SARS Nucleocapsid (1-49)

What is the structural composition of SARS-CoV-2 Nucleocapsid (1-49) and how does it relate to the full nucleocapsid protein?

The SARS-CoV-2 Nucleocapsid (1-49) constitutes the N-terminal portion of the N-terminal domain (NTD) of the nucleocapsid protein. The full nucleocapsid protein contains both N-terminal (NTD) and C-terminal domains (CTD) connected by a serine-arginine-rich (SR) linker region . The crystal structure of the N-NTD has been solved at 1.8 Å resolution, revealing conserved features with other coronavirus N proteins while displaying distinct charge distribution patterns that may alter RNA-binding modes .

Within the full nucleocapsid protein architecture, the N-terminal region including residues 1-49 participates in the protein's primary function of RNA binding. This region contains flexible elements that contribute to the recognition of specific RNA motifs in the viral genome . The NTD works in concert with other domains to facilitate the nucleocapsid protein's multiple roles, including viral RNA transcription, genome packaging, and interactions with host cellular machinery .

How do the structural characteristics of the SARS-CoV-2 Nucleocapsid (1-49) contribute to its function in viral replication?

The N-terminal region of the SARS-CoV-2 nucleocapsid protein contains dynamic disordered regions that house putatively transiently-helical binding motifs, enabling its adaptability in RNA recognition . These flexible regions within residues 1-49 are critical for reading the intrinsic signature of preferred RNA elements for selective and stable complex formation within the large pool of available motifs in the viral genome .

The flexibility of this region contributes to the multivalent nature of the full nucleocapsid protein, which is important for various functions including phase separation with RNA and genome packaging . NMR studies have shown that the N-terminal domain preferentially interacts with smaller RNA fragments compared to the C-terminal domain, suggesting that the N-terminal region including residues 1-49 plays a crucial role in the initial recognition and binding of viral RNA . This initial binding may then facilitate recruitment of additional nucleocapsid proteins, ultimately leading to the formation of ribonucleoprotein complexes essential for viral replication and assembly.

What experimental evidence demonstrates the RNA binding specificity of SARS-CoV-2 Nucleocapsid (1-49)?

NMR spectroscopy studies have systematically analyzed the interactions of the nucleocapsid protein's N-terminal RNA-binding domain (NTD), which includes residues 1-49, with individual cis RNA elements clustering in the SARS-CoV-2 regulatory 5'-genomic end . These studies have unraveled the NTD RNA-binding preferences in the natural genome context, showing that the domain's flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation .

Comparative binding studies using NMR titrations have shown that the N-terminal region (N 1-209, which includes residues 1-49) binds a seven-nucleotide RNA fragment derived from the 5'-UTR of the SARS-CoV-2 genome with approximately 2-3 fold higher affinity than the C-terminal region (N 251-419) . While the estimated binding affinity for the C-terminal region is around 300-400 μM, the N-terminal region shows stronger binding with chemical shift perturbations (CSPs) indicating specific residues involved in RNA recognition .

Cross-linking studies have further demonstrated that the N protein is enriched within specific linear regions of the viral genome , supporting its role in recognizing particular RNA sequences or structures, with the N-terminal domain including residues 1-49 likely playing a crucial role in this specificity.

How does phosphorylation modulate the RNA binding properties of the SARS-CoV-2 Nucleocapsid (1-49) region?

Phosphorylation of the N-terminal region of the SARS-CoV-2 nucleocapsid protein has been shown to reduce its RNA binding affinity . NMR studies demonstrate that phosphorylation of N 1-209, which encompasses residues 1-49, leads to reduced chemical shift perturbations (CSPs) upon RNA binding, indicating weaker interaction with RNA substrates .

The phosphorylation state of the nucleocapsid protein has also been shown to quench an inherent dynamic exchange process within the serine-arginine-rich (SR) region , which may indirectly affect the RNA binding properties of the N-terminal region including residues 1-49. This post-translational modification appears to serve as a molecular switch that regulates the balance between different functions of the nucleocapsid protein during viral infection, potentially transitioning the protein between RNA binding, phase separation, and interaction with host proteins.

