Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions - PubMed (original) (raw)

[Preprint]. 2022 Mar 17:rs.3.rs-1394331.

doi: 10.21203/rs.3.rs-1394331/v1.

Jasmine R Mueller 2, En-Ching Luo 2, Brian A Yee 2, Danielle Schafer 2, Jonathan C Schmok 2, Frederick E Tan 2, Katherine Rothamel 2, Rachael N McVicar 3, Elizabeth M Kwong 3, Ben A Croker 4, Krysten L Jones 2, Hsuan-Lin Her 2, Chun-Yuan Chen 2, Anthony Q Vu 2, Wenhao Jin 2, Samuel S Park 2, Phuong Le 2, Kristopher W Brannan 2, Eric R Kofman 2, Yanhua Li 5, Alexandra T Tankka 2, Kevin D Dong 2, Yan Song 2, Alex E Clark 6, Aaron F Carlin 6, Eric L Van Nostrand 7, Sandra L Leibel 4, Gene W Yeo 2

Affiliations

Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions

Joy S Xiang et al. Res Sq. 2022.

Abstract

The COVID-19 pandemic is caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2). The betacoronvirus has a positive sense RNA genome which encodes for several RNA binding proteins. Here, we use enhanced crosslinking and immunoprecipitation to investigate SARS-CoV-2 protein interactions with viral and host RNAs in authentic virus-infected cells. SARS-CoV-2 proteins, NSP8, NSP12, and nucleocapsid display distinct preferences to specific regions in the RNA viral genome, providing evidence for their shared and separate roles in replication, transcription, and viral packaging. SARS-CoV-2 proteins expressed in human lung epithelial cells bind to 4773 unique host coding RNAs. Nine SARS-CoV-2 proteins upregulate target gene expression, including NSP12 and ORF9c, whose RNA substrates are associated with pathways in protein N-linked glycosylation ER processing and mitochondrial processes. Furthermore, siRNA knockdown of host genes targeted by viral proteins in human lung organoid cells identify potential antiviral host targets across different SARS-CoV-2 variants. Conversely, NSP9 inhibits host gene expression by blocking mRNA export and dampens cytokine productions, including interleukin-1α/β. Our viral protein-RNA interactome provides a catalog of potential therapeutic targets and offers insight into the etiology of COVID-19 as a safeguard against future pandemics.

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Conflict of interest statement

Competing interests J.S.X, F.E.T, J.C.S and G.W.Y declare a pending patent application. ELVN is co-founder, member of the Board of Directors, on the SAB, equity holder, and paid consultant for Eclipse BioInnovations. ELVN’s interests have been reviewed and approved by the Baylor College of Medicine in accordance with its conflict-of-interest policies. The authors declare no other competing interests.

Figures

Figure 1.

Figure 1.. Genome maps of SARS-CoV-2 protein interactions with viral RNA.

a) Schematic showing eCLIP performed on SARS-CoV-2 proteins in virus infected Vero E6 cells. Proteins in infected cells are UV crosslinked to bound transcripts, which are immunoprecipitated (IP) with antibodies that recognize NSP8 (primase), NSP12 (RNA dependent RNA polymerase, RdRp) and N (nucleocapsid) proteins. Protein-RNA IP product and Input lysate are resolved by SDS-PAGE and membrane transferred, followed by band excision at the estimated protein size to 75kDa above in both IP and Input lanes. Excised bands are subsequently purified, and library barcoded for Illumina sequencing. b) Mean fold change of eCLIP read density mapped to the positive sense SARS-CoV-2 genome in immunoprecipitated (IP) compared to input samples. Mean is taken from n = 2 independent biological samples. c) NSP12 eCLIP zoomed into yellow highlighted regions in b. Top row, NSP12 eCLIP; bottom row, SHAPE Shannon entropy with a sliding median of 55 nt. Shaded region in bottom row is partitioned at the global median entropy. d) Correlation between normalized SHAPE entropy and normalized log2(Fold Change) of IP over INPUT eCLIP read density i.e. eCLIP enrichment for NSP12 (left), NSP8 (middle) and N (right). R, Pearson’s coefficient. e) Secondary structure of the NSP12 eCLIP peak region from position 7412–7545. f) Fraction of RNA bound to NSP12 from filter binding assay, using hairpin RNA from position 7414–7555 and scrambled RNA as negative control. g) Correlation matrix of mean fold change of eCLIP read density: Bottom left panels, 2D density plots; diagonal, density plot corresponding to samples in bottom labels; top right panels, Pearson’s coefficient between samples.

