Defining distinct RNA-protein interactomes of SARS-CoV-2 genomic and subgenomic RNAs

Figures

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  Figure 1.. HyPR-MSSG for purification of SARS-CoV-2 sgRNA interactomes. (A) Capture oligonucleotides (COs) were designed to bind to specific regions of the SARS-CoV-2 gRNA or three individual sgRNAs to allow for simultaneous, specific capture of all four RNAs individually from a single infected cell lysate. (B) Description of the HyPR-MSSG procedure. (C) Capture efficiency was determined by measuring the extent of the depletion of the target RNA from the pre-capture lysate using RT-qPCR. (D) Capture specificity was measured by comparing the ratio of the targeted RNA to non-targeted RNAs in each capture using RT-qPCR. Error bars in (C) and (D) represent standard deviations of the mean from the four biological replicates.

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  Figure 2.. Determination of SARS-CoV-2 RNA protein interactomes. (A) Volcano plots displaying the distribution of all proteins for each SARS-CoV-2 RNA capture with relative protein abundance (log2 target RNA capture/scrambled control capture) plotted against significance level (−log10 p-value), with significantly enriched proteins indicated by colored dots. Red dots indicate proteins that passed a 5% FDR cutoff using a student’s t-test (tier 1) and purple dots indicate proteins that passed the less rigorous fold-change and p-value cutoff metrics (tier 2). (B) Graph displaying the number of proteins found in each individual or subset of interactomes for each of the two time points.

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  Figure 3.. Analysis of SARS-CoV-2 RNA Protein Interactomes. (A) Gene ontology enrichment analysis of the genomic, S, N, and ORF8 RNA protein interactomes at both 8- and 24-hour time points. Refer to supplementary table 3 for complete data. (B) Proteins identified as related to “Posttranscriptional Gene Silencing” according to GO analysis using STRING plotted to show which interactomes they were identified in at the 8- and 24-hour time points. (C) Proteins identified as related to “Cytoplasmic Ribonucleoprotein Granule” according to GO analysis plotted to show which interactomes they were identified in at the 8- and 24-hour time points. Colors in (B) and (C) distinguish proteins unique to or shared among the indicated viral RNA interactomes.

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  Figure 4.. Effects of siRNA knockdowns on viral replication readouts. Calu3 cells were transfected with two independent siRNAs targeting PPP1CC, A3F, MSI2, TRIM71 and LIN28B as well as a non-targeting siRNA control. 48h post-siRNA, cells were infected with SARS-CoV-2 and harvested at the indicated time points for analysis of viral RNA expression (A), viral protein expression (B) or the percentage of NG positive cells (C). (A) RT-qPCR results showing the fold-change for each SARS-CoV-2 RNA 48- and 96-hpi normalized to 18S RNA after siRNA-mediated knockdown of the indicated targets. Fold-changes are relative to a non-targeting (NT) control set to 1. (B) Western blot analysis detecting SARS-CoV-2 N protein after siRNA-mediated knockdown of the indicated targets. (C) Flow cytometry analysis of infected cells showing the percentage of NG-positive cells at the indicated time points post-infection. For (A) and (C) data relative to a non-targeting siRNA control (set to 1) and error bars represent the standard error of the mean (SEM).