What are the optimal expression and purification strategies for studying SARS-CoV-2 Nucleocapsid (1-49) in isolation?

For structural and functional studies of SARS-CoV-2 Nucleocapsid (1-49), bacterial expression systems using E. coli have proven effective. Based on methodologies implied in the research literature, an optimal expression and purification strategy would include:

• Vector design: Cloning the gene segment encoding residues 1-49 into an expression vector with an appropriate affinity tag (His6 or GST) and a cleavable linker.

• Expression conditions: Transformation into E. coli BL21(DE3) or similar strains, with expression induced at lower temperatures (16-18°C) to enhance solubility and proper folding.

• Cell lysis: Sonication or pressure-based cell disruption in buffers containing salt (typically 300-500 mM NaCl) and reducing agents to prevent aggregation.

• Affinity purification: Initial purification using Ni-NTA (for His-tagged constructs) or glutathione sepharose (for GST-tagged constructs).

• Tag removal: Cleavage of the affinity tag using specific proteases (TEV, thrombin, or PreScission) followed by reverse affinity chromatography.

• Size exclusion chromatography: Final purification step to ensure homogeneity and remove any aggregates.

For NMR studies, isotope labeling with 15N and/or 13C is achieved by growing bacteria in minimal media containing 15NH4Cl and/or 13C-glucose as the sole nitrogen and carbon sources . This enables detailed structural analysis and characterization of RNA interactions using various NMR experiments.

For crystallography studies that yielded high-resolution structures of the N-NTD, protein samples were purified to high homogeneity through multiple chromatography steps followed by concentration to ~10-20 mg/ml for crystallization trials .

What NMR spectroscopy approaches are most effective for analyzing SARS-CoV-2 Nucleocapsid (1-49) interactions with RNA?

NMR spectroscopy has proven to be a powerful technique for analyzing the interactions between SARS-CoV-2 Nucleocapsid (1-49) and RNA. Several complementary NMR approaches have been particularly effective:

• 2D 1H-15N HSQC titration experiments: This approach monitors chemical shift perturbations (CSPs) of backbone amide resonances upon RNA binding. By gradually adding RNA to 15N-labeled N protein constructs and recording spectra at each titration point, researchers can identify residues involved in RNA binding and estimate binding affinities .

• Triple-resonance experiments: These experiments (HNCA, HNCACB, CBCA(CO)NH, etc.) are essential for backbone assignment of the protein, which is a prerequisite for precisely identifying the specific residues involved in RNA interactions.

• Relaxation measurements: T1, T2, and heteronuclear NOE experiments provide information about the dynamics of different regions of the protein, both free and in complex with RNA, revealing how flexibility contributes to binding.

• Paramagnetic relaxation enhancement (PRE): By incorporating paramagnetic tags into either the protein or RNA, this technique can provide long-range distance restraints and information about transient interactions.

• Residual dipolar coupling (RDC): This technique provides angular constraints that are valuable for determining the relative orientation of different regions of the protein when bound to RNA.

These NMR approaches have been used to systematically analyze the interactions of the nucleocapsid protein's N-terminal RNA-binding domain with individual cis RNA elements . The research has successfully demonstrated how the domain's flexible regions, including those in residues 1-49, read the intrinsic signature of preferred RNA elements for selective and stable complex formation .

What are the characteristic conformational dynamics of SARS-CoV-2 Nucleocapsid (1-49) in both free and RNA-bound states?

The SARS-CoV-2 nucleocapsid protein, including its N-terminal region with residues 1-49, exhibits significant conformational dynamics that are crucial for its function . The protein contains dynamic disordered regions that house putatively transiently-helical binding motifs , suggesting that residues 1-49 may sample multiple conformations in the free state.

In the free state, the N-terminal domain likely displays a high degree of flexibility, particularly in regions that are involved in RNA binding. This flexibility allows the domain to adapt to different RNA structures and sequences, enabling both specific and non-specific RNA interactions essential for viral replication .

