Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase

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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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Figure 1: RACK1 regulates poxvirus protein synthesis.
Figure 2: VacV B1 kinase phosphorylates RACK1.
Figure 3: RACK1 modification correlates with enhanced polyA-leader activity.
Figure 4: Plant RACK1 loop mimicry controls selectivity towards viral RNAs.

Change history

  • 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.

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Authors

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.

Corresponding author

Correspondence to Derek Walsh.

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The authors declare no competing financial interests.

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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 figures and tables

Extended Data Figure 1 Annotated MS/MS spectra for RACK1 modifications detected in VacV-infected or B1–mCherry-expressing NHDFs.

a, Fully annotated MS/MS spectra including scan and charge state of indicated RACK1 peptides from VacV-infected cells. b, Fully annotated MS/MS spectra including scan and charge state of indicated RACK1 peptides from B1–mCherry-expressing cells. MS analysis represents two (a) and one (b) replicate(s).

Extended Data Figure 2 PKCβII inhibition suppresses RACK1 modification.

a, Effects of RACK1 or PKCβII depletion, or PKCβII inhibition using PMA, on the formation of RACK1 doublets and RACK1 mobility in uninfected or VacV-infected NHDFs. Doublets and migration shifts to majority of RACK1 in the slower-migrating form are indicated. b, c, To compensate for natural distortion of bands in the outer lanes in SDS–PAGE gels when analysing multiple samples, portions of the uncropped blot in a are aligned to illustrate the presence of doublets and the migration of phosphorylated (P) and non-phosphorylated (non-P) species of RACK1 under each condition. Data are representative of 3 independent replicates. For raw gel data see Supplementary Fig. 1.

Extended Data Figure 3 PKCβII inhibition prevents host shut-off and formation of viral factories associated with late stages of VacV infection.

a, Inhibition of PKCβII using 50 nM PMA reduces rates of late viral protein synthesis and prevents the inhibition of host translation (host shut-off) associated with late stages of VacV infection. PMA does not affect HSV-1. b, Depletion of PKCβII using three independent siRNAs results in a dose-dependent effect on host shut-off and the rates of late viral protein synthesis. c, Representative images of the effects of PMA treatment on viral factory formation relative to DMSO solvent controls. Fixed samples were stained with Hoechst to detect nuclei and viral factories, and anti-I3 antibody. d, Zoomed regions highlighted in c. Mature viral factories (MVFs) stain intensely for DNA and I3. Failed viral factories (fVFs) exhibit punctate staining for I3, characteristic of early viral factories but do not stain strongly for DNA. e, The percentage of mature and failed viral factories in cells treated with DMSO solvent control or PMA. n > 45 viral factories per condition. Note, although small I3 puncta are seen in DMSO-treated cells, these stain strongly for DNA unlike PMA-treated samples. These may represent new factories forming, or fragments of growing factories but failed factories could not be detected. All data represents 3 or more replicates. For raw gel data, see Supplementary Fig. 1.

Extended Data Figure 4 RACK1 is not modified by other viruses, and its modification in VacV-infected cells is dependent upon the viral B1 kinase.

ad. RACK1 is not modified in uninfected cells, or cells infected by other DNA or RNA viruses. a, RACK1 doublets are only detectable in NHDFs infected with VacV, but not in uninfected cells or cells infected with HSV-1, EMCV or VSV. Mobility shifts in 4E-BP1 indicative of hypo- or hyper-phosphorylation illustrate expected mTOR activation upon VacV or HSV-1 infection, and mTOR suppression by EMCV or VSV. b, c, Patterns of protein synthesis further confirm that samples analysed in a, were infected with EMCV (b) or VSV (c). d, Summary table displaying MS/MS results of unmodified and phosphorylated peptides of RACK1 from uninfected NHDFs, NHDFs infected with the indicated viruses, or B1–mCherry expressing NHDFs. %AA, percentage of amino acids detected; peptide #, number of peptides identified; spectral #, number of MS/MS spectral counts. In all analyses, the protein FDR is ≤1% based on the target decoy strategy. e, B1 mutants fail to form RACK1 doublets at permissive and non-permissive temperatures. Western blot analysis showing that B1 mutants do not produce B1 protein at either permissive (32 °C) or non-permissive (37 °C) temperatures. In line with unique B1 substrates not complemented by host kinases, RACK1 doublets (arrows) are not detected at either temperature. Notably, the extent of doublet formation correlates with B1 expression levels. At 32 °C, wild-type virus produces less B1 than at 37 °C, modifying RACK1 to a lesser extent. F10 mutants produce more B1 than wild-type virus at 32 °C and modify RACK1 more efficiently. This suggests that F10 has the potential to negatively regulate B1, in line with results from luciferase assays in Fig. 3e. Data represents 3 or more replicates; MS/MS represents 2 replicates (d). For raw gel data see Supplementary Fig. 1.

