Sequestration by IFIT1 Impairs Translation of 2′O-unmethylated Capped RNA

<div><p>Viruses that generate capped RNA lacking 2′O methylation on the first ribose are severely affected by the antiviral activity of Type I interferons. We used proteome-wide affinity purification coupled to mass spectrometry to identify human and mouse proteins specifically binding to capped RNA with different methylation states. This analysis, complemented with functional validation experiments, revealed that IFIT1 is the sole interferon-induced protein displaying higher affinity for unmethylated than for methylated capped RNA. IFIT1 tethers a species-specific protein complex consisting of other IFITs to RNA. Pulsed stable isotope labelling with amino acids in cell culture coupled to mass spectrometry as well as <i>in vitro</i> competition assays indicate that IFIT1 sequesters 2′O-unmethylated capped RNA and thereby impairs binding of eukaryotic translation initiation factors to 2′O-unmethylated RNA template, which results in inhibition of translation. The specificity of IFIT1 for 2′O-unmethylated RNA serves as potent antiviral mechanism against viruses lacking 2′O-methyltransferase activity and at the same time allows unperturbed progression of the antiviral program in infected cells.</p></div

Human and mouse IFIT1 bind directly to unmethylated capped RNA.

<p>(<b>a</b>) Isolation of luciferase-tagged human IFIT (hIFIT) proteins from transfected 293T cells with beads coated with 250 ng RNA bearing 5′ OH, PPP or CAP. The graphs show luciferase activity after affinity purification (AP) with PPP-RNA and CAP-RNA (normalized to OH-RNA) and the activity of 10% of the input lysates. (<b>b</b>) Data obtained (as in <b>a</b>) for luciferase-tagged murine Ifit (mIfit) proteins affinity purified with PPP-RNA and CAP-RNA. (<b>c</b>) Recombinant His-tagged hIFIT1, -2, -3, and -5 were incubated with beads only or beads coated with OH-RNA or CAP-RNA. Bound proteins were detected by western blotting. Input shows 1/10^th of the amount incubated with beads. (<b>d</b>) Purification of luciferase-tagged wild-type (WT) and hIFIT1 mutants with CAP-RNA-coated beads. The graphs show luciferase activity after affinity purification and the activity of 10% of the input lysates. (<b>e</b>) Ratios of LFQ intensities of proteins identified by mass spectrometry in precipitates of CAP-RNA vs. OH-RNA in IFN-α-treated MEFs from wild-type (Ifit1^+/+, grey bars) and Ifit1-deficient (Ifit1^−/−, black bars) C57BL/6 mice. Error bars indicate means (±SD) from three independent affinity purifications. Asterisks indicate ratios with negative values.</p

IFIT1 specifically blocks translation of 2′-O-unmethylated capped viral RNA.

<p>(<b>a</b>) Experimental design used to assess the stability of MHV RNA in infected cells. Bone marrow-derived macrophages (Mφ) from C57/BL6 mice were treated with 50 U of IFN-α for 2 h prior to infection with wild-type MHV (WT) or 2′O methyltransferase-deficient MHV (DA) at 4°C for 1 h. Directly after infection, cells were treated with 100 µg/ml cycloheximide (CHX) or DMSO. Total RNA was harvested at 0, 4, and 8 h post infection and analysed by quantitative RT-PCR. (<b>b</b>) MHV nucleoprotein (MHV-N) RNA in cells infected with MHV WT (grey) or DA mutant (red), treated with DMSO (solid lines) or CHX (dashed lines). Data from one representative experiment of three are depicted, showing means ±SD after normalization to a known amount of in vitro transcribed <i>Renilla</i> luciferase RNA (Ren) added to cell lysates. (<b>c</b>) Experimental design for pulsed SILAC coupled to mass spectrometry to determine relative changes in protein translation during infection. Macrophages from C75/BL6 (Ifit1^+/+) and Ifit1-deficient (Ifit1^−/−) mice grown in normal growth medium containing light (L) amino acids were infected at 4°C for 1 h with wild-type MHV (WT) or 2′O methyltransferase-deficient MHV (DA). Five hours post infection cells were incubated with starvation medium (lacking Lys and Arg) for 30 min, then SILAC medium containing heavy (H) labelled amino acids (Lys8, Arg10) was added, and 2 h later total protein lysate was prepared and subjected to LC-MS/MS analysis. (<b>d</b>) Translation rates for 721 cellular proteins, as determined by heavy (H) to light (L) ratios from LC-MS/MS, were plotted as box-whisker plots (whiskers from 10th to 90th percentile). Individual ratios for the MHV nucleoprotein (MHV-N) and membrane protein (MHV-M) in WT- (grey) and DA-infected (red) Ifit1^+/+ (circles) and Ifit1^−/− (triangles) macrophages are plotted separately. Data are from three independent experiments. (<b>e,f</b>) Principal Component Analysis based on valid H/L ratios of all measurements from (<b>d</b>) showing clustering of the individual samples of the entire dataset (<b>e</b>). Panel (<b>f</b>) shows all proteins plotted for their contribution to the variation in components 1 and 2. MHV proteins are indicated in blue.</p

IFIT1 inhibits viral RNA and protein synthesis in cells infected with 2′O methyltransferase-deficient coronavirus.