Figure 2.

Figure 2.. SARS-CoV-2 protein interactions with host cell RNAs in virus infected cells

a) Bar plot showing number of all genes, number of all peaks, number of coding genes and number of peaks mapping to coding genes from n = 2 biologically independent replicates of NSP12, NSP8 and N eCLIP of SARS-CoV-2 infected cells. Target genes have at least one reproducible peak (by IDR) associated with each protein. b) Stacked bar plot showing TPM of reads mapped to the Vero E6 genome or SARS-CoV-2 genome in each of NSP12, NSP8 and N eCLIP. c) Venn diagram showing number of African Green Monkey (host) genes targeted by NSP8 and NSP12. d) Violin plot showing the distribution of Log2FoldChange in transcript levels in Vero E6 cells infected by SARS-CoV-2, for significantly differentially expressed genes (adjusted P < 0.05). Kolmogorov–Smirnov test p-values between eCLIP targets of NSP12 and NSP8 versus all differentially expressed genes are indicated above the plot. e) Top 25 Enriched Gene Ontology (GO) processes (adjusted p < 0.01) for NSP12 target host genes. Box plot indicates quartiles of differential expression (log2(Fold Change)) of target genes (grey dots).

Figure 3.

Figure 3.. The SARS-CoV-2 proteome interacts with thousands of host transcripts.

a) Schematic showing SARS-CoV-2 proteins individually tagged and expressed in human lung epithelial cells BEAS-2B to assay with eCLIP. b) Bar plot indicating number of all genes, number of all peaks, number of coding genes and number of coding peaks found to interact with each protein from n = 2 biologically independent experiments. In addition to SARS-CoV-2 proteins, ENCODE eCLIP data for example human RNA-binding proteins (hRBPs) are included for comparison. Target genes have at least one reproducible peak (by IDR) associated with each protein. c) Clustermap showing unique host coding genes (columns) targeted by each SARS-CoV-2 protein (rows). d) Example genome browser tracks for NSP3, NSP12, N and NSP2 mapping to DYNCH1, TUSC3, CXCL5 and NAP1L4 respectively. e & f) Western blots showing viral (pink background) and human (blue background) proteins enriched via CLASP (e) and RIC (f), with total cell lysate showed in input column (IN). g) Enriched Gene Ontology (GO) processes (adjusted p-value < 10–5) of unique eCLIP target coding genes for various SARS-CoV-2 proteins.

Figure 4.

Figure 4.. SARS-CoV-2 proteins specifically upregulate target gene expression.

