Phase separation by the SARS-CoV-2 nucleocapsid protein: Consensus and open questions
2022; Elsevier BV; Volume: 298; Issue: 3 Linguagem: Inglês
10.1016/j.jbc.2022.101677
ISSN1083-351X
AutoresSean M. Cascarina, Eric D. Ross,
Tópico(s)RNA and protein synthesis mechanisms
ResumoIn response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein—an essential viral protein required for viral replication—and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs. In response to the recent SARS-CoV-2 pandemic, a number of labs across the world have reallocated their time and resources to better our understanding of the virus. For some viruses, including SARS-CoV-2, viral proteins can undergo phase separation: a biophysical process often related to the partitioning of protein and RNA into membraneless organelles in vivo. In this review, we discuss emerging observations of phase separation by the SARS-CoV-2 nucleocapsid (N) protein—an essential viral protein required for viral replication—and the possible in vivo functions that have been proposed for N-protein phase separation, including viral replication, viral genomic RNA packaging, and modulation of host-cell response to infection. Additionally, since a relatively large number of studies examining SARS-CoV-2 N-protein phase separation have been published in a short span of time, we take advantage of this situation to compare results from similar experiments across studies. Our evaluation highlights potential strengths and pitfalls of drawing conclusions from a single set of experiments, as well as the value of publishing overlapping scientific observations performed simultaneously by multiple labs. SARS-CoV-2, the virus responsible for COVID-19 and the ongoing pandemic, has exacted an enormous toll on human health, with >5.6 million deaths and >350 million infections currently attributed to the virus (according to the World Health Organization data, https://covid19.who.int/, accessed on 1/26/22). The pandemic had already led to an estimated $16 trillion in global economic costs by October 2020 (1Cutler D.M. Summers L.H. The COVID-19 pandemic and the $16 trillion virus.JAMA. 2020; 324: 1495-1496Google Scholar) and disrupted nearly every economic sector, including science. Over the past year and a half, extraordinary progress has been made in improving our understanding of this novel virus. Emerging experimental results implicate the SARS-CoV-2 nucleocapsid (N) protein as a critical viral factor mediating viral replication, viral genomic RNA (gRNA) packaging, and modulation of host-cell response to infection. Intriguingly, the N protein has the ability to undergo phase separation (PS), which is now considered a pervasive phenomenon organizing a broad diversity of biological processes in cells. In this review, we discuss emerging models, experimental results, and possible in vivo functions related to PS by the SARS-CoV-2 N protein. Additionally, given the remarkable number of related publications on this topic within the span of ∼ 1 year, we leverage this unusual situation to evaluate how overlapping work, performed and published in parallel by independent groups, may shape resulting conclusions. In practice, science is often performed sequentially: one published discovery typically precedes, informs, and directs subsequent experimentation. The incentive structure in science, which rewards novelty, promotes this sequential model, and disincentivizes studies focused on replication and validation. One limitation of this sequential model is that subtle differences in experimental design can sometimes have a significant impact on experimental results and thus influence the direction of subsequent experiments. However, the sequential publication model was punctuated by the SARS-CoV-2 pandemic. Many labs with historically little or no prior experience in virology applied their respective areas of expertise to questions related to SARS-CoV-2. This abrupt reallocation of resources to the same topic of study by many labs organically created a scientific question of its own: what happens when multiple labs perform and publish closely related experiments in parallel? Here, we compare a set of recent and related studies reporting PS by the SARS-CoV-2 N protein to examine this question. The prototypical function for coronaviral N proteins is to condense and organize gRNA in nascent virions (2Chang C. Hou M.