Extended Data Figure 5 B1–mCherry localizes to viral factories along with RACK1.

a, Immunofluorescence analysis of cells expressing B1–mCherry and RACK1–eGFP, either mock-infected or infected with VacV. Hochest was used to stain DNA. Zoomed images of orange boxed region are presented in lower panels. iVF, immature viral factory; mVF, mature viral factory with cavities formed; nuc, nucleus. b, c, Additional examples of B1 and RACK1 localization in different immature and mature factories. Data represents three independent experiments.

Extended Data Figure 6 RACK1 localization to viral factories in living cells.

a, eGFP alone localizes to both nuclei and the cytoplasm, with no notable localization to viral factories (yellow arrows). be, Still images from Supplementary Videos 1, 2, 3, 4, 5, 6, 7, 8, 9, highlighting the dynamic behaviour of RACK1 within viral factories. b, Still image from Supplementary Video 1 showing immature viral factories (iVFs) that lack RACK1 accumulation, and a mature viral factory (mVF) with large cavity filled with RACK1. In some cells, what appear to be old viral factories (oVFs) or Golgi structures that do not stain strongly for active DNA synthesis using Cro–mCherry and appear disorganized can also be seen. c, Still images from Supplementary Videos 2 and 3 showing smaller, immature viral factories (iVF) in which a cavities form and fill with RACK1. In larger mature factories (mVF), cavities dynamically grow and change shape, but retain RACK1. d, Still images from Supplementary Videos 4 and 5, showing additional examples of mVFs with peripheral and cavity accumulations of RACK1. e, Still images from Supplementary Videos 6, 7, 8, 9 showing zoomed viral factories and the dynamic behaviour of RACK1 within viral cavities (yellow arrows). This includes the formation of new cavities, which is accompanied by the appearance of RACK1 at these sites. Data represent 3 independent experiments.

Extended Data Figure 7 VacV infection does not alter RACK1 interactions with ribosomal proteins or polyA-binding protein isoforms.

a, Table summarizing ribosomal and polyA-binding protein interactions identified by LC–MS/MS proteomic analysis of uninfected or VacV-infected NHDFs expressing RACK1–eGFP or eGFP. Samples sets are: (1) uninfected NHDFs expressing RACK1–eGFP; (2) VacV-infected NHDFs expressing RACK1–eGFP; (3) VacV-infected NHDFs expressing eGFP. Yellow, 40S ribosomal subunits; green, 60S ribosomal subunits; blue, polyA-binding protein isoforms. b, To validate results obtained from LC–MS/MS analysis in a, NHDFs expressing eGFP or RACK1–eGFP were either mock-infected or infected with VacV. eGFP or RACK1–eGFP complexes were isolated from soluble cell extracts and analysed by western blotting with the indicated antibodies. In line with LC–MS/MS analysis, the association of RACK1 with representative large and small ribosomal subunits was unaffected by infection. Demonstrating specificity, β-actin was not recovered in eGFP or RACK1–eGFP complexes. c, To determine whether modified RACK1 associates with ribosomes, NHDFs were treated with RACK1-targeting siRNA and infected with VacV. Under these conditions, all remaining RACK1 in VacV-infected cells is in the modified state (Fig. 1c, f). Free, 40S, 60S, 80S and polysome fractions from VacV-infected NHDFs were analysed by western blotting and show RACK1 is only detected in ribosomal fractions and not free fractions. All data represents 3 independent replicates, except a, MS represents 2 replicates. For raw gel data see Supplementary Fig. 1.

Extended Data Figure 8 RACK1 co-localizes with RPS10 within viral factories.

Mock or infected samples were fixed and stained for RPS10, along with Hoechst to detect nuclei and viral factories. RACK1–eGFP around and within cavities of viral factories co-localized with RPS10, a validated interacting partner of RACK1 in Extended Data Fig. 7a, b. Data represents 3 independent experiments.