Upon RNA binding, the flexible regions within the N-terminal domain, including parts of residues 1-49, likely undergo conformational changes to accommodate the RNA structure. NMR studies have shown that the domain's flexible regions read the intrinsic signature of preferred RNA elements for selective and stable complex formation , suggesting that these regions adopt more ordered conformations in the RNA-bound state.

The conformational dynamics of the nucleocapsid protein also contribute to its ability to undergo liquid-liquid phase separation when mixed with RNA . This phase separation behavior depends on the multivalent interactions between the protein and RNA, with the N-terminal domain playing a crucial role in these interactions . The flexibility of residues 1-49 likely contributes to these multivalent interactions by allowing the domain to engage with multiple RNA sites simultaneously.

How does the sequence conservation of SARS-CoV-2 Nucleocapsid (1-49) compare across different coronavirus strains?

The sequence conservation of the N-terminal domain (NTD) of the nucleocapsid protein, including residues 1-49, shows a pattern of both conserved and variable regions across different coronavirus strains. This conservation pattern reflects the functional importance of certain residues in RNA binding and protein-protein interactions.

The N-NTD structure shows conserved features across coronaviruses , suggesting that the core functions of this domain, potentially including key residues within the 1-49 region, are maintained throughout evolution. These conserved features likely include residues involved in fundamental RNA binding mechanisms that are essential for all coronaviruses.

The conservation of binding sites targeted by small molecules against other coronaviruses like HCoV-OC43 and MERS-CoV suggests that these sites, which may include residues within the 1-49 region, are potential targets for broad-spectrum antivirals . At the same time, the distinct antigenic characteristics of different regions of the N protein are critical for developing specific immune-based rapid diagnostic tests .

Antibody cross-reactivity studies indicate that the N protein is a primary target of both SARS-CoV-2-specific and HCoV crossreactive antibodies . While the C-terminus region of SARS-CoV-2 N with minimal sequence homology with HCoV was found to be more specific for SARS-CoV-2 , this suggests that the N-terminal region, potentially including residues 1-49, may have higher sequence conservation and greater potential for crossreactivity with other coronaviruses.

What structural and functional variations exist in the Nucleocapsid (1-49) region between SARS-CoV-2 and other beta-coronaviruses?

The SARS-CoV-2 nucleocapsid N-terminal domain, which includes residues 1-49, shares structural similarities with other beta-coronaviruses but also displays important variations that may affect its function. The crystal structure of the N-NTD has been solved at 1.8 Å resolution , allowing for detailed comparisons with other coronaviruses.

Functionally, these structural variations may translate into differences in RNA binding specificity and affinity. The N-terminal region of the SARS-CoV-2 nucleocapsid protein preferentially interacts with smaller RNA fragments relative to the C-terminal region , but whether this preference is shared with other beta-coronaviruses or represents a unique feature of SARS-CoV-2 requires further investigation.

Immunologically, there are interesting differences in cross-reactivity patterns. Cross-reactivity with SARS-CoV-2 was stronger for alpha-HCoV rather than beta-HCoV despite having less sequence identity, revealing the importance of conformational recognition rather than just sequence homology . This suggests that the N-terminal domain, including residues 1-49, may have structural features that are recognized differently by antibodies against different coronavirus groups.

The binding sites targeted by small molecules against other beta-coronaviruses like HCoV-OC43 and MERS-CoV are relatively conserved in the SARS-CoV-2 N protein structure , suggesting potential commonalities in drug binding sites that could be exploited for broad-spectrum antiviral development.

What host proteins interact with SARS-CoV-2 Nucleocapsid (1-49), and how do these interactions affect viral pathogenesis?

The N-terminal region of the SARS-CoV-2 nucleocapsid protein, including residues 1-49, interacts with several host proteins that play roles in innate immunity and cellular signaling. Based on the available research, key interactions include:

• Cyclophilin-A: The unstructured termini of the nucleocapsid protein, potentially including parts of the 1-49 region, engage with host cyclophilin-A . Cyclophilin-A is involved in protein folding and immune regulation, suggesting that this interaction may modulate the host immune response to infection.