-H. Chang C.-F. Hsiao C.-D. Huang T.-H. The SARS coronavirus nucleocapsid protein - forms and functions.Antiviral Res. 2014; 103: 39-50Google Scholar). Virion formation occurs via the accumulation of the SARS-CoV-2 structural proteins [the spike (S), envelope (E), membrane (M), and N proteins] and gRNA at the ER-Golgi intermediate compartment (ERGIC) membrane. Multiple studies suggest that a single strand of SARS-CoV-2 gRNA forms dense, locally ordered ribonucleoprotein (RNP) regions consisting predominantly of N protein associated with the gRNA strand (3Klein S. Cortese M. Winter S.L. Wachsmuth-Melm M. Neufeldt C.J. Cerikan B. Stanifer M.L. Boulant S. Bartenschlager R. Chlanda P. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.Nat. Commun. 2020; 11: 5885Google Scholar, 4Yao H. Song Y. Chen Y. Wu N. Xu J. Sun C. Zhang J. Weng T. Zhang Z. Wu Z. Cheng L. Shi D. Lu X. Lei J. Crispin M. et al.Molecular architecture of the SARS-CoV-2 virus.Cell. 2020; 183: 730-738.e13Google Scholar, 5Cao C. Cai Z. Xiao X. Rao J. Chen J. Hu N. Yang M. Xing X. Wang Y. Li M. Zhou B. Wang X. Wang J. Xue Y. The architecture of the SARS-CoV-2 RNA genome inside virion.Nat. Commun. 2021; 12: 3917Google Scholar). Locally ordered RNPs may be further organized into more complex arrangements via clustering of RNPs in particular stoichiometries and geometries (3Klein S. Cortese M. Winter S.L. Wachsmuth-Melm M. Neufeldt C.J. Cerikan B. Stanifer M.L. Boulant S. Bartenschlager R. Chlanda P. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.Nat. Commun. 2020; 11: 5885Google Scholar, 4Yao H. Song Y. Chen Y. Wu N. Xu J. Sun C. Zhang J. Weng T. Zhang Z. Wu Z. Cheng L. Shi D. Lu X. Lei J. Crispin M. et al.Molecular architecture of the SARS-CoV-2 virus.Cell. 2020; 183: 730-738.e13Google Scholar), although other evidence and prior models of the SARS-CoV N protein suggest a more linear, helical RNP arrangement (2Chang C. Hou M.-H. Chang C.-F. Hsiao C.-D. Huang T.-H. The SARS coronavirus nucleocapsid protein - forms and functions.Antiviral Res. 2014; 103: 39-50Google Scholar, 5Cao C. Cai Z. Xiao X. Rao J. Chen J. Hu N. Yang M. Xing X. Wang Y. Li M. Zhou B. Wang X. Wang J. Xue Y. The architecture of the SARS-CoV-2 RNA genome inside virion.Nat. Commun. 2021; 12: 3917Google Scholar, 6Filho H.V.R. Jara G.E. Batista F.A.H. Schleder G.R. Tonoli C.C. Soprano A.S. Guimarães S.L. Borges A.C. Cassago A. Bajgelman M.C. Marques R.E. Trivella D.B.B. Franchini K.G. Figueira A.C.M. Benedetti C.E. et al.Structural dynamics of SARS-CoV-2 nucleocapsid protein induced by RNA binding.bioRxiv. 2021; ([preprint])https://doi.org/10.1101/2021.08.27.457964Google Scholar). These RNPs preferentially accumulate on curved membranes (3Klein S. Cortese M. Winter S.L. Wachsmuth-Melm M. Neufeldt C.J. Cerikan B. Stanifer M.L. Boulant S. Bartenschlager R. Chlanda P. SARS-CoV-2 structure and replication characterized by in situ cryo-electron tomography.Nat. Commun. 2020; 11: 5885Google Scholar), indicating either that RNP association aids in membrane curvature during nascent virion formation, or that curved membranes are the preferred recruitment surface for RNPs. The N protein also interacts with a luminal domain (i.e., in the interior of virions) of the M protein, which was proposed as a possible mechanism for mediating recruitment of N-containing RNPs to the ERGIC membrane (7Lu S. Ye Q. Singh D. Cao Y. Diedrich J.K. Yates J.R. Villa E. Cleveland D.W. Corbett K.D. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.Nat. Commun. 