Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Fluorescence microscopy analysis of A549 WT and XRN1-KO cells infected with SINV for 18 h (MOI 2). n = 3. Cellular mRNA was detected by smFISH with an Oligo(dT) 25 probe (polyA RNA), nuclei were stained with DAPI, and SINV capsid was visualized by immunofluorescence. Yellow arrows indicate the accumulation of polyA RNA in the viral factories marked with SINV capsid antibody. (B) MA plot comparing the read coverage and the log2 fold change between SINV infected (8 hpi top; or 18 hpi bottom) and uninfected condition of each transcript detected in the RNA sequencing experiment for HEK293 WT, XRN1 partial, and full KO cells. Blue dots represent RNAs enriched with p.adj < 0.05 while grey dots represent non-significant changes. Dashed lines: black indicates the zero log2 fold change while grey represents the median fold changes across all transcripts. (C) Proportion of normalized human and SINV reads from RNAseq experiment at 18 hpi. (D) Boxplot of the Synonymous dinucleotide usage (SDU) of the three most important features in upregulated (pos) and downregulated (neg) transcripts upon SINV infection. (E) Comparison of the SDU of the indicated dinucleotides between SINV genome and cellular transcripts upregulated and downregulated upon SINV infection. (F) Luciferase activity of Rluc-WT or reporters with altered dinucleotide codon usage (Rluc-up and Rluc-down) measured at 6 hours post transfection (hpt) relative to the luciferase levels at 4 hpt in SINV infected conditions. n = 4; error bars: standard error; * p < 0.05.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Fluorescence, Microscopy, Infection, Staining, Immunofluorescence, RNA Sequencing Assay, Comparison, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Western blot analysis of samples from (MOI = 1. n = 3). (B) Correlation of the RNAseq and RT-qPCR data from for randomly selected transcripts. Error bars represent standard error of three independent experiments. (C) GO term enrichment for upregulated transcripts in SINV infected WT (left) and XRN1-KO (right) cells. (D) MA plot comparing the read coverage and the log2 fold change between WT and XRN1-KO uninfected cells of each gene detected in the RNAseq experiment from . Red, blue, and grey dots represent significantly upregulated, downregulated, and unchanged transcripts levels, respectively. Purple dots represent ISGs in HEK293 cells, as defined in . Density plots displaying distribution of fold changes among each colour group are shown at the right side of the figure. (E) GO term enrichment of transcripts upregulated in XRN1-KO cells in non-infected conditions (XRN1 WT versus KO cells in non-infected conditions, left) and transcripts upregulated in XRN1-KO cells 18 hpi (XRN1 WT versus KO cells in infected conditions, right). (F) Western blot analysis of antiviral response proteins OAS1, OAS3, and IFIT1 in WT and XRN1/5’-3’ RNA decay components KO cells in non-infected conditions. n = 3 (G) Top 20 most important features and their corresponding importance scores identified by Synonymous Dinucleotide Usage (SDU) and Codon Usage comparing up versus down-regulated transcripts upon SINV infection (RNAseq experiment fig 3B). (H) Comparison of the SDU between SINV genome and cellular transcripts upregulated and downregulated upon SINV infection. (I) Luciferase activity of Rluc-WT or reporters with altered dinucleotide codon usage (Rluc-up and Rluc-down) measured at 6 hours post transfection (hpt) relative to the luciferase levels at 4 hours post transfection (hpt) in mock conditions. n = 4; error bars: standard error. (J-K) RNA fold change of RLuc-WT or reporters with altered dinucleotide codon usage (RLuc-up and RLuc-down) measured at 6 hours post transfection (hpt) relative to RNA levels at 4 hours post transfection (hpt) in mock (J) and infected conditions (K). n = 3; error bars: standard error.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Western Blot, Quantitative RT-PCR, Infection, Comparison, Luciferase, Activity Assay, Transfection

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Principal component analysis of data from immunoprecipitated (IP) and size match input (SMI) samples from iCLIP2 experiments in uninfected and SINV-infected cells at 4 or 18 hpi. (B) Visualisation of IR adaptor of size match input (SMI) and immunoprecipitated (IP) samples from iCLIP2 experiment, in mock and infected conditions. (C) MA plot comparing the read coverage and the log2 fold change between the differentially regulated transcripts in XRN1-KO cells pre- and post-infection with the XRN1 targets (left) and not targets (right) identified by iCLIP2. Blue and red dots represent significantly downregulated and upregulated RNAs, respectively. (D) Bar plots reflecting the same data shown in . Instead of absolute counts, the percentage of transcripts is plotted to better visualize the effect seen in KO cells. (E) Comparison of binding profile of TRIM25 and XRN1on SINV genome at 9 and 18hpi, respectively. (F) SINV genome coverage from RNAseq (WT and XRN1-KO samples) and iCLIP2 data with inset showing a ‘zoomed in’ view of the 3’ end of the viral genome. (G) Silver staining analysis of samples from anti-GFP-beads for mass spectrometry analysis from . (H-I) Volcano plots showing XRN1 interactor proteins enriched in XRN1-IP compared to WCL in mock (H) and infected (I) conditions.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Immunoprecipitation, Infection, Comparison, Binding Assay, Silver Staining, Mass Spectrometry

