Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes

Nature Protocols volume 16, pages 516–531 (2021)Cite this article

Subjects

Abstract

The initial interactions between incoming, pre-replicated virion RNA and host protein factors are important in infection and immunity. Yet currently there are no methods to study these crucial events. We established VIR-CLASP (VIRal Cross-Linking And Solid-phase Purification) to identify the primary viral RNA–host protein interactions. First, host cells are infected with 4-thiouridine (4SU)-labeled RNA viruses and irradiated with 365 nm light to crosslink 4SU-labeled viral genomes and interacting proteins from host or virus. The crosslinked RNA binding proteins (RBPs) are purified by solid-phase reversible immobilization (SPRI) beads with protein-denaturing buffers, and then identified by proteomics. With VIR-CLASP, only the incoming virion RNAs are labeled with 4SU, so crosslinking events specifically occur between proteins and pre-replicated virion RNA. Since solid-phase purification under protein-denaturing conditions, rather than sequence-specific nucleic acid purification, is used to pull-down total RNA and crosslinked RBPs, this method facilitates investigation of potentially all RNA viruses, regardless of RNA sequence. Preparation of 4SU-labeled virus takes ∼7 days and VIR-CLASP takes 1 day.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Outline of VIR-CLASP.
Fig. 2: Anticipated results for VIR-CLASP samples.
Fig. 3: Proteomic Analysis of VIR-CLASP.

Similar content being viewed by others

Data availability

Raw and semi-processed proteomics data used to generate Fig. 3 can be accessed from the PRIDE repository with the dataset identifier PXD015863 and was originally published in5.

Code availability

All code used for proteomic analysis and figure generation can be accessed under the GNU General Public License v3.0 at https://github.com/Ascano-Lab.

References

  1. Phillips, S. L., Soderblom, E. J., Bradrick, S. S. & Garcia-Blanco, M. A. Identification of proteins bound to dengue viral RNA in vivo reveals new host proteins important for virus replication. MBio 7, e01865–15 (2016).

    Article  CAS  Google Scholar 

  2. Lenarcic, E. M., Landry, D. M., Greco, T. M., Cristea, I. M. & Thompson, S. R. Thiouracil cross-linking mass spectrometry: a cell-based method to identify host factors involved in viral amplification. J. Virol. 87, 8697–8712 (2013).

    Article  CAS  Google Scholar 

  3. Garcia-Moreno, M. et al. System-wide profiling of RNA-binding proteins uncovers key regulators of virus infection. Mol. Cell 74, 196–211.e11 (2019).

    Article  CAS  Google Scholar 

  4. Zhou, Y. & Routh, A. Mapping RNA-capsid interactions and RNA secondary structure within virus particles using next-generation sequencing. Nucleic Acids Res. 48, e12–e12 (2020).

    Article  Google Scholar 

  5. Kim, B. et al. Discovery of widespread host protein interactions with the pre-replicated genome of CHIKV using VIR-CLASP. Mol. Cell 78, 624–640.e7 (2020).

    Article  CAS  Google Scholar 

  6. Barrows, N. J. et al. Dual roles for the ER membrane protein complex in flavivirus infection: viral entry and protein biogenesis. Sci. Rep. 9, 9711 (2019).

    Article  Google Scholar 

  7. Hafner, M. et al. Transcriptome-wide identification of RNA-binding protein and microRNA target sites by PAR-CLIP. Cell 141, 129–141 (2010).

    Article  CAS  Google Scholar 

  8. Favre, A. et al. 4-Thiouridine photosensitized RNA-protein crosslinking in mammalian cells. Biochem. Biophys. Res. Commun. 141, 847–854 (1986).

    Article  CAS  Google Scholar 

  9. DeAngelis, M. M., Wang, D. G. & Hawkins, T. L. Solid-phase reversible immobilization for the isolation of PCR products. Nucleic Acids Res. 23, 4742–4743 (1995).

    Article  CAS  Google Scholar 

  10. Licatalosi, D. D. et al. HITS-CLIP yields genome-wide insights into brain alternative RNA processing. Nature 456, 464–469 (2008).

    Article  CAS  Google Scholar 

  11. Van Nostrand, E. L. et al. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat. Methods 13, 508–514 (2016).

    Article  Google Scholar 

  12. König, J. et al. iCLIP reveals the function of hnRNP particles in splicing at individual nucleotide resolution. Nat. Struct. Mol. Biol. 17, 909–915 (2010).

    Article  Google Scholar 

  13. Ascano, M., Hafner, M., Cekan, P., Gerstberger, S. & Tuschl, T. Identification of RNA-protein interaction networks using PAR-CLIP. Wiley Interdiscip. Rev. RNA 3, 159–177 (2012).

    Article  CAS  Google Scholar 

  14. Trendel, J. et al. The human RNA-binding proteome and its dynamics during translational arrest. Cell 176, 391–403.e19 (2019).

    Article  CAS  Google Scholar 

  15. Asencio, C., Chatterjee, A. & Hentze, M. W. Silica-based solid-phase extraction of cross-linked nucleic acid-bound proteins. Life Sci. Alliance 1, e201800088 (2018).

    Article  Google Scholar 

  16. Shchepachev, V. et al. Defining the RNA interactome by total RNA-associated protein purification. Mol. Syst. Biol. 15, e8689 (2019).

    Article  Google Scholar 

  17. Berensmeier, S. Magnetic particles for the separation and purification of nucleic acids. Appl. Microbiol. Biotechnol. 73, 495–504 (2006).

    Article  CAS  Google Scholar 

  18. Miller, C. et al. Dynamic transcriptome analysis measures rates of mRNA synthesis and decay in yeast. Mol. Syst. Biol. 7, 458 (2011).

