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The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA-binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA-binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction. SARS-CoV-2 nucleocapsid (N) protein is responsible for viral genome packaging. Here the authors employ single-molecule spectroscopy with all-atom simulations to provide the molecular details of N protein and show that it undergoes phase separation with RNA.

The Nucleocapsid protein (N) of SARS-CoV-2 plays a critical role in the viral lifecycle by regulating RNA replication and by packaging the viral genome. N and RNA phase separate to form condensates that may be important for these functions. Both functions occur at membrane surfaces, but how N toggles between these two membrane-associated functional states is unclear. Here, we reveal that phosphorylation switches how N condensates interact with membranes, in part by modulating condensate material properties. Our studies also show that phosphorylation alters N’s interaction with viral membrane proteins. We gain mechanistic insight through structural analysis and molecular simulations, which suggest phosphorylation induces a conformational change in N that softens condensate material properties. Together, our findings identify membrane association as a key feature of N condensates and provide mechanistic insights into the regulatory role of phosphorylation. Understanding this mechanism suggests potential therapeutic targets for COVID infection. The SARS-CoV-2 Nucleocapsid (N) protein serves multiple roles in the viral lifecycle. Phosphorylation toggles N between these roles by altering N’s conformation, its material properties when phase separated with RNA, and its membrane interactions.

The COVID pandemic has affected over 760,000,000 individuals worldwide since late 2019. Understanding how SARS-CoV-2, the virus responsible for the disease, functions at a mechanistic level is essential to develop therapeutics and vaccines. SARS-CoV-2 utilizes four structural proteins that work together during the viral life cycle to ensure the spread of infections: spike (S), envelope (E), membrane (M) and nucleocapsid (N). Though much work has focused on the S protein for the purpose of vaccines, the N protein plays a key function in the viral life cycle as well. Nucleocapsid is responsible for packaging the viral genome and incorporating the viral ribonucleocapsid into the virion. In SARS-CoV-2, the packaged viral genome adopts a more “beads on a string” organization than the helical configuration previously observed in other coronaviruses. Little is known about how N protein controls the packaging of the viral genome. N protein is composed of five domains: a folded RNA binding domain, a folded dimerization domain, and three flanking intrinsically disordered regions that were proposed to modulate interaction with RNA. Despite their potential role in modulating genome compaction, properties of corresponding disordered regions in nucleocapsid proteins from other coronaviruses remains largely understudied. At the beginning of the COVID pandemic, there was no insight on whether predicted disordered regions in SARS-CoV-2 remain disordered in the context of the full-length protein and how they modulated protein-RNA interactions.In my thesis work, I made use of single-molecule confocal fluorescence spectroscopy, and in particular, single-molecule Förster Resonance Energy Transfer (FRET) to close this knowledge gap and investigate conformations, dynamics, and interactions of the disordered regions within the SARS-CoV-2 nucleocapsid protein.I first determined that the three predicted disordered regions of N protein are disordered in the context of full-length protein. The combination of single-molecule FRET experiments and all-atom Monte Carlo simulations revealed that the monomeric full-length protein is flexible and dynamic. In addition, we observed that the protein undergoes phase separation when mixed with RNA.Having characterized the monomeric form of the protein, I next investigated the protein-protein interactions that lead to dimerization. I found that the dimerization domain is partially disordered and flexible when N protein is monomeric. I further determined the concentration under which dimerization occurs (KD = 11 ± 3 nM) to be in good agreement with previous AUC experiments and found that even in the dimeric state, N protein retains some of the dynamic nature of the monomer. I also quantified that dimer formation does not alter the conformations of the disordered NTD and folded RBD, but causes an expansion of the disordered linker and CTD. These observations were consistent with my previous determination of interactions of the linker and CTD with the dimerization domain. As a next step, I started to investigate the interactions of the N protein with RNA. To understand the role of a disordered region in aiding the recruitment of RNA, I started to investigate whether the NTD enhances the affinity of RNA to the RBD. For this, I focused on using truncations of the NTD-RBD and RBD in isolation. My experiments showed that the presence of the NTD enhances the affinity by over 50-fold compared to the RBD in isolation. Furthermore, when in complex with RNA, the NTD forms a dynamic fuzzy complex, as seen also in coarse-grained simulations. Comparison of single- and double-stranded RNA provided evidence that the NTD-RBD preferentially binds to single-strandedRNA.Finally, I examined how a crowded environment (mimicked by polyethylene glycol molecules) can modify binding properties of the NTD-RBD to RNA and found that the NTD binding is sensitive to the solution environment. Comparison of Omicron and wildtype (Wuhan-Hu-1) variants revealed that significant differences in binding affinity observed in absence of crowding are equalized in presence of the crowders. In conclusion, single-molecule fluorescence spectroscopy has offered a powerful toolbox for investigating protein conformations and interactions of disordered regions. The work has provided new insights on the molecular interactions encoded in the SARS-CoV-2 N protein and paves the way to quantitative studies of interactions with other binding partners, viral genome RNA, and small molecules.

The Nucleocapsid protein (N) of SARS-CoV-2 plays a critical role in the viral lifecycle by regulating RNA replication and packaging the viral genome. N and RNA phase separate to form condensates that may be important for these functions. Both functions occur at membrane surfaces, but how N toggles between these two membrane-associated functional states is unclear. Here, we reveal that phosphorylation switches how N condensates interact with membranes, partly by modulating condensate material properties. Phosphorylated N forms liquid condensates that wet membranes, reminiscent of N's role in RNA replication. In contrast, unmodified N forms viscoelastic condensates that can be engulfed by membranes, evoking viral genome packaging. These results suggest that phosphorylation serves as a regulatory switch within the viral replication cycle by modulating N's association with membranes. We gained mechanistic insight through structural analysis and molecular simulations, which suggest phosphorylation induces a conformational change that softens condensate material properties. Our studies also show that phosphorylation alters N's interaction with viral membrane proteins. Together, our findings uncover a novel aspect of SARS-CoV-2 biology by identifying membrane association as a key feature of N condensates and providing mechanistic insights into the regulatory role of phosphorylation. Understanding this mechanism suggests potential therapeutic targets for COVID infection.