• 14-3-3τ: The nucleocapsid protein also interacts with host 14-3-3τ through its unstructured termini . The 14-3-3 proteins are involved in various signaling pathways and cell cycle regulation, suggesting that this interaction may disrupt normal cellular processes to favor viral replication.

• Pin1: While the primary interaction with Pin1 occurs through the serine-arginine-rich (SR) region, the N-terminal domain may also contribute to this interaction . Pin1 is a proline isomerase that targets phosphorylated Ser-Pro or Thr-Pro sequences and has been implicated in mediating infection through interactions with the N protein .

These host protein interactions likely affect viral pathogenesis in several ways:

• Immune modulation: Since these host proteins play roles in innate immunity, the SARS-CoV-2 nucleocapsid protein may block the host response by competing for interactions , potentially contributing to immune evasion.

• Viral replication: Interactions with host proteins may facilitate viral replication by recruiting cellular factors required for RNA synthesis or by disrupting host processes that would otherwise limit viral replication.

• Cellular stress response: By interacting with key host regulatory proteins, the nucleocapsid protein may alter cellular stress responses, potentially contributing to the cytopathic effects observed during infection.

These diverse interactions highlight the multifunctional nature of the nucleocapsid protein and suggest that the N-terminal region, including residues 1-49, may play important roles beyond RNA binding in the viral life cycle .

How can the interactions between SARS-CoV-2 Nucleocapsid (1-49) and host proteins be targeted for therapeutic intervention?

The interactions between the SARS-CoV-2 nucleocapsid N-terminal region and host proteins present several opportunities for therapeutic intervention. Potential strategies include:

• Small molecule inhibitors: The crystal structure of the N-NTD at 1.8 Å resolution provides a structural basis for the design of small molecules that could disrupt interactions with host proteins. The binding sites targeted by small molecules against other coronaviruses are relatively conserved in the SARS-CoV-2 N protein structure , suggesting that similar approaches could be effective.

• Peptide-based inhibitors: Peptides that mimic the binding interfaces between the N-terminal region and host proteins like cyclophilin-A or 14-3-3τ could competitively inhibit these interactions. Such peptides could be designed based on the structural characterization of these protein-protein interactions.

• Targeting phosphorylation: Since phosphorylation of the N protein modulates its interactions with host proteins like Pin1 , inhibitors of the kinases responsible for N protein phosphorylation could indirectly disrupt these interactions. Alternatively, phosphomimetic compounds could be designed to interfere with phosphorylation-dependent interactions.

• Allosteric modulators: Compounds that bind to allosteric sites on the N-terminal domain could induce conformational changes that prevent host protein interactions without directly competing for the binding site.

• RNA aptamers: Since the N-terminal domain primarily functions in RNA binding , RNA aptamers designed to bind with high affinity to this region could prevent its interaction with host proteins by occupying the binding surface or inducing conformational changes.

These therapeutic strategies would aim to disrupt the virus's ability to manipulate host cell processes, potentially limiting viral replication and pathogenesis. The specificity of such interventions would depend on targeting interactions that are unique to SARS-CoV-2 or at least not shared with human proteins, to minimize off-target effects.

How does SARS-CoV-2 Nucleocapsid (1-49) contribute to phase separation, and what is the functional significance of this property?

The SARS-CoV-2 nucleocapsid protein undergoes liquid-liquid phase separation when mixed with RNA , forming condensates that may serve as sites for viral genome replication and packaging. While the search results don't specifically address the role of residues 1-49 in this process, the N-terminal domain (NTD) likely contributes significantly to phase separation due to its RNA-binding properties.

The nucleocapsid protein has been characterized as a flexible and multivalent RNA-binding protein , with the N-terminal domain preferentially interacting with smaller RNA fragments . These properties are conducive to phase separation, as multivalent interactions between proteins and RNA can drive the formation of condensates. The flexibility of the N-terminal region, which includes residues 1-49, may allow it to engage in multiple weak interactions that collectively contribute to phase separation.