<p>(<b>a–b</b>) HeLa cells were cotransfected for 48 h with an expression construct for the HCoV-229E receptor, human aminopeptidase N, and siRNAs targeting IFIT1 or the green fluorescent protein (GFP). Cells were then treated with 20 U IFN-α and infected with wild-type HCoV-229E (229E-WT; grey bars) or the 2′O methyltransferase-deficient HCoV-229E (D129A) mutant (229E-DA; red bars). Total RNA and protein were harvested 24 h post infection and analysed by quantitative RT-PCR (<b>a</b>) and western blotting (<b>b</b>), respectively. Quantitative RT-PCR data are from one of three representative experiments showing means ±SD for HCoV-229E nucleoprotein (229E-N) RNA after normalization to cyclin B (CycB) mRNA. (<b>c–d</b>) Bone marrow-derived macrophages (Mφ) derived from C57BL/6 (Ifit1^+/+) and Ifit1-deficient (Ifit1^−/−) mice were treated or not with 50 U of IFN-α for 2 h and infected with wild-type MHV (WT; grey bars) or 2′O methyltransferase-deficient MHV (DA; red bars). RNA and protein were harvested 8 h post infection and analysed by quantitative RT-PCR (<b>c</b>) and western blotting (<b>d</b>). Quantitative RT-PCR results are from one of three representative experiments, showing means ±SD for MHV nucleoprotein (MHV-N) RNA after normalization to the TATA-binding protein (TBP) mRNA. (<b>e</b>) Ifit1^+/+ and Ifit1^−/− mice were infected intraperitoneally with 5,000 plaque-forming units of MHV WT (grey bars) or DA (red bars). Viral titers in the spleens of 12 mice per condition were measured 48 h after infection. Data are shown as Tukey box-whisker plots (ND, not detectable; outlier indicated as black dot).</p

Mass spectrometry-based identification of human and murine interactors of capped RNA.

<p>(<b>a</b>) Schematic depiction of the experimental approach used for mass spectrometry (MS)-based identification of cellular RNA binding proteins. Biotinylated RNA with different 5′ end structures (OH, PPP, CAP, CAP0, CAP1) was coupled to streptavidin beads, and incubated with lysates obtained from cells that had been left untreated or treated with 1000 U/ml IFN-α for 16 h. Bound proteins were denatured, alkylated and directly digested with trypsin. The resulting peptides were subjected to shotgun liquid chromatography-tandem MS (LC-MS/MS). Three independent experiments were performed for each RNA bait, and the data were analysed with the MaxQuant software <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#ppat.1003663-Cox1" target="_blank">[37]</a> using the label-free quantification algorithm <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#ppat.1003663-Luber1" target="_blank">[38]</a>. (<b>b–d</b>) Proteins obtained from lysates of IFN-α-treated HeLa cells using the indicated biotinylated RNA baits were analysed by LC-MS/MS. Volcano plots show the degrees of enrichment (ratio of label-free quantitation (LFQ) protein intensities; x-axis) and p-values (t-test; y-axis) by PPP-RNA (<b>b</b>), CAP-RNA (<b>c</b>), and CAP1-RNA (<b>d</b>) baits as compared to OH-RNA. Significantly enriched interactors (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#s4" target="_blank">Materials and Methods</a>) are separated by a hyperbolic curve (dotted line) from background proteins (blue dots), known cap-binding proteins (dark-green), and proteins known to associate with capped RNA (light green). Interferon-induced proteins <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#ppat.1003663-Shapira1" target="_blank">[21]</a> detected in the significantly enriched fractions (IFIT1-3 and 5, DDX58) are highlighted (red triangles). (<b>e–g</b>) As in (<b>b–d</b>) but for lysates of IFN-α-treated mouse embryo fibroblasts (MEFs). The interferon-induced proteins Ifit1 and Ifit1c <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#ppat.1003663-Liu1" target="_blank">[42]</a> in significantly enriched and non-enriched fractions are highlighted.</p

IFIT1 binds capped RNA in a methylation state-dependent manner.

