance changes and significance was calculated using one-sided Student’s t-tests with a null hypothesis of 0 fold change. Supplementary Table 3: Reactome network protein–protein interactome analysis results. Reactome FI network analysis results of the proteins that exhibited increased protein-based ubiquitination (n = 120). The Reactome FI clustering algorithm was used to divide the interactome into modules (tab 1). Reactome FI was used to perform GO and pathway enrichment analysis. The GO ‘Molecular Functions’ is included for modules (tab 2) and the entire network (tab 5). The GO ‘Biological Processes’ is included for modules (tab 3) and the entire network (tab 6). The Reactome Pathway analysis is included for modules (tab 4) and the entire network (tab 7). Data xwere generated from three biologically independent experiments. Supplementary Table 4: SILAC ratios for chymotryptic and tryptic peptides from targeted hnRNP-C RBR-ID analysis. Heavy/light ratios indicate the proportion of peptides crosslinked to RNA after UV light treatment. Data represent three biological replicates and two technical replicates (n = 6) per digestion condition. Chymotryptic (tab 1) and tryptic (tab 2) data were processed independently in Proteome Discoverer v.2.3. Results were filtered to exclude peptides with any missing values across replicates. Supplementary Table 5: hnRNP-C interactome derived from RBR-ID data. The non-crosslinked sample from the hnRNP-C RBR-ID was used to generate the hnRNP-C interactome. Normalized, log2-transformed data quantification for proteins identified in association with hnRNP-C. Data were generated from three biological replicates and two technical replicates (n = 6) per digestion condition. Fold changes were calculated in log2 and P values were calculated using unpaired, two-sided Student’s t-tests.

Source data

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Herrmann, C., Dybas, J.M., Liddle, J.C. et al. Adenovirus-mediated ubiquitination alters protein–RNA binding and aids viral RNA processing. Nat Microbiol 5, 1217–1231 (2020). https://doi.org/10.1038/s41564-020-0750-9

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