Fig. 4: RNA-binding and Affinity Purification Mass-Spectrometry (AP-MS) analysis of mutant and control SARS-CoV-2 N protein. | Nature Communications

Fig. 4: RNA-binding and Affinity Purification Mass-Spectrometry (AP-MS) analysis of mutant and control SARS-CoV-2 N protein.

From: SARS-CoV-2 genomes from Saudi Arabia implicate nucleocapsid mutations in host response and increased viral load

Fig. 4

a A schematic diagram showing the SARS-CoV-2 N protein different domains (Upper: control, Lower: mutant) and highlighting the mutation site (R203K and G204R) and the linker region (LKR) containing a serine-arginine rich motif (SR-motif). The bar-plot (lower panel) indicates the SIFT37 predicted deleteriousness score of substitution at position 203 and 204 from R to K and G to R respectively. b Sketch of In vitro RNA immunoprecipitation (RIP) procedure used for analysis of viral RNA interaction with mutant and control N protein (See methods for details). Isolated RNAs were analyzed by RT-qPCR using specific viral N gene (N1 and N2), E gene, S gene, and ORF1ab region. c Bar chart shows level of viral RNA retrieval (% input) with mutant and control N protein (± SD from n = 3 independent experiments, [two-sided t-test, p-values N1:0.00080 (***), N2:0.00088 (***), E:0.008 (**), S:0.00059 (***), and ORF1ab:0.002 (**)]). d Identification of host-interacting partners of mutant and control SARS-CoV-2 N protein by Affinity Mass-Spectrometry. Heatmap showing significantly differentially changed human proteins (3 replicates) interactome in mutant versus control N protein AP-MS analysis. e Gene Ontology (GO)-enrichment analysis of significantly changed terms between mutant and control proteins in terms of biological process and pathway enrichment. The scale shows p-value adjusted Log2 of odds ratio mutant versus control. f Profiling of phosphorylation status of mutant and control N protein by Mass-Spectrometry. Sketch showing part of SR-rich motif of SARS-CoV-2 N protein containing the KR mutation site (R203K and G204R) (Lower). The hyper-phosphorylated serine 206 (as shown in (g)) in the mutant N protein near the KR mutation site is indicated in orange color. g Phosphorylation status of mutant and control N protein was analyzed by mass spectrometry (±SD from n = 3 biologically independent experiments per affinity condition). Bar-plot shows the Log2 intensities of phosphorylated peptide (Serine 206) in control and mutant condition (see Supplementary Data 4).

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