2021; 12: 502Google Scholar). Some evidence suggests that the N protein of both SARS-CoV and SARS-CoV-2 may also interact with the E protein (8Tseng Y.-T. Wang S.-M. Huang K.-J. Wang C.-T. SARS-CoV envelope protein palmitoylation or nucleocapid association is not required for promoting virus-like particle production.J. Biomed. Sci. 2014; 21: 34Google Scholar, 9Li J. Guo M. Tian X. Wang X. Yang X. Wu P. Liu C. Xiao Z. Qu Y. Yin Y. Wang C. Zhang Y. Zhu Z. Liu Z. Peng C. et al.Virus-host interactome and proteomic survey reveal potential virulence factors influencing SARS-CoV-2 pathogenesis.Med. (N. Y.). 2021; 2: 99-112.e7Google Scholar). While precise detail regarding the interactions and arrangements of individual molecules within intact virions is still forthcoming, the N protein clearly plays a central role in gRNA compaction and organization in SARS-CoV-2 virions. PS by a protein involves the formation of two distinct yet coexisting phases from a well-mixed protein solution: a dense phase of high protein concentration and a dilute phase of low protein concentration (10Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: Organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Google Scholar). Subsequent to initial PS, the dense phase may also undergo additional phase transitions that change its material properties (11Patel A. Lee H.O. Jawerth L. Maharana S. Jahnel M. Hein M.Y. Stoynov S. Mahamid J. Saha S. Franzmann T.M. Pozniakovski A. Poser I. Maghelli N. Royer L.A. Weigert M. et al.A liquid-to-solid phase transition of the ALS protein FUS accelerated by disease mutation.Cell. 2015; 162: 1066-1077Google Scholar, 12Molliex A. Temirov J. Lee J. Coughlin M. Kanagaraj A.P. Kim H.J. Mittag T. Taylor J.P. Phase separation by low complexity domains promotes stress granule assembly and drives pathological fibrillization.Cell. 2015; 163: 123-133Google Scholar). Consequently, condensates can exhibit material properties consistent with liquids, gels, or solids (13Boeynaems S. Alberti S. Fawzi N.L. Mittag T. Polymenidou M. Rousseau F. Schymkowitz J. Shorter J. Wolozin B. Van Den Bosch L. Tompa P. Fuxreiter M. Protein phase separation: A new phase in cell biology.Trends Cell Biol. 2018; 28: 420-435Google Scholar), and these properties can be influenced by many factors including protein sequence, protein concentration, the presence and concentrations of other molecules, and physical and chemical environment. One of the key features of proteins associated with PS is “multivalency,” which describes proteins with multiple binding sites for partner molecules. PS can occur in either single-component or multicomponent systems (14Dignon G.L. Best R.B. Mittal J. Biomolecular phase separation: From molecular driving forces to macroscopic properties.Annu. Rev. Phys. Chem. 2020; 71: 53-75Google Scholar, 15Ruff K.M. Dar F. Pappu R.V. Polyphasic linkage and the impact of ligand binding on the regulation of biomolecular condensates.Biophys. Rev. 2021; 2021302Google Scholar). In a single-component system, PS is driven by homotypic interactions (i.e., between two identical biopolymers), whereas co-PS in a multicomponent system is driven by heterotypic interactions (i.e., between different biopolymers) or a combination of homotypic and heterotypic interactions. While no single type of domain is required in multivalent proteins to observe PS, certain domains appear to be more common among proteins known to phase separate. For example, a number of phase separating proteins contain RNA-binding domains, intrinsically disordered regions (IDRs), oligomerization domains, and low-complexity domains. PS has gained recent attention in biology due to its connection with “biomolecular condensates” (10Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: Organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Google Scholar, 13Boeynaems S. Alberti S. Fawzi N.L. Mittag T. Polymenidou M. Rousseau F. Schymkowitz J. Shorter J. Wolozin B. Van Den Bosch L. Tompa P. Fuxreiter M. Protein phase