Evidence for Substrate Binding-Induced Zwitterion Formation in the Catalytic Cys-His Dyad of the SARS-CoV Main Protease

The coronavirus main protease (M^pro) represents an attractive drug target for antiviral therapy of coronavirus (CoV) infections, including severe acute respiratory syndrome (SARS). The SARS-CoV M^pro and related CoV proteases have several distinct features, such as an uncharged Cys-His catalytic dyad embedded in a chymotrypsin-like protease fold, that clearly separate these enzymes from archetypical cysteine proteases. To further characterize the catalytic system of CoV main proteases and to obtain information about improved inhibitors, we performed comprehensive simulations of the proton-transfer reactions in the SARS-CoV M^pro active site that lead to the Cys^–/His⁺ zwitterionic state required for efficient proteolytic activity. Our simulations, comprising the free enzyme as well as substrate–enzyme and inhibitor–enzyme complexes, lead us to predict that zwitterion formation is fostered by substrate binding but not inhibitor binding. This indicates that M^pro employs a substrate-induced catalytic mechanism that further enhances its substrate specificity. Our computational data are in line with available experimental results, such as X-ray geometries, measured p<i>K</i>_a values, mutagenesis experiments, and the measured differences between the kinetic parameters of substrates and inhibitors. The data also provide an atomistic picture of the formerly postulated electrostatic trigger involved in SARS-CoV M^pro activity. Finally, they provide information on how a specific microenvironment may finely tune the activity of M^pro toward specific viral protein substrates, which is known to be required for efficient viral replication. Our simulations also indicate that the low inhibition potencies of known covalently interacting inhibitors may, at least in part, be attributed to insufficient fostering of the proton-transfer reaction. These findings suggest ways to achieve improved inhibitors

Genome-wide distribution and regulation of p65 NF-κB DNA binding sites in response to HCoV-229E compared to IL-1.

<p>(A) Total number of constitutive and HCoV-229E- or IL-1 regulated p65 ChIP-seq peaks. Regulation was defined by a ratio of normalized read counts derived from treated / untreated cells of at least 2-fold and a p value below 0.01. The likelihood of overlaps between HCoV-229E-or IL-1-regulated peaks occurring by chance is shown by the odds ratio and by the corresponding hypergeometric p value. (B) Quantification of normalized read counts for 50 HCoV-229E-specific, 353 IL-1-specific or 82 jointly regulated groups of p65 DNA-binding events. (C) Genome-wide localization pattern of p65 ChIP-seq peaks relative to the next annotated TSS. (D) Quantification of histone acetylation patterns of genomic regions containing regulated p65 peaks. (E) Examples of an intergenic p65 peak regulated by both HCoV-229E and IL-1, along with profiles of histone modifications observed in these cases. Shown are browser profiles of chr.14. The red horizontal bar marks the p65 peak region, vertical bars indicate predicted p65 DNA-binding motifs. Statistics for box plots (B, D) are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s014" target="_blank">S4 Table</a>. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s007" target="_blank">S7</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s008" target="_blank">S8</a> Figs.</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

Differential activation or repression of regulators of NF-κB signaling by HCoV-229E or IL-1.

<p>(A) HuH7 cells were infected with HCoV-229E for the indicated times, or were treated with IL-1 for 1 h or were left untreated. Expression and phosphorylation (P) of the indicated proteins was analyzed by immunoblotting of whole cell extracts. Tubulin or β-actin antibodies were used to control for equal loading. (B) Graphs show quantification of relative protein/P-protein levels including means +/- s.d. from five to seven independent experiments. For viral proteins the 6 h (nsp8) or 9 h (N protein) protein levels were set as one. Asterisks indicate significance of differences as obtained from one-tailed t-tests comparing cells infected for 24 h with untreated cells (**** p<0.0001, *** p<0.001, ** p<0.005, * p<0.05). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s005" target="_blank">S5 Fig</a>.</p

Comparison of HCoV-229E- and IL-1-regulated transcriptomes reveals common and coronavirus-specific sets of genes belonging to specific biological pathways.

