Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2

Abstract

Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2 Dear EditorTo date, tens of millions of people have been infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the outbreak of the respiratory disease named the coronavirus disease 2019 . As a newly emerged member of the coronavirus family, SARS-CoV-2 is an enveloped positive-strand RNA virus, which has probably the largest genome (approximately 30 kb) among all RNA viruses. The nucleocapsid (N) protein of SARS-CoV-2 is mainly responsible for recognizing and wrapping viral RNA into helically symmetric structures (Malik, 2020). It was also reported that N protein can boost the efficiency of transcription and replication of viral RNA, implying its vital and multifunctional roles in the life cycle of coronavirus (Surjit and Lal, 2008;Chang et al., 2014). Recently, several independent research teams have reported that N protein of SARS-CoV-2 is capable of undergoing liquid-liquid phase separation (LLPS) (Iserman et al., 2020;Perdikari et al., 2020;Savastano et al., 2020).Here, we comprehensively determined the characteristics of the phase separation driven by the N protein of SARS-CoV-2 (termed SARS-CoV-2 N), and found that LLPS is involved in the interplay between the N protein-viral RNA complex of SARS-CoV-2 (termed SARS-CoV-2 N-RNA) and other viral proteins, such as nsp12. Importantly, we identified two small molecules targeting the SARS-CoV-2 N protein, which can intervene the phase separation properties of the N protein-viral RNA-nsp12 (termed SARS-CoV-2 N-RNA-nsp12) complex, thus probably improving the accessibility of other antiviral drugs (e.g., remdesivir) to their viral targets (e. g., nsp12/RdRp).First, IUPred2 (Erdos and Dosztanyi, 2020) and PLAAC (Lancaster et al., 2014) programs showed that SARS-CoV-2 N is highly disordered, and contains three intrinsically disordered regions (IDRs), with one also displaying prion-like activity (Figs. 1A and S1). Then, we expressed and purified the recombinant SARS-CoV-2 N protein with an mEGFP-tag (a monomeric variant of EGFP, A206K) or a His-tag using a prokaryotic expression system to understand the properties of N-driven LLPS in vitro (Fig. S2A and S2B). Confocal fluorescence microscopy showed that SARS-CoV-2 N was readily self-associated to form numerous micron-sized spherical condensates (Fig. 1B and 1C). Further time-lapse observations revealed that the SARS-CoV-2 N condensates fused and coalesced into larger ones upon their intersections (Fig. 1D and Video S1), verifying the liquid-like properties of SARS-CoV-2 N condensates. We also used fluorescence recovery after photobleaching (FRAP) to deeply study the dynamics of internal molecules within the N protein condensates. Recovery of fluorescence within the bleached regions (Fig. 1E) showed that SARS-CoV-2 N can partially freely diffuse within the condensed phase, consistent with their liquid-like behavior. In addition, phase condensation of SARS-CoV-2 ...

Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids

“…The importance of LLPS in vivo in the viral life cycle is stressed by results from an evolutionary analysis of SARS-CoV-2 (Gussow et al, 2020), which found localization in the nucleolus, a macromolecular condensate ll OPEN ACCESS iScience Article of proteins and RNA, and is correlated with increased pathogenicity and case fatality rate. LLPS may be regulated by small molecules, and is thereby susceptible to small molecule inhibitors, some of which have already been identified in different laboratories (Carlson et al, 2020;Cubuk et al, 2021;Iserman et al, 2020;Jack et al, 2020;Zhao et al, 2021). We believe this strategy of therapeutic development will be supported by increasing detailed knowledge of molecular configurations and binding processes involved in N-protein functions.…”

“…Structures and dynamics of both CTD and NTD, isolated or in complex with NA, have been studied and reveal significant molecular details (Ahamad et al, 2020;Caruso et al, 2020;Chen et al, 2007;Dinesh et al, 2020;Kang et al, 2020;Takeda et al, 2008;Zinzula et al, 2020). Intriguingly, it was reported recently by several laboratories that N-protein undergoes liquid-liquid phase separation (LLPS) (Carlson et al, 2020;Cascarina and Ross, 2020;Chen et al, 2020a;Cubuk et al, 2021;Iserman et al, 2020;Jack et al, 2020;Lu et al, 2021;Perdikari et al, 2020;Savastano et al, 2020;Wang et al, 2021;Zhao et al, 2021). Such demixing is commonly mediated by ultra-weak but highly multi-valent interactions (Alberti et al, 2019;Vernon et al, 2018).…”

Liquid-Liquid Phase Separation: Unraveling the Enigma of Biomolecular Condensates in Microbial Cells

“…Multi-stimuli-responsive carriers (thermal or pH-responsive reversible coacervate droplets) can also be imbued with the ability to package and deliver drugs ( Gabryelczyk et al, 2019 ). Furthermore, microfluidic techniques can be employed to create monodisperse coacervate droplets, making it possible to mimic diverse intracellular activities within uniform unilamellar lipid vesicles ( Deshpande et al, 2001 ; Van Swaay et al, 2015 ; Deng and Huck, 2017 ; Love et al, 2020 ; Zhao et al, 2021 ). However, the intrinsic properties and functions of these coacervate droplets may differ dramatically as a function of size, and it remains unclear how large a condensate must grow before specific functions can arise ( Lyon et al, 2021 ).…”

Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions

“…Shortly after the emergence of SARS-CoV-2, we proposed that the SARS-CoV-2 N protein would undergo PS in vitro, and that similar biophysical behavior in vivo might mediate the formation of RNA-protein condensates during viral RNA packaging into new virions, or modulate host-cell condensates (namely, stress granules) via direct physical interaction (19). In the ensuing months, many studies examining various aspects of the PS behavior of the SARS-CoV-2 N protein, including its role in viral RNA packaging, stress granule modulation, regulation of host-cell innate immune pathways, and regulation by host-cell kinases, were formally published (7,(20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36).…”

“…PS of proteins is often sensitive to salt concentrations and types of salts used (48), which is generally presumed to reflect electrostatic driving forces for PS. Lower salt concentrations were typically associated with enhanced N-protein PS (20,25,28,29,32,33,35,36), suggesting that electrostatic interactions play an important role in PS. Additionally, in the few studies that examined N-protein PS across a range of pH conditions, lower pH was associated with enhanced PS (20,32).…”