separation: A new phase in cell biology.Trends Cell Biol. 2018; 28: 420-435Google Scholar, 16Hyman A.A. Weber C.A. Jülicher F. Liquid-liquid phase separation in biology.Annu. Rev. Cell Dev. Biol. 2014; 30: 39-58Google Scholar), which are membraneless organelles that are typically enriched in certain proteins and nucleic acids. Much like the dense phase observed in vitro, biomolecular condensates consist of a network of interactions between multivalent proteins and partner molecules (often nucleic acids, proteins, or other biopolymers). Many types of biomolecular condensates have been described, including (but not limited to) stress granules, P-bodies, nucleoli, nuclear speckles, germ granules, and Cajal bodies (10Banani S.F. Lee H.O. Hyman A.A. Rosen M.K. Biomolecular condensates: Organizers of cellular biochemistry.Nat. Rev. Mol. Cell Biol. 2017; 18: 285-298Google Scholar). Each type of biomolecular condensate is associated with distinct sets of constituent molecules, material properties, biological functions, stability, and regulation. Regardless of these differences, biomolecular condensation represents an elegant biological solution for organizing and concentrating groups of molecules in a regulatable and sensitive fashion. Given the prevalence, diversity, and importance of biomolecular condensates in eukaryotes, it is perhaps no surprise that some viruses are able to interact with and manipulate endogenous condensates or trigger the formation of entirely new viral condensates in host cells (17Gaete-Argel A. Márquez C.L. Barriga G.P. Soto-Rifo R. Valiente-Echeverría F. Strategies for success. Viral infections and membraneless organelles.Front. Cell. Infect. Microbiol. 2019; 9: 336Google Scholar, 18Etibor T.A. Yamauchi Y. Amorim M.J. Liquid biomolecular condensates and viral lifecycles: Review and perspectives.Viruses. 2021; 13: 366Google Scholar). 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 (19Cascarina S.M. Ross E.D. A proposed role for the SARS-CoV-2 nucleocapsid protein in the formation and regulation of biomolecular condensates.FASEB J. 2020; 34: 9832-9842Google Scholar). 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 (7Lu S. Ye Q. Singh D. Cao Y. Diedrich J.K. Yates J.R. Villa E. Cleveland D.W. Corbett K.D. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.Nat. Commun. 2021; 12: 502Google Scholar, 20Zhao M. Yu Y. Sun L.-M. Xing J.-Q. Li T. Zhu Y. Wang M. Yu Y. Xue W. Xia T. Cai H. Han Q.-Y. Yin X. Li W.-H. Li A.-L. et al.GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein.Nat. Commun. 2021; 12: 2114Google Scholar, 21Iserman C. Roden C.A. Boerneke M.A. Sealfon R.S.G. McLaughlin G.A. Jungreis I. Fritch E.J. Hou Y.J. Ekena J. Weidmann C.A. Theesfeld C.L. Kellis M. Troyanskaya O.G. Baric R.S. Sheahan T.P. et al.Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid.Mol. Cell. 2020; 80: 1078-1091.e6Google Scholar, 22Cubuk J. Alston J.J. Incicco J.J. Singh S. Stuchell-Brereton M.D. Ward M.D. Zimmerman M.I. Vithani N. Griffith D. Wagoner J.A. Bowman G.R. Hall K.B. Soranno A. Holehouse A.S. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.Nat. Commun. 2021; 12: 1936Google Scholar, 23Dang M. Li Y. Song J. ATP biphasically modulates LLPS of SARS-CoV-2 nucleocapsid protein and specifically binds its RNA-binding domain.Biochem. Biophys. Res. Commun. 2021; 541: 50-55Google Scholar, 24Zhao H. Wu D. Nguyen A. Li Y. Adão R.C. Valkov E. Patterson G.H. Piszczek G. Schuck P. Energetic and structural features of SARS-CoV-2 N-protein co-assemblies with nucleic acids.iScience. 2021; 24: 102523Google Scholar, 25Wang S. Dai T. Qin Z. Pan T. Chu F. Lou L. Zhang L. Yang B. Huang H. Lu H. Zhou F. Targeting liquid–liquid phase separation of SARS-CoV-2 nucleocapsid protein promotes innate antiviral immunity by elevating MAVS activity.Nat. Cell Biol. 2021; 23: 718-732Google Scholar, 26Huang W. Ju X. Tian M. Li X. Yu Y. Sun Q. Ding Q. Jia D. Molecular determinants for regulation of G3BP1/2 phase separation by the SARS-CoV-2 nucleocapsid protein.Cell Discov. 