Extended Data Figure 9 Analysis of reporter RNA levels and phylogenetic comparison of RACK1 from different species.

ac, Luciferase reporters RNA levels are not significantly affected by VacV infection and do not correlate with enhancer effects. a, Agarose gels showing the specificity of primers used to detect luciferase and β-actin mRNAs in samples. b, qPCR analysis of RNA levels represented as fold-change in infected samples over mock-infected samples. Minimal changes in RNA abundance are detected in infected cells and do not correlate with enhancer selectivity towards polyA-leaders. Statistical analysis demonstrated no significant differences in RNA abundance between mock versus infected. Bars represent s.e.m. n = 3 per group. β-actin-luciferase, P value = 0.176376; polyU-luciferase, P value = 0.194325; polyA-luciferase group, P value = 0.765344. *P < 0.05; two tailed t-test. c, B1 or F10 mutants do not alter polyA-luciferase RNA levels relative to wild-type infection in a manner that would explain their differential effects on polyA-enhancer function. Data shows qPCR analysis of RNA levels represented as fold-change in Condit temperature sensitive (Cts)-mutant-infected samples over wild-type VacV-infected samples. Error bars represent standard error of the mean. Statistical analysis demonstrated no significant differences in RNA abundance (one-way ANOVA, n = 3 per group, P value = 0.9271. *P value <0.05 relative to wild-type VacV). Post-hoc analysis was performed between wild type and B1 mutant; P value 0.8999947, wild-type VacV and F10 mutant; P value 0.899994. In all groups P > 0.05 relative to wild-type VacV, further confirming that the mutant viral infection does not affect the abundance of polyA-luciferase RNA. d, Phylogenetic comparisons reveal variability in charge and structure in the RACK1 loop that is modified by VacV. RACK1 is highly conserved with the notable exception of the extended loop highlighted by the green box. See also Fig. 4b. β, beta sheets. For raw data see Supplementary Fig. 1. Source data

Extended Data Figure 10 Negative charge in the RACK1 loop confers a selective advantage to 5′ polyA-RNAs.

a, Recovery of β-actin and 5′ polyA-luciferase RNAs associated with wild-type or loop mutant forms of RACK1–eGFP in reporter-expressing cells. RACK1 phosphomimetics or the plant-loop chimaera select against β-actin but not polyA-luciferase RNAs, recapitulating the selective advantage for viral RNAs observed in infected cells when the RACK1 loop is phosphorylated. Similar to viral RNAs, the STSS-EEEE mutant retains selectivity against β-actin RNA but with suboptimal association with 5′ polyA-luciferase RNA. This assay also enabled examination of 5′ polyA-RNA recovery in an uninfected context, and demonstrated that 5′ polyA-RNA also efficiently associates with wild-type RACK1 complexes. This provides further support for the overall model whereby VacV customizes host ribosomes to regulate sliding on its unusual 5′ polyA RNAs, but in doing so simultaneously suppresses translation of canonical host RNAs as illustrated in b. Data represents 3 independent experiments. For raw gel data see Supplementary Fig. 1. b, Model for ribosome customization by VacV-induced modifications to the RACK1 loop. Left, the translation system in uninfected cells is optimized for efficient β-actin RNA translation. This involves binding, scanning, efficient AUG recognition and formation of 80S ribosomes that translate the open reading frame. Upon VacV-mediated phosphorylation of RACK1 (or in cells expressing RACK1 phosphomimetics or the plant-loop chimaera), the canonical host initiation process is suppressed, reducing RNA recovery. Right, in uninfected cells non-canonical 5′ polyA-RNAs are efficiently loaded with 40S subunits. However, sliding (arrows) reduces the efficiency of AUG recognition, with 40S subunits frequently scanning past the start site rather than assembling 80S subunits to initiate translation of the open reading frame. As such, these unusual RNAs will associate with wild-type RACK1 despite the fact that their translation is sub-optimal. Upon VacV infection (or in cells expressing RACK1 phosphomimetics or the plant loop chimaera) negative charge in the RACK1 loop off-sets sliding, generating an optimized scanning and AUG recognition process for 5′ polyA-RNAs. Under modified RACK1 conditions in which 5′ polyA-RNAs are initiating efficiently, excessive charge clustering (STSS-EEEE mutant) is suboptimal and is evident in reduced 5′ polyA or viral RNA recovery.

Supplementary information

Supplementary information

This file contains Supplementary Figures 1-2. (PDF 1073 kb)

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). (MOV 16640 kb)

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). (MOV 18122 kb)

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). (MOV 8509 kb)

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). (MOV 10520 kb)

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). (MOV 12118 kb)

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). (MOV 6736 kb)

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). (MOV 22715 kb)

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). (MOV 1661 kb)

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). (MOV 8882 kb)

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Jha, S., Rollins, M., Fuchs, G. et al. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase. Nature 546, 651–655 (2017). https://doi.org/10.1038/nature22814

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