<p>(<b>a</b>) Ratio of LFQ intensities of proteins identified by LC-MS/MS as significantly enriched in CAP1-RNA relative to CAP-RNA affinity purifications from IFN-treated HeLa cells, after filtering against the set of proteins that showed enrichment relative to 5′ OH-RNA(see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#ppat.1003663.s002" target="_blank">Fig. S2b</a>). Error bars indicate means (±SD) from three independent affinity purifications. (<b>b</b>) Precipitation of endogenous proteins from lysates of IFN-α treated HeLa cells with biotinylated RNA bearing 5′ OH, PPP, CAP, CAP0 or CAP1 structures. Human IFIT1 (hIFIT1), EIF4E and ILF3 in precipitates were detected by western blotting. Input shows 1/10^th (mIFIT1, EIF4E) and 1/30^th (hIFIT1) of the amount incubated with beads. (<b>c</b>) Affinity purification of luciferase-tagged human (hIFIT1) and murine (mIfit1) IFIT1 expressed in 293T cells on beads bearing 5′ OH, CAP, CAP0, or CAP1 RNA. (<b>d</b>) Binding of recombinant IFIT1 to capped RNAs. As in (<b>c</b>), but RNA-coated beads were incubated with recombinant His-tagged mouse Ifit1 (His-mIfit1), human His-hIFIT1 or human His-EIF4E and bound protein was quantified by western blotting. (<b>e</b>) Binding of recombinant His-tagged hIFIT1 and EIF4E to chemically synthesized, biotinylated RNA oligomers. Synthetic triphosphorylated RNAs with (CAP1) or without (CAP0) 2′O-methyl group on the first ribose were capped in vitro using recombinant vaccinia virus capping enzyme (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003663#s4" target="_blank">Materials and Methods</a>). As control we used a synthetic RNA harbouring a 5′ hydroxyl group (OH). Synthetic RNAs were coupled to beads, incubated with recombinant proteins and bound proteins detected by western blotting. Input shows 1/10^th of the amount incubated with beads.</p

Alterations detected in the K22 resistant variants of HCoV-229E.

a<p>Detected by comparison of the nucleotide sequences of HCoV-229E subjected to 10–13 passages in the presence of K22 including its plaque purified variants A-R with those of initial virus or mock-passaged virus (accession number KF293665).</p>b<p>Plaque purified HCoV-229E that served as initial material for the virus passages.</p>c<p>IC50 (µM).</p>d<p>Fold resistance to K22 as related to initial virus is shown in parentheses.</p>e<p>Virus preparation and its plaque purified variants M-R obtained in separate K22 selection experiment.</p>f<p>The virus used for preparation of recombinant nsp6 mutants.</p>g<p>K22 resistant recombinant viruses.</p

Analysis of recombinant HCoV-229E nsp6 mutants.

<p>(<b>A</b>) Predicted topological structure of HCoV-229E nsp6 indicating the location of K22 resistance mutations. Concerning transmembrane domains VI and VII two proposed topologies are shown. (<b>B-C</b>) Recombinant nsp6 mutant viruses are resistant to K22. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at a moi of 0.05 for 45 min at 4°C, and K22 (10 µM) was added at specific time points relative to the end of inoculation period. The infectious cell culture medium and cells were harvested after 24 h of incubation at 37°C, and copy numbers of cell-associated (CA) or extracellular (EX) viral RNA was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments. (<b>D-F</b>) Replication kinetics of recombinant nsp6 mutant viruses. MRC-5 cells were inoculated with nsp6 recombinant HCoV-229E^H121L, HCoV-229E^M159V, HCoV-229E^H121L/M159V or wild-type HCoV-229E at an moi of 0.05 for 1 h at 4°C. The infectious cell culture medium and cells were harvested at specific time points relative to the end of inoculation period, and copy numbers of cell-associated (CA; <b>D</b>) or extracellular (EX; <b>E</b>) viral RNA and infectivity (<b>F</b>) was determined. Data shown are means (±SD) of duplicate determinations from two independent experiments.</p

K22 affects formation of double membrane vesicles (DMVs).

<p>MRC-5 cells growing on Melinex polyester film were infected with wild type HCoV-229E (WT) or with K22-resistant recombinant nsp6 mutant HCoV-229E^M159V (M159V) and incubated for 18 h at 37°C with or without K22. The cells were then fixed with glutaraldehyde and processed for electron microscopy without their scrapping or pelleting. (<b>A</b>) Electron micrographs of cells infected with WT virus show presence of perinuclear clusters of DMVs (arrow) and viral particles (arrowhead), and the lack of their production upon K22 treatment (4 µM). (<b>B</b>) Note presence of DMVs and viral particles in cells infected with K22-resistant nsp6 recombinant HCoV-229E^M159V (M159V) irrespective of the addition of K22. Each image shown was selected from a pool of over 30 images captured in three separate experiments.</p

Alignment of coronavirus nsp6 sequences.

<p>Alignment of nsp6 sequences derived from coronaviruses used in this study was performed with Geneious Software (Biomatters Ltd, New Zealand). Coronavirus species and corresponding GenBank accession numbers are indicated. Membrane domains predicted by TMHMM Server v. 2.0 (<a href="http://www.cbs.dtu.dk/services/TMHMM/" target="_blank">http://www.cbs.dtu.dk/services/TMHMM/</a>) are indicated by cyan shading while conserved amino acid residues are highlighted by black/grey shading. K22 resistance-conferring mutations in HCoV-229E nsp6, identified in this study, are depicted.</p