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) XRN1 binding site and target gene counts from iCLIP2 analysis of HEK293-Flp-In T-REx-XRN1-eGFP cells. Cells were either uninfected or infected with SINV for 4 or 18 h (MOI 10). n = 3. (B) Venn diagram representing the overlap of target genes identified in iCLIP2 in mock and SINV infected cells (4 h and 18 h). (C) Density plot of distribution of XRN1 binding sites on mature target mRNAs. (D) MA plots from (of WT 18 hpi sample) showing XRN1 targets (left, 4hpi) and non-targets (right) identified by iCLIP2. Blue and red dots represent significantly downregulated and upregulated RNAs, respectively. (E) Bar plots showing the percentage of XRN1 target genes (based on iCLIP2) that are downregulated, unchanged, and upregulated (based on RNAseq) in WT, XRN1 partial (PKO), and full KO (KO). (F) Bar plots showing the number of XRN1 target genes and non-target genes (based on iCLIP2) that are upregulated and downregulated (based on RNAseq) in WT, XRN1 partial (PKO), and full KO (KO). (G) Top: Binding profile of XRN1 on SINV genome at 4 hpi and 18 hpi. Bottom: schematic representation of SINV genome features. (H) Volcano plot showing proteins enriched in HEK293-Flp-In T-REx-XRN1-eGFP IP in mock versus SINV-infected cells (18 hpi, MOI 10). n = 3. (I) Volcano plot showing proteins enriched in HEK293-Flp-In T-REx-XRN1-eGFP IP compared to WCL in SINV-infected cells (18 hpi, MOI 10). n = 3. (J) Western blot analysis of input and anti-GFP-beads IP (pull down of GFP-CTRL and XRN1-GFP) in mock and infected conditions. n = 3.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Binding Assay, Infection, Western Blot

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Fluorescence microscopy analysis of mock and SINV-infected cells at the indicated time-points post infection (MOI 1). N = 3. NME3, HPRT1, and APRT were detected by immunofluorescence, SINV-nsP3 was visualized by tagging with mScarlet, and nuclei were stained with DAPI. Yellow arrows indicate colocalization of nsP3 with NME3 (top) HPRT1 (middle), and APRT (bottom). (B) Western blot analysis of A549-WT cells (CTRL siRNA) and cells depleted of components of the nucleotide salvage pathway with two different siRNAs mix (Pool-1: HPRT1, CTPS1/2, APRT, UPRT, CDA, and NME3; and Pool-2: HPRT1, CTPS1, APRT, CDA, and UPRT), infected with SINV for 18 h (MOI 0.1). n = 3. (C) Western blot analysis of HEK293-Flp-In T-REx WT and XRN1 KO cells complemented with the indicated XRN1 mutants, in presence or absence of externally supplemented nucleosides and infected with SINV for 18 h (MOI 0.1). n = 3. Below the blots the average normalized capsid level of three independent experiments, in bracket standard error. * p < 0.05. Expression of XRN1 constructs was induced by doxycycline treatment (Dox) for at least 96 hours prior infection. (D) Western blot analysis of HEK293T KO cells of the indicated member of the RNA decay machinery, supplemented with nucleosides (2X) and infected with SINV for 18 h (MOI 0.01). n = 3.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Fluorescence, Microscopy, Infection, Immunofluorescence, Staining, Western Blot, Expressing, Construct

Journal: bioRxiv

Article Title: XRN1 supplies free nucleotides to feed alphavirus replication

doi: 10.1101/2024.12.09.625895

Figure Lengend Snippet: (A) Fluorescence microscopy analysis of mock and SINV-infected cells at the indicated time-points post infection (MOI 1). n = 3. PPAT or NME1 were detected by immunofluorescence, SINV-nsP3 was visualized by tagging with mScarlet, and nuclei were stained with DAPI. Yellow arrows indicate colocalization of nsP3 with PPAT (top), or NME1 (bottom). (B) Cell count of WT cells and cells depleted of components of the nucleotide salvage pathway with two different siRNA mixes (Pool-1: HPRT1, CTPS1/2, APRT, UPRT, CDA, and NME3; and Pool-2: HPRT1, CTPS1, APRT, CDA, and UPRT), infected with SINV for 18 h (MOI 0.1). n = 3. Error bars: standard error. (C) Western blot analysis of HEK293 WT and XRN1 partial KO (XRN1-PKO) cells supplemented with nucleosides and infected with SINV for 18 h. (MOI 0.1). n = 5. Below the blots the average normalized capsid level of four independent experiments, in bracket standard error. * p < 0.05. (D) Western blot analysis of DDX6-KO cells, supplemented with nucleosides and infected with SINV for 18 h. (MOI 0.01). n = 3. (E) Schematic model representing the role of the RNA decay machinery and nucleotide salvage pathway in regulating alphavirus infection. XRN1 and the 5-3DM localize to SINV viral factories and degrade cellular transcripts in proximity to the replication centre to provide high local concentration of nucleotides to sustain viral replication.

Article Snippet: The pcDNA5-Rluc-WT, up, or down constructs containing the wild-type DNA Renilla luciferase sequence from pRL-CMV (Promega AF025843) or the di-nucleotide optimized Rluc sequence optimised to resemble SINV (Upregulated transcripts, up) or Mock (downregulated transcripts, down) conditions were purchased from GeneScript and inserted into pcDNA™/FRT/TO (ThermoFisher # V652020) via BamHI and NotI sites.

Techniques: Fluorescence, Microscopy, Infection, Immunofluorescence, Staining, Cell Counting, Western Blot, Concentration Assay