Natural product sennoside B disrupts liquid-liquid phase separation of SARS-CoV-2 nucleocapsid protein by inhibiting its RNA-binding activity

Abstract

The nucleocapsid protein (NP) of SARS-CoV-2, an RNA-binding protein, is capable of undergoing liquid-liquid phase separation (LLPS) during viral infection, which plays a crucial role in virus assembly, replication, and immune regulation. In this study, we developed a homogeneous time-resolved fluorescence (HTRF) method for identifying inhibitors of the SARS-CoV-2 NP-RNA interaction. Using this HTRF-based approach, we identified two natural products, sennoside A and sennoside B, as effective blockers of this interaction. Bio-layer interferometry assays confirmed that both sennosides directly bind to the NP, with binding sites located within the C-terminal domain. Additionally, fluorescence recovery after photobleaching (FRAP) experiments revealed that sennoside B significantly inhibited RNA-induced LLPS of the NP, while sennoside A displayed comparatively weaker activity. Thus, the developed HTRF-based assay is a valuable tool for identifying novel compounds that disrupt the RNA-binding activity and LLPS of the SARS-CoV-2 NP. Our findings may facilitate the development of antiviral drugs targeting SARS-CoV-2 NP.

Keywords: HTRF; SARS-CoV-2; liquid-liquid phase separation; nucleocapsid protein; sennoside B.

Figure 1.

The sequence of the SARS-CoV-2 nucleocapsid protein (NP) is highly conserved across various variants of the novel coronavirus (A) Schematic representation of the domain structure of the SARS-CoV-2 NP. (B) Multiple sequence alignment of the N protein of coronaviruses. (C) Structures of representative small-molecule compounds targeting the SARS-CoV-2 NP.

Figure 2.

Recombinant SARS-CoV-2 NP binds to RNA in vitro. (A) SDS-PAGE analysis of purified full-length and truncated forms of NP. (B) Secondary structure of the 5′ leader region sequences of the viral genome. (C) Representative binding kinetics of recombinant NP to RNA as determined by biolayer interferometry (BLI). The data are representative of three independent experiments. (D) The binding curve generated by fitting steady-state response levels at the end of the association phase versus NP concentration, with a dissociation constant (K_D) of 3.8 nM. Kinetic values calculated from n = 3 experiments.

Figure 3.

Development of a HTRF-based assay for screening inhibitors of the SARS-CoV-2 NP-RNA interaction. (A) Schematic representation of the HTRF assay principle. (B) Cross-titration of His₆-NP binding to biotin-labelled RNA, showing HTRF signal intensity from low (white) to high (blue). (C) Signal-to-background (S/B) ratio comparison generated from NP-RNA cross-titration. (D) and (E) Chemical structure of GCG and PJ34. (F) Comparison of the inhibitory effects of GCG and PJ34 at 50 μM on NP-RNA interaction. (G) Dose-response curves of GCG for inhibition of NP-RNA interaction, presented as mean ± SD from three independent experiments. (H) PJ34 disrupts the NP-RNA interaction at a higher concentration (100 μM). (I) GCG disrupts the NP-RNA interaction at a lower concentration (6.25 μM). (H) and (I) biosensors immobilised with RNA were incubated with NP in the presence of control DMSO or compound, and binding to the biosensor was monitored for 120 s via wavelength shift. (J) Binding affinity determined using a BLI assay between GCG and SARS-CoV-2 NP.

Figure 4.

Sennoside A and Sennoside B inhibit the SARS-CoV-2 NP-RNA interaction. (A) Comparison of the inhibitory effects of various compounds on the NP-RNA interaction, presented as averages ± standard deviation for n = 3 independent experiments. (B) and (C) Chemical structure of Sennoside A and Sennoside B. (D) and (H) Dose-response curves of Sennoside A and Sennoside B for the inhibition of NP-RNA interaction. (E), (F) and (G) Representative binding sensorgrams depicting the interaction of Sennoside A with full-length NP, CTD and NTD as assessed by BLI. (I), (J), and (K) Representative binding sensorgrams depicting the interaction of Sennoside B with full-length NP, CTD and NTD as assessed by BLI. The results were determined from three independent experiments.

Figure 5.

Sennoside B inhibits the liquid-liquid phase separation (LLPS) of NP. (A) Confocal microscopy images of NP droplets in the absence and presence of Sennoside A or Sennoside B, with DMSO (1%, v/v) used as the vehicle control. (B) Quantification of NP droplet diameter in the absence and presence of Sennoside A, Sennoside B, or DMSO, with a minimum of 150 droplets counted for each treatment group (***p < 0.001).