The cardiac troponin complex, composed of troponins I, T, and C, plays a central role in regulating the calcium-dependent interactions between myosin and the thin filament. Mutations in troponin can cause cardiomyopathies; however, it is still a major challenge to connect how changes in sequence affect troponin's function. Recent high-resolution structures of the thin filament revealed critical insights into the structure-function relationship of troponin, but there remain large, unresolved segments of troponin, including the troponin-T linker region that is a hotspot for cardiomyopathy mutations. This linker region is predicted to be intrinsically disordered, with behaviors that are not well described by traditional structural approaches; however, this proposal has not been experimentally verified. Here, we used a combination of single-molecule Förster resonance energy transfer (FRET), molecular dynamics simulations, and functional reconstitution assays to investigate the troponin-T linker region. We show that in the context of both isolated troponin and the fully regulated troponin complex, the linker behaves as a dynamic, intrinsically disordered region. This region undergoes polyampholyte expansion in the presence of high salt and distinct conformational changes during the assembly of the troponin complex. We also examine the ΔE160 hypertrophic cardiomyopathy mutation in the linker and demonstrate that it does not affect the conformational dynamics of the linker, rather it allosterically affects interactions with other troponin complex subunits, leading to increased molecular contractility. Taken together, our data clearly demonstrate the importance of disorder within the troponin-T linker and provide new insights into the molecular mechanisms driving the pathogenesis of cardiomyopathies.

The SARS-CoV-2 Nucleocapsid (N) is a 419 amino acids protein that drives the compaction and packaging of the viral genome. This compaction is aided not only by protein-RNA interactions, but also by protein-protein interactions that contribute to increasing the valence of the nucleocapsid protein. Here, we focused on quantifying the mechanisms that control dimer formation. Single-molecule Förster Resonance Energy Transfer enabled us to investigate the conformations of the dimerization domain in the context of the full-length protein as well as the energetics associated with dimerization. Under monomeric conditions, we observed significantly expanded configurations of the dimerization domain (compared to the folded dimer structure), which are consistent with a dynamic conformational ensemble. The addition of unlabeled protein stabilizes a folded dimer configuration with a high mean transfer efficiency, in agreement with predictions based on known structures. Dimerization is characterized by a dissociation constant of ~ 12 nM at 23 °C and is driven by strong enthalpic interactions between the two protein subunits, which originate from the coupled folding and binding. Interestingly, the dimer structure retains some of the conformational heterogeneity of the monomeric units, and the addition of denaturant reveals that the dimer domain can significantly expand before being completely destabilized. Our findings suggest that the inherent flexibility of the monomer form is required to adopt the specific fold of the dimer domain, where the two subunits interlock with one another. We proposed that the retained flexibility of the dimer form may favor the capture and interactions with RNA, and that the temperature dependence of dimerization may explain some of the previous observations regarding the phase separation propensity of the N protein.

Abstract The SARS-CoV-2 nucleocapsid (N) protein is an abundant RNA binding protein critical for viral genome packaging, yet the molecular details that underlie this process are poorly understood. Here we combine single-molecule spectroscopy with all-atom simulations to uncover the molecular details that contribute to N protein function. N protein contains three dynamic disordered regions that house putative transiently-helical binding motifs. The two folded domains interact minimally such that full-length N protein is a flexible and multivalent RNA binding protein. N protein also undergoes liquid-liquid phase separation when mixed with RNA, and polymer theory predicts that the same multivalent interactions that drive phase separation also engender RNA compaction. We offer a simple symmetry-breaking model that provides a plausible route through which single-genome condensation preferentially occurs over phase separation, suggesting that phase separation offers a convenient macroscopic readout of a key nanoscopic interaction. Competing Interest Statement The authors have declared no competing interest. Footnotes * None of the conclusions arrived at in our original submission have changed. We have performed a large body of additional controls, expanded the scope of simulations to obtain better conformational sampling, and updated references to reflect the ever-evolving literature.

The SARS-CoV-2 Nucleocapsid (N) protein is responsible for condensation of the viral genome. Characterizing the mechanisms controlling nucleic acid binding is a key step in understanding how condensation is realized. Here, we focus on the role of the RNA Binding Domain (RBD) and its flanking disordered N-Terminal Domain (NTD) tail, using single-molecule Förster Resonance Energy Transfer and coarse-grained simulations. We quantified contact site size and binding affinity for nucleic acids and concomitant conformational changes occurring in the disordered region. We found that the disordered NTD increases the affinity of the RBD for RNA by about 50-fold. Binding of both nonspecific and specific RNA results in a modulation of the tail configurations, which respond in an RNA length-dependent manner. Not only does the disordered NTD increase affinity for RNA, but mutations that occur in the Omicron variant modulate the interactions, indicating a functional role of the disordered tail. Finally, we found that the NTD-RBD preferentially interacts with single-stranded RNA and that the resulting protein:RNA complexes are flexible and dynamic. We speculate that this mechanism of interaction enables the Nucleocapsid protein to search the viral genome for and bind to high-affinity motifs.Competing Interest StatementThe authors have declared no competing interest.