2021; 7: 69Google Scholar, 27Prakash Somasekharan S. Gleave M. SARS-CoV-2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets.FEBS Lett. 2021; 595: 2872-2896Google Scholar, 28Jack A. Ferro L.S. Trnka M.J. Wehri E. Nadgir A. Nguyenla X. Fox D. Costa K. Stanley S. Schaletzky J. Yildiz A. SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.PLoS Biol. 2021; 19e3001425Google Scholar, 29Savastano A. Ibáñez de Opakua A. Rankovic M. Zweckstetter M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.Nat. Commun. 2020; 11: 6041Google Scholar, 30Carlson C.R. Asfaha J.B. Ghent C.M. Howard C.J. Hartooni N. Safari M. Frankel A.D. Morgan D.O. Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a biophysical basis for its dual functions.Mol. Cell. 2020; 80: 1092-1103.e4Google Scholar, 31Luo L. Li Z. Zhao T. Ju X. Ma P. Jin B. Zhou Y. He S. Huang J. Xu X. Zou Y. Li P. Liang A. Liu J. Chi T. et al.SARS-CoV-2 nucleocapsid protein phase separates with G3BPs to disassemble stress granules and facilitate viral production.Sci. Bull. 2021; 66: 1194-1204Google Scholar, 32Perdikari T.M. Murthy A.C. Ryan V.H. Watters S. Naik M.T. Fawzi N.L. SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.EMBO J. 2020; 39e106478Google Scholar, 33Wang J. Shi C. Xu Q. Yin H. SARS-CoV-2 nucleocapsid protein undergoes liquid–liquid phase separation into stress granules through its N-terminal intrinsically disordered region.Cell Discov. 2021; 7: 5Google Scholar, 34Wu Y. Ma L. Cai S. Zhuang Z. Zhao Z. Jin S. Xie W. Zhou L. Zhang L. Zhao J. Cui J. RNA-induced liquid phase separation of SARS-CoV-2 nucleocapsid protein facilitates NF-κB hyper-activation and inflammation.Signal Transduct. Target. Ther. 2021; 6: 167Google Scholar, 35Chen H. Cui Y. Han X. Hu W. Sun M. Zhang Y. Wang P.H. Song G. Chen W. Lou J. Liquid–liquid phase separation by SARS-CoV-2 nucleocapsid protein and RNA.Cell Res. 2020; 30: 1143-1145Google Scholar, 36Zhao D. Xu W. Zhang X. Wang X. Ge Y. Yuan E. Xiong Y. Wu S. Li S. Wu N. Tian T. Feng X. Shu H. Lang P. Li J. et al.Understanding the phase separation characteristics of nucleocapsid protein provides a new therapeutic opportunity against SARS-CoV-2.Protein Cell. 2021; 12: 734-740Google Scholar). Figure 1 highlights the factors affecting N-protein PS in vitro and the proposed functions of N-protein PS in vivo, each of which is discussed in the ensuing sections. Additionally, we compare the N-protein domains purported to be critical for PS and, more broadly, what can be learned from a “consensus” view resulting from many related studies published in a short timeframe. We would like to note that while we have done our best to faithfully interpret the available data, not all studies present rigorous quantification of PS and quantification methods differed between studies; therefore, our conclusions are based at least to some degree on our subjective interpretation. PS has often been associated with RNA-binding proteins containing prion-like or other low-complexity domains (37Fomicheva A. Ross E.D. From prions to stress granules: Defining the compositional features of prion-like domains that promote different types of assemblies.Int. J. Mol. Sci. 2021; 22: 1251Google Scholar, 38March Z.M. King O.D. Shorter J. Prion-like domains as epigenetic regulators, scaffolds for subcellular organization, and drivers of neurodegenerative disease.Brain Res. 2016; 1647: 9-18Google Scholar, 39Harrison A.F. Shorter J. RNA-binding proteins with prion-like domains in health and disease.Biochem. J. 2017; 474: 1417-1438Google Scholar). RNA itself is capable of undergoing PS (40Van Treeck B. Protter D.S.W. Matheny T. Khong A. Link C.D. Parker R. RNA self-assembly contributes to stress granule formation and defining the stress granule transcriptome.Proc. Natl. Acad. Sci. U. S. A. 2018; 115: 2734-2739Google Scholar), can often induce PS of specific proteins at lower protein concentrations (41Lin Y. Protter