    Article  CAS  Google Scholar 

  19. Hoffmann, M. et al. SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271–280.e8 (2020).

    Article  CAS  Google Scholar 

  20. Jiang, P., Liu, Y., Ma, H.-C., Paul, A. V. & Wimmer, E. Picornavirus morphogenesis. Microbiol. Mol. Biol. Rev. 78, 418–437 (2014).

    Article  Google Scholar 

  21. Quail, M. A., Swerdlow, H. & Turner, D. J. Improved protocols for the illumina genome analyzer sequencing system. Curr. Protoc. Hum. Genet. Chapter 18, Unit 18, 2 (2009).

    Google Scholar 

  22. Telesnitsky, A. & Wolin, S. L. The host RNAs in retroviral particles. Viruses 8, 235 (2016).

    Article  Google Scholar 

  23. Noda, T. et al. Importance of the 1+7 configuration of ribonucleoprotein complexes for influenza A virus genome packaging. Nat. Commun. 9, 54 (2018).

    Article  Google Scholar 

  24. Kaur, P., Lee, R. C. H. & Chu, J. J. H. Infectious viral quantification of chikungunya virus-virus plaque assay. Methods Mol. Biol. 1426, 93–103 (2016).

    Article  CAS  Google Scholar 

  25. Pedersen, I. R. Density gradient centrifugation studies on lymphocytic choriomeningitis virus and on viral ribonucleic acid. J. Virol. 6, 414–420 (1970).

    Article  CAS  Google Scholar 

  26. Lim, C.-K. Virus isolation and preparation of sucrose-banded chikungunya virus samples for transmission electron microscopy. Methods Mol. Biol. 1426, 153–162 (2016).

    Article  CAS  Google Scholar 

  27. Mortz, E., Krogh, T. N., Vorum, H. & Görg, A. Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption/ionization-time of flight analysis. Proteomics 1, 1359–1363 (2001).

    Article  CAS  Google Scholar 

  28. Link, A. J. & Labaer, J. In-gel trypsin digest of gel-fractionated proteins. Cold Spring Harb. Protoc. 2009, pdb.prot5110–pdb.prot5110 (2009).

    Article  Google Scholar 

  29. Smyth, G. K. Linear models and empirical bayes methods for assessing differential expression in microarray experiments. Stat. Appl. Genet. Mol. Biol. 3, Article3 (2004).

    Article  Google Scholar 

  30. Sysoev, V. O. et al. Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. Nat. Commun. 7, 12128–11 (2016).

    Article  CAS  Google Scholar 

  31. Conway, J. R., Lex, A. & Gehlenborg, N. UpSetR: an R package for the visualization of intersecting sets and their properties. Bioinformatics 33, 2938–2940 (2017).

    Article  CAS  Google Scholar 

  32. Dickson, A. M. et al. Dephosphorylation of HuR protein during alphavirus infection is associated with HuR relocalization to the cytoplasm. J. Biol. Chem. 287, 36229–36238 (2012).

    Article  CAS  Google Scholar 

  33. Daffis, S. et al. 2′-O methylation of the viral mRNA cap evades host restriction by IFIT family members. Nature 468, 452–456 (2010).

    Article  CAS  Google Scholar 

  34. Lex, A., Gehlenborg, N., Strobelt, H., Vuillemot, R. & Pfister, H. UpSet: visualization of intersecting sets. IEEE Trans. Vis. Comput. Graph 20, 1983–1992 (2014).

    Article  Google Scholar 

Download references

Acknowledgements

We thank K. L. Rose and W. H. McDonald at the Vanderbilt Mass Spectrometry Research Center for processing of the mass spectrometry samples; M. Albertolle for technical help in the mass spectrometry analysis; T. Voss and J. E. Crowe Jr. (Vanderbilt University Medical Center) for IAV; and T. S. Dermody (University of Pittsburgh School of Medicine) for CHIKV. Finally, we thank members of the Ascano laboratory for their support, collegiality, and critical review of the manuscript. This work was supported by the National Institutes of Health 1R35GM119569-01 (M.A.), CTSA award No.UL1TR000445 from the National Center for Advancing Translational Sciences (B.K. and M.A.), Vanderbilt University Dept. of Biochemistry start-up funds (M.A.), the Chemical Biology of Infectious Disease training grant 5T32AI11254-02 (S.A.), and the Chemistry-Biology Interface training grant 5T32GM065086-14.

Author information

Author notes

  1. Byungil Kim

    Present address: Infectious Disease Therapeutic Research Center, Korea Research Institute of Chemical Technology (KRICT), Daejeon, Republic of Korea

  2. These authors contributed equally: Byungil Kim, Sarah Arcos.

Authors and Affiliations

  1. Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA

    Byungil Kim, Sarah Arcos, Katherine Rothamel & Manuel Ascano

Authors
  1. Byungil Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Sarah Arcos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Katherine Rothamel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manuel Ascano
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.K. and M.A. designed, and B.K. optimized the VIR-CLASP technique. S.A. performed the proteomic analysis and data visualization. S.A., B.K., and M.A. wrote the paper.

Corresponding author

Correspondence to Manuel Ascano.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information Nature Protocols thanks Encarnacion Martinez-Salas, Ding Shou-wei and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Kim, B. et al. Mol. Cell 78, 624–640.e7 (2020): https://doi.org/10.1016/j.molcel.2020.04.013

Barrows, N. et al. Sci. Rep. 9, 9711 (2019): https://doi.org/10.1038/s41598-019-45910-9

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Kim, B., Arcos, S., Rothamel, K. et al. Viral crosslinking and solid-phase purification enables discovery of ribonucleoprotein complexes on incoming RNA virus genomes. Nat Protoc 16, 516–531 (2021). https://doi.org/10.1038/s41596-020-00429-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41596-020-00429-6

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research