<p>HuH7 cells were infected with HCoV-229E for 24 h or were left uninfected. In parallel, cell cultures were treated with IL-1 for 1 h. Transcriptome analyses were performed from total RNA using Agilent microarrays. Data from four independent experiments were pooled of which the last two included IL-1 stimulation. (A) Shown is the overlap of HCoV-229E-regulated genes in HuH7 cells with the 37 genes regulated in A549 cells as identified in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.g001" target="_blank">Fig 1</a>. Black arrows indicate seven genes whose expression changes were validated by RT-qPCR as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s003" target="_blank">S3A Fig</a>. (B) Venn diagram of all genes regulated more than 2-fold that were significantly expressed over background in response to HCoV-229E or IL-1. The left heatmap shows the averaged ratios of the 61 genes that were regulated by both HCoV-229E and IL-1. The right heatmap shows the top 50 genes that were regulated by HCoV-229E only. Individual gene expression ratios for the CoV-specific regulated genes are shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s003" target="_blank">S3B Fig</a>. (C) Gene set enrichment analyses were used to identify HCoV-229E-regulated KEGG pathways from the four independent experiments. Shown are the ten most strongly down- (blue colors) or upregulated (red colors) pathways as indicated by the adjusted p values. Sizes of bubbles correspond to the total number of genes annotated to each KEGG pathway which varied from 127 genes (KEGG_05012) up to 1113 genes (KEGG_01100). The x-axis indicates the relative number of genes of each pathway that were found to be expressed in HuH7 cells. D) Relative expression data for the top ten up- and downregulated genes of the indicated pathways in response to virus infection or IL-1 treatment were selected. Gene lists were merged and heatmaps show ratios of expression and provide a comparison of virus-infected cells with IL-1 treated cells. Gray colors in (A) and (D) show genes with no detectable expression in a particular condition. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s003" target="_blank">S3</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s004" target="_blank">S4</a> Figs and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s013" target="_blank">S3 Table</a>.</p

Identification of host cell genes directly regulated in response to HCoV-229E replication in human lung epithelial cells.

<p>(A) A549 lung epithelial cells were infected with HCoV-229E and collected for further analyses at 16, 24 and 48 h p.i.. As controls, cells were mock infected or incubated with heat-inactivated virus and collected at the time points indicated above. Transcriptomes for the respective cells were determined using Agilent microarrays. Graphs represent MA plot visualizations of changes in gene expression (M, log₂ ratios) versus mRNA abundance (A, log₂ average fluorescence intensity). An intensity-based cut off was calculated and red dots above or below the red lines represent genes with differential expression in response to virus. (B) Scheme of cell populations isolated from HCoV-229E-infected A549 cells at 48 h p.i. or from uninfected cells by laser microdissection. (C) Two independent series of microarray experiments were performed from laser microdissected cells and data were averaged. MA plots showing transcriptome changes in cells infected with HCoV-229E compared to adjacent or distant cells or to noninfected cells collected from a separate mock-infected culture. D) The 37 genes that were specifically regulated by HCoV-229E as shown in (A), lower right panel, were selected and their expression in both independent experiments of laser microdissected cell populations as determined in (C) was visualized by heatmaps. Arrows indicate seven genes with differential expression levels that were further validated by RT-qPCR as shown in (E). Bar graphs displayed in (E) represent mean changes +/- s.e.m. from 3 independent experiments. RNA1 represents the HCoV-229E genome RNA as determined using replicase gene (nsp8)-specific primers. See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s001" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s002" target="_blank">S2</a> Figs and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s011" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s012" target="_blank">S2</a> Tables.</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

Suppression of p65 NF-κB or TNFAIP3 (A20) affect HCoV-229E replication and transcriptional regulation of NF-κB target genes.

<p>HeLa cells were transiently transfected with empty pSuper (vector), pSuper encoding shRNAs directed against p65 (B-D) or pLKO.1 encoding shRNAs against TNFAIP3 (E, F). One day later, cells were selected for 48 h (shp65) or 72 h (shTNFAIP3) in 1 μg/ml puromycin. Then, cells were infected with HCoV-229E for 24 h or were treated with IL-1 for 1 h. (A) Equal amounts of proteins from whole cell extracts were analyzed for the expression of p65 NF-κB, IκBα and viral N protein. β-actin antibodies were used to control for equal loading. Right graph: Quantification of relative N protein levels (mean +/- s.e.m.) from six independent experiments. (B) Mean relative changes +/- s.e.m. of mRNA expression of HCoV-229E or IL-1 target genes from six independent experiments of cells treated as in (A). (C) Recruitment of phosphorylated RNA pol II or p65 to the promoters of these target genes. Shown are results from two independent ChIP-PCR experiments, IgG precipitations served as negative controls. (D) Viral titers (mean TCID50/ml +/- s.e.m.) were determined in the supernatants of the same cells used for the experiments described in (C). (E) Equal amounts of proteins from whole cell extracts were analyzed for the expression of TNFAIP3 and viral N protein. β-actin antibodies were used to control for equal loading. Right graph: Quantification of relative N protein levels (mean +/- s.e.m.) from five independent experiments. (F) Mean relative changes +/- s.e.m. of mRNA expression of HCoV-229E or IL-1 target genes from five independent experiments. The asterisks indicate significance of differences as obtained from t-tests (* p<0.05). See also <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006286#ppat.1006286.s006" target="_blank">S6 Fig</a>.</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

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