D.S.W. Rosen M.K. Parker R. formation and maturation of phase-separated liquid droplets by RNA-binding proteins.Mol. Cell. 2015; 60: 208-219Google Scholar), and can regulate the material properties of condensates in a variety of ways [reviewed in (42Roden C. Gladfelter A.S. RNA contributions to the form and function of biomolecular condensates.Nat. Rev. Mol. Cell Biol. 2020; 22: 183-195Google Scholar)]. The SARS-CoV-2 N protein contains two structured domains capable of binding RNA (43Zhou R. Zeng R. von Brunn A. Lei J. Structural characterization of the C-terminal domain of SARS-CoV-2 nucleocapsid protein.Mol. Biomed. 2020; 1: 2Google Scholar, 44Yang M. He S. Chen X. Huang Z. Zhou Z. Zhou Z. Chen Q. Chen S. Kang S. Structural insight into the SARS-CoV-2 nucleocapsid protein C-terminal domain reveals a novel recognition mechanism for viral transcriptional regulatory sequences.Front. Chem. 2021; 8: 624765Google Scholar, 45Peng Y. Du N. Lei Y. Dorje S. Qi J. Luo T. Gao G.F. Song H. Structures of the SARS-CoV-2 nucleocapsid and their perspectives for drug design.EMBO J. 2020; 39e105938Google Scholar, 46Kang S. Yang M. Hong Z. Zhang L. Huang Z. Chen X. He S. Zhou Z. Zhou Z. Chen Q. Yan Y. Zhang C. Shan H. Chen S. Crystal structure of SARS-CoV-2 nucleocapsid protein RNA binding domain reveals potential unique drug targeting sites.Acta Pharm. Sin. B. 2020; 10: 1228-1238Google Scholar, 47Wu C. Qavi A.J. Hachim A. Kavian N. Cole A.R. Moyle A.B. Wagner N.D. Sweeney-Gibbons J. Rohrs H.W. Gross M.L. Peiris J.S.M. Basler C.F. Farnsworth C.W. Valkenburg S.A. Amarasinghe G.K. et al.Characterization of SARS-CoV-2 nucleocapsid protein reveals multiple functional consequences of the C-terminal domain.iScience. 2021; 24: 102681Google Scholar), as well as multiple flanking IDRs that enhance RNA binding (44Yang M. He S. Chen X. Huang Z. Zhou Z. Zhou Z. Chen Q. Chen S. Kang S. Structural insight into the SARS-CoV-2 nucleocapsid protein C-terminal domain reveals a novel recognition mechanism for viral transcriptional regulatory sequences.Front. Chem. 2021; 8: 624765Google Scholar, 47Wu C. Qavi A.J. Hachim A. Kavian N. Cole A.R. Moyle A.B. Wagner N.D. Sweeney-Gibbons J. Rohrs H.W. Gross M.L. Peiris J.S.M. Basler C.F. Farnsworth C.W. Valkenburg S.A. Amarasinghe G.K. et al.Characterization of SARS-CoV-2 nucleocapsid protein reveals multiple functional consequences of the C-terminal domain.iScience. 2021; 24: 102681Google Scholar). While N protein alone typically exhibited weak or undetectable PS in the majority of studies (7Lu S. Ye Q. Singh D. Cao Y. Diedrich J.K. Yates J.R. Villa E. Cleveland D.W. Corbett K.D. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.Nat. Commun. 2021; 12: 502Google Scholar, 20Zhao M. Yu Y. Sun L.-M. Xing J.-Q. Li T. Zhu Y. Wang M. Yu Y. Xue W. Xia T. Cai H. Han Q.-Y. Yin X. Li W.-H. Li A.-L. et al.GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein.Nat. Commun. 2021; 12: 2114Google Scholar, 21Iserman C. Roden C.A. Boerneke M.A. Sealfon R.S.G. McLaughlin G.A. Jungreis I. Fritch E.J. Hou Y.J. Ekena J. Weidmann C.A. Theesfeld C.L. Kellis M. Troyanskaya O.G. Baric R.S. Sheahan T.P. et al.Genomic RNA elements drive phase separation of the SARS-CoV-2 nucleocapsid.Mol. Cell. 2020; 80: 1078-1091.e6Google Scholar, 27Prakash Somasekharan S. Gleave M. SARS-CoV-2 nucleocapsid protein interacts with immunoregulators and stress granules and phase separates to form liquid droplets.FEBS Lett. 2021; 595: 2872-2896Google Scholar, 29Savastano A. Ibáñez de Opakua A. Rankovic M. Zweckstetter M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.Nat. Commun. 