IFN induction by NSV vRNAs depends on RIG-I and the 5′ triphosphate group.

<p>(A) Verification of knockdowns. Human 293T cells were treated with retroviral shRNA constructs directed against either RIG-I or MDA5, and cotransfected with expression constructs for HA-tagged MDA5 (left panels) or GFP-fused RIG-I (right panels). Western blot analysis using antibodies against the respective fusion tags is shown. Detection of cellular β-tubulin was used as an internal control. (B) Effect of shRNA knockdowns on IFN induction by viral RNAs, using the reporter constructs and RNA transfection protocols as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002032#pone-0002032-g001" target="_blank">Fig. 1A</a>. The negative control shRNA construct (CTRL) targets the heat shock 70 interacting protein and was tested to have no effect on IFN induction (data not shown). (C) Genomic RNAs from ZEBOV and NiV were either mock treated, treated with SAP, or treated with SAP in the presence of the phosphatase inhibitor EDTA. IFN-β reporter assays and RNA transfections were performed as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002032#pone-0002032-g001" target="_blank">Fig. 1A</a>. Mean values and standard deviations from 3 independent experiments are shown.</p

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

Negative-strand RNA viruses (NSVs).

<p>Negative-strand RNA viruses (NSVs).</p

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

Non-inducing viral RNAs contain a 5′ monophosphate.

<p>(A) vRNAs of RVFV (panel 1), HTNV (panel 2) and CCHFV (panel 3) were incubated with a 5′monophosphate-specific 5′-3′ exonuclease. After 4 h of incubation, digestion efficacy was tested by RT-PCR analysis using primer pairs specific for the viral S segment. A comparison with untreated vRNAs is shown (lane input RNA). As additional controls, RNA was incubated without enzyme (lane buffer) or H₂O was used for RT-PCR. The faint residual RT-PCR bands obtained after digestion of HTNV or CCHFV vRNAs are most likely caused by a minority of RNAs containing exonuclease-resistant 5′-OH ends. Such 5′-OH ends were previously observed for HTNV vRNA and thought to represent a preparation artifact <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002032#pone.0002032-Garcin1" target="_blank">[18]</a>. (B) Activation of the IFN-β promoter by genomic RNAs isolated from MV and BDV particles. Mean values and standard deviations from 3 independent experiments are shown. (C) Treatment of MV and BDV genomic RNAs with a 5′ monophosphate-specific 5′-3′ exonuclease and subsequent RT-PCR analysis.</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

vRNA binding by RIG-I.

<p>GFP-RIG-I expressed by 293T cells was coupled to protein G Sepharose beads via a GFP-specific antiserum. Beads were incubated with vRNAs of either RVFV, HTNV, CCHFV, or BDV. After extensive washing, RNAs were extracted from the precipitates and cDNA synthesis was performed using random hexanucleotide oligomers. An aliquot of 10% of the input RNA was kept as RT-PCR control (first lane). All precipitated RNAs were subjected to RT-PCR specific for sequences of RVFV (panel 1), HTNV (panel 2), CCHFV (panel 3), and BDV (panel 4). H₂O was used as negative control.</p

Genomic RNAs of ZEBOV and NiV activate the IFN response similar to FLUAV RNA.

<p>Human 293T cells were transfected with luciferase reporter plasmids to measure activation of the inducible IFN-β promoter and the constitutively active SV40 promoter, respectively. At 6 h post-transfection, cells were either mock treated or transfected with 1 µg viral genomic RNA (vRNA) of ZEBOV (A), NiV (B), or FLUAV (C). After overnight incubation, cells were lysed and promoter activities were normalised to the mock-induced samples. Mean values and standard deviations from 3 independent experiments are shown. (D) Detection of mRNAs for IFN-β (panel 1) and the IFN-stimulated genes IP-10, ISG56, and OAS (panels 2 to 4). Detection of γ-actin mRNA served as control (panel 5). 293T cells were transfected with 1 µg vRNA or 5 µg of the dsRNA analog poly(IC) and monitored 18 h later for gene upregulation by RT-PCR analysis.</p

Genomic RNAs of segmented NSVs.

<p>RNAs isolated from virus particles of LASV (A) and RVFV (B) were tested for their ability to activate the IFN-β promoter in dependency of RIG-I or MDA5 (left panels) or 5′ triphosphate groups (right panels). (C) IFN-β promoter activation by genomic RNAs of the bunyaviruses RVFV, HTNV, and CCHFV. Mean values and standard deviations from 3 independent experiments are shown. (D) Purified virion RNAs (5 µg) of RVFV, HTNV, and CCHFV were separated on denaturing formaldehyde agarose gels. The genome segments (L, large; M, middle; S, small) are labeled on the right, and asterisks indicate 28S and 18S rRNA bands. Contamination of virus preparations with ribosomal RNAs are often observed <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0002032#pone.0002032-Staunton1" target="_blank">[49]</a> but have no consequences for IFN induction. As a control RNA isolated from ultracentrifuged supernatants of uninfected cells is shown (mock).</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