Letter

Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase

  • Nature volume 546, pages 651655 (29 June 2017)
  • doi:10.1038/nature22814
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Abstract

Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements1. However, beyond differential subunit expression during development2,3, evidence for regulated ribosome specification within individual cells has remained elusive1. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5′ untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase4 confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders4.

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Change history

  • Updated online 23 June 2017

    Citations to four references in the Methods were corrected to fix misnumbering.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (NIH) R01AI127456 and R21AI105330 to D.W., R00DC013805 to J.N.S., R01AI099506 to P.S., and Catalyst Award C-068 from the Chicago Biomedical Consortium to J.N.S. M.G.R. was supported by training grant T32GM008061. We thank G. McFadden, R. Condit, P. Traktman, D. Evans and Y. Xiang for reagents.

Author information

Author notes

    • Sujata Jha
    •  & Madeline G. Rollins

    These authors contributed equally to this work.

Affiliations

  1. Department of Microbiology-Immunology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA

    • Sujata Jha
    • , Madeline G. Rollins
    • , Dean J. Procter
    •  & Derek Walsh
  2. The RNA Institute, Department of Biological Sciences, University at Albany-SUNY, Albany, New York 12222, USA

    • Gabriele Fuchs
  3. Department of Neurology, Feinberg School of Medicine, Northwestern University, Chicago, Illinois 60611, USA

    • Elizabeth A. Hall
    • , Kira Cozzolino
    •  & Jeffrey N. Savas
  4. Department of Microbiology & Immunology, Stanford University School of Medicine, Stanford, California 94305, USA

    • Peter Sarnow

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Contributions

S.J. and M.G.R. contributed equally to this work. S.J. and M.G.R. generated RACK1 mutants, performed knockout and knockdown experiments, and isolation and analysis of GFP complexes. G.F. and P.S. generated RACK1 knockouts. S.J. and D.J.P. performed and analysed luciferase assays and imaging. K.C. and E.A.H. prepared samples, performed MS and analysed data. J.N.S. analysed and prepared the figures. D.W. designed and analysed experiments. D.W. wrote the manuscript. S.J., G.F., J.N.S. and P.S. edited the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Derek Walsh.

Reviewer Information Nature thanks J. Dinman 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.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary information

    This file contains Supplementary Figures 1-2.

Videos

  1. 1.

    Video 1: RACK1 localization to immature and mature VacV viral factories in living cells

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). RACK1 does not localize to smaller, immature viral factories (in the lower field of view) but accumulates within cavities that form in larger, mature factories (center-left). Cro-mCherry does not efficiently label nuclei of non-dividing primary cells or enlarged, disorganized older viral factories (adjacent to nucleus in center field) as rates of DNA synthesis are low. Timestamp is in minutes. Bar = 20µm. Acquisition rate = 2 frames per minute (fpm). Playback = 7 frames per second (fps).

  2. 2.

    Video 2: RACK1 accumulation within cavity domains of viral factories

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). RACK1 appears in smaller factories as cavities form, and RACK1 puncta enlarge to fill cavities as they grow. Timestamp is in minutes. Bar = 20µm. Acquisition rate = 4 frames per minute (fpm). Playback = 7 frames per second (fps).

  3. 3.

    Video 3: Dynamic reorganization and accumulation of RACK1 relative to cavity domains of viral factories.

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). Large RACK1 accumulations dynamically track with and mirror the behavior of large cavity domains in mature viral factories. RACK1 can also be seen to appear within new cavities as they form. Timestamp is in minutes. Bar = 20µm. Acquisition rate = 1 frames per minute (fpm). Playback = 7 frames per second (fps).

  4. 4.

    Video 4: RACK1 accumulation within cavity domains of viral factories

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). An independent example of the accumulation of RACK1 within cavities within mature viral factories that are actively synthesizing DNA (strongly staining with cro-mCherry), and the disorganized structure of older factories (poorly stained for cro-mCherry). Timestamp is in minutes. Bar = 20µm. Acquisition rate = 4 frames per minute (fpm). Playback = 7 frames per second (fps).

  5. 5.

    Video 5: RACK1 accumulation at peripheral and cavity regions of viral factories

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). RACK1 accumulates at peripheral and cavity subdomains of viral factories. Timestamp is in minutes. Bar = 20µm. Acquisition rate = 4 frames per minute (fpm). Playback = 7 frames per second (fps).

  6. 6.

    Video 6: RACK1 accumulation within viral factories

    Zoomed images from Video 3. Events are marked with green arrows (RACK1) or Red Arrows (VF Cavities): First Arrow Set highlights RACK1 concentrations within large cavities that mirror the dynamic behavior of VF cavities. Second and Third Arrow Sets highlight the appearance of new cavities within factories that contain RACK1. Timestamp is in minutes. Bar = 5µm. Acquisition rate = 1 frames per minute (fpm). Playback = 7 frames per second (fps).

  7. 7.

    Video 7: RACK1 dynamics within viral factories

    Zoomed images from Video 2. Events are marked with green arrows (RACK1) or Red Arrows (VF Cavities): First Arrow Set highlights a small, immature VF with no RACK1 accumulation (upper arrow) and a mature VF cavity containing RACK1 (lower arrow). Second Arrow Set highlights the appearance of RACK1 within the smaller, maturing factory as a cavity forms (upper arrow) and the expansion of RACK1 in the mature VF as its cavity grows (lower arrow). Timestamp is in minutes. Bar = 5µm. Acquisition rate = 4 frames per minute (fpm). Playback = 7 frames per second (fps).

  8. 8.

    Video 8: RACK1 dynamics within viral factories

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). Timestamp is in minutes. Bar = 5µm. Acquisition rate = 1 frame per minute (fpm). Playback = 7 frames per second (fps).

  9. 9.

    Video 9: RACK1 accumulation within viral factories

    Live cell imaging of VacV-infected NHDFs expressing RACK1-eGFP (Left panel; green in merge) and Cro-mCherry (middle panel; red in merge). Timestamp is in minutes. Bar = 5µm. Acquisition rate = 4 frames per minute (fpm). Playback = 7 frames per second (fps).

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