2020; 11: 6041Google Scholar, 30Carlson C.R. Asfaha J.B. Ghent C.M. Howard C.J. Hartooni N. Safari M. Frankel A.D. Morgan D.O. Phosphoregulation of phase separation by the SARS-CoV-2 N protein suggests a biophysical basis for its dual functions.Mol. Cell. 2020; 80: 1092-1103.e4Google Scholar, 31Luo L. Li Z. Zhao T. Ju X. Ma P. Jin B. Zhou Y. He S. Huang J. Xu X. Zou Y. Li P. Liang A. Liu J. Chi T. et al.SARS-CoV-2 nucleocapsid protein phase separates with G3BPs to disassemble stress granules and facilitate viral production.Sci. Bull. 2021; 66: 1194-1204Google Scholar, 32Perdikari T.M. Murthy A.C. Ryan V.H. Watters S. Naik M.T. Fawzi N.L. SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.EMBO J. 2020; 39e106478Google Scholar, 33Wang J. Shi C. Xu Q. Yin H. SARS-CoV-2 nucleocapsid protein undergoes liquid–liquid phase separation into stress granules through its N-terminal intrinsically disordered region.Cell Discov. 2021; 7: 5Google Scholar), RNA almost universally induced PS of the SARS-CoV-2 N protein across all studies evaluated in depth (Fig. 1Ai). RNAs of varying lengths and sequences can induce N-protein PS to varying degrees, suggesting that this process is somewhat nonspecific in vitro (though the formation of N+RNA condensates in vivo may exhibit greater sequence specificity, as discussed in a later section). For studies that tested a wide range of RNA concentrations, exceedingly high amounts of RNA tended to inhibit PS (7Lu S. Ye Q. Singh D. Cao Y. Diedrich J.K. Yates J.R. Villa E. Cleveland D.W. Corbett K.D. The SARS-CoV-2 nucleocapsid phosphoprotein forms mutually exclusive condensates with RNA and the membrane-associated M protein.Nat. Commun. 2021; 12: 502Google Scholar, 22Cubuk J. Alston J.J. Incicco J.J. Singh S. Stuchell-Brereton M.D. Ward M.D. Zimmerman M.I. Vithani N. Griffith D. Wagoner J.A. Bowman G.R. Hall K.B. Soranno A. Holehouse A.S. The SARS-CoV-2 nucleocapsid protein is dynamic, disordered, and phase separates with RNA.Nat. Commun. 2021; 12: 1936Google Scholar, 28Jack A. Ferro L.S. Trnka M.J. Wehri E. Nadgir A. Nguyenla X. Fox D. Costa K. Stanley S. Schaletzky J. Yildiz A. SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.PLoS Biol. 2021; 19e3001425Google Scholar, 32Perdikari T.M. Murthy A.C. Ryan V.H. Watters S. Naik M.T. Fawzi N.L. SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.EMBO J. 2020; 39e106478Google Scholar), which is consistent with re-entrant phase behavior due to an imbalance in the stoichiometries of constituent molecules. Electrostatic forces were consistently implicated across studies in mediating or regulating RNA-dependent PS (Fig. 1Aii). PS of proteins is often sensitive to salt concentrations and types of salts used (48Alberti S. Gladfelter A. Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates.Cell. 2019; 176: 419-434Google Scholar), which is generally presumed to reflect electrostatic driving forces for PS. Lower salt concentrations were typically associated with enhanced N-protein PS (20Zhao M. Yu Y. Sun L.-M. Xing J.-Q. Li T. Zhu Y. Wang M. Yu Y. Xue W. Xia T. Cai H. Han Q.-Y. Yin X. Li W.-H. Li A.-L. et al.GCG inhibits SARS-CoV-2 replication by disrupting the liquid phase condensation of its nucleocapsid protein.Nat. Commun. 2021; 12: 2114Google Scholar, 25Wang S. Dai T. Qin Z. Pan T. Chu F. Lou L. Zhang L. Yang B. Huang H. Lu H. Zhou F. Targeting liquid–liquid phase separation of SARS-CoV-2 nucleocapsid protein promotes innate antiviral immunity by elevating MAVS activity.Nat. Cell Biol. 2021; 23: 718-732Google Scholar, 28Jack A. Ferro L.S. Trnka M.J. Wehri E. Nadgir A. Nguyenla X. Fox D. Costa K. Stanley S. Schaletzky J. Yildiz A. SARS-CoV-2 nucleocapsid protein forms condensates with viral genomic RNA.PLoS Biol. 2021; 19e3001425Google Scholar, 29Savastano A. Ibáñez de Opakua A. Rankovic M. Zweckstetter M. Nucleocapsid protein of SARS-CoV-2 phase separates into RNA-rich polymerase-containing condensates.Nat. Commun. 2020; 11: 6041Google Scholar, 32Perdikari T.M. Murthy A.C. Ryan V.H. Watters S. Naik M.T. Fawzi N.L. SARS-CoV-2 nucleocapsid protein phase-separates with RNA and with human hnRNPs.EMBO J. 2020; 39e106478Google Scholar, 33Wang J. Shi C. Xu Q. Yin H. SARS-CoV-2 nucleocapsid protein undergoes liquid–liquid phase separation into stress granules throu
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