a) Stacked bar plot showing fraction of reproducible peaks (by IDR14) mapping to different regions of coding genes. 3ss, 3′ splice site; 3utr, 3′ untranslated region (UTR), 5ss, 5′ splice site; 5utr, 5′ UTR; CDS, coding sequence. b) Clustermap showing read density of target RNA by each SARS-CoV-2 protein scaled to a metagene profile containing 5′ UTR, CDS and 3′ UTR regions. c) Schematic showing the Renilla-MS2 and Firefly dual luciferase reporter constructs, where individual SARS-CoV-2 proteins fused to MCP are recruited to the Renillia-MS2 mRNA. d & e) Bar plot showing luciferase reporter activity ratios (d) and reporter RT-qPCR ratios (e) for the indicated coexpressed SARS-CoV-2 protein, known human regulators of RNA stability (CNOT7, BOLL) and negative control (FLAG peptide). Ratios are normalized to the negative control (mean ± s.e.m., n = 3 biologically independent replicate transfections; * p<0.05, ** p<0.005, *** p<0.0005, **** p<0.0001, two-tailed multiple t-test; ns, not significant). f) Bar plot showing the fold change of luciferase activity ratio and RT-qPCR ratio (mean ± s.e.m, n = 3; * p<0.05, *** p<0.001, two-tailed Welch’s t-test). g) Cumulative distributive plot (CDF) of log2(Fold Change) of gene expression in HEK293T cells transfected with a plasmid overexpressing NSP12 versus an empty vector plasmid. KS test p values indicate significance of difference in differential expression of NSP12 target genes versus non-eCLIP target genes. h) Enriched Gene Ontology (GO) processes (adjusted p < 10–4) of NSP12 target genes, with box plots indicating quartiles of differential expression (log2(Fold Change)) of target genes (black dots). i) CDF plot of ∆log2(Fold Change) of polysomal mRNA levels in BEAS-2B cells nucleofected with a plasmid overexpressing ORF9c versus an empty vector plasmid. KS test p values indicate significance of difference in differential expression of ORF9c target genes versus non-eCLIP target genes. j) Enriched BioPlanet pathways (adjusted p < 0.01) of ORF9c target genes, with box plots indicating quartiles of differential expression (∆log2(Fold Change) of polysomal mRNA levels) of target genes (black dots). k) Immunofluorescence images (40X) of SARS-CoV-2 infected A549-ACE2 cells stained for SARS-CoV-2 NSP8 (red), endogenous genes (green), DNA content (blue). l) Heat map showing infection rate as measured by the integrated intensity of immunofluorescence staining of SARS-CoV-2 nucleocapsid protein in human iPSC derived lung organoid cells. Cells are treated with siRNAs targeting different host genes prior to viral infection by three different variants of SARS-CoV-2. Significant differences in infection rates are given by two-tailed t-test, * p<0.05, **p<0.01, ns, not significant, as compared to scrambled siRNA control for n = 3 biologically independent samples.

Figure 5.

Figure 5.. NSP9 interacts with U2AF2 substrates and inhibits mRNA export.

a) Pie charts showing distribution of eCLIP peaks across different coding RNA regions for NSP2, NSP5, NSP7 and NSP9. Genomic content and exonic content are based on the hg19 human reference genome. b) Jaccard index similarity of NSP9 target genes as compared with all 223 ENCODE RBP datasets. c) Metadensity of eCLIP reads truncation sites averaged across all RNA targets by SARS-CoV-2 NSP2, NSP5, NSP7 and NSP9, and U2AF1/2 from the ENCODE consortium, zoomed into the region 150 nt upstream of 3′ splice sites, and the region 150 nt downstream of the 5′ end of the last exon. d) Schematic illustrating a model of NSP9 interacting with nuclear pore complex proteins NUP62, NUP214, NUP58, NUP88 and NUP54, and inhibiting U2AF2 substrate recognition in preventing NXF1 facilitated transport. e) Cumulative distributive plot (CDF) of log2(Fold Change) of BEAS-2B cells overexpressing NSP9 versus wildtype BEAS-2B cells in each of nuclear, cytosolic, and total mRNA fractions. Solid line indicate NSP9 target genes, dashed lines indicate genes that are not NSP9 targets. f) Genome browser tracks of NSP9 eCLIP target RNA mapped to IL-1α, IL-1β, ANXA2 and UPP1. g) Bar plot showing ratios of cytosolic to total fraction of mRNA levels measured by RT-qPCR, in wild type (WT) BEAS-2B cells, and BEAS-2B cells transduced to express NSP9 (*p<0.05, **p<0.0005, two-tailed multiple t-test with pooled variance, n = 2 biologically independent replicates). h) Bar plot showing mean concentration of IL-1α in culture media from WT and NSP9 expressing BEAS-2B cells, 48h after induction by cytokines indicated on the x-axis (US, unstimulated; mean ± s.e.m, n = 3 biologically independent replicates; *p < 0.05, Tukey’s multiple comparisons test). i) Bar plot showing mean concentration of IL-1α in culture media from WT and NSP9 expressing BEAS-2B cells, 48h after induction by different levels of TNFα (mean ± s.e.m, n = 3 biologically independent replicates, *p<0.05, **p<0.005, two-tailed t-test). j) Bar plot showing mean concentration of IL-1β in culture media from WT and NSP9 expressing BEAS-2B cells, 48h after induction by 0 or 100 ng/ml TNFα (mean ± s.e.m, n = 3 biologically independent replicates, *p<0.05, **p<0.005, two-tailed t-test).

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