The functional significance of nucleocapsid-mediated phase separation may include:

• Compartmentalization of viral replication: Phase-separated condensates could create microenvironments that concentrate viral and host factors needed for genome replication , enhancing the efficiency of this process.

• Selective packaging of viral RNA: Phase separation may facilitate the selective recognition and packaging of viral genomic RNA among the abundant cellular RNAs . A symmetry-breaking model has been proposed that provides a plausible route through which single-genome condensation preferentially occurs over phase separation .

• Protection from host defenses: Phase-separated compartments may shield viral RNA from host immune sensors or nucleases, contributing to immune evasion.

• Regulation of viral gene expression: By concentrating viral RNA and regulatory proteins, phase separation may modulate the timing and level of viral gene expression during different stages of infection.

Understanding the contribution of residues 1-49 to phase separation could provide insights into potential therapeutic strategies targeting this process, potentially disrupting viral replication and assembly.

What experimental approaches can effectively characterize the phase separation properties of SARS-CoV-2 Nucleocapsid (1-49)?

Characterizing the phase separation properties of SARS-CoV-2 Nucleocapsid (1-49) requires a combination of experimental approaches that probe both macroscopic and microscopic aspects of this phenomenon:

• Light microscopy techniques:

  • Differential interference contrast (DIC) microscopy to visualize droplet formation

  • Fluorescence microscopy using fluorescently labeled protein and RNA to monitor co-localization

  • Fluorescence recovery after photobleaching (FRAP) to assess the liquid-like properties of condensates

• Biophysical characterization:

  • Single-molecule spectroscopy combined with all-atom simulations to uncover molecular details of phase separation

  • Turbidity assays to quantify phase separation propensity under various conditions

  • Dynamic light scattering (DLS) to measure the size distribution of condensates

• Molecular determinants:

  • Mutagenesis studies to identify key residues within the 1-49 region that contribute to phase separation

  • Truncation analysis to determine if residues 1-49 alone can undergo phase separation or require other domains

  • Post-translational modification analysis to assess how phosphorylation affects phase separation properties

• RNA contribution:

  • Varying RNA length, sequence, and structure to determine how these parameters affect phase separation with the N-terminal domain

  • Competition assays between different RNA species to assess selectivity

  • In vitro transcription within condensates to evaluate functional significance

• Advanced techniques:

  • NMR spectroscopy to characterize the molecular environment within condensates

  • Cryo-electron microscopy to visualize the organization of proteins and RNA within condensates

  • Atomic force microscopy to assess the mechanical properties of condensates

These experimental approaches would provide complementary information about how residues 1-49 contribute to phase separation, the molecular interactions driving this process, and its functional significance in the viral life cycle. The multivalent interactions that drive phase separation also engender RNA compaction , suggesting that these techniques could reveal both macroscopic phase separation and nanoscopic RNA-protein interactions relevant to viral genome packaging.

How does the antigenic profile of SARS-CoV-2 Nucleocapsid (1-49) influence diagnostic test development and immunological cross-reactivity?

The antigenic profile of the SARS-CoV-2 nucleocapsid N-terminal region, including residues 1-49, has significant implications for diagnostic test development and immune response evaluation. Based on the research findings:

• Cross-reactivity concerns: The N protein is a primary target of both SARS-CoV-2-specific and human coronavirus (HCoV) crossreactive antibodies . This cross-reactivity is particularly relevant for the N-terminal region, which may share more sequence and structural conservation with other coronaviruses compared to the C-terminal region.

• Diagnostic implications: While the N protein is highly immunogenic in COVID-19 patients, the detection of binding antibodies in pre-pandemic individuals indicates potential crossreactivity with common cold human coronaviruses . This crossreactivity questions the utility of N-protein-based tests, particularly those targeting the N-terminal region, in seroprevalence studies.

• Specificity vs. sensitivity trade-off: Research has shown that the C-terminus region of SARS-CoV-2 N with minimal sequence homology with HCoV was more specific for SARS-CoV-2 and highly immunogenic . This suggests that diagnostic tests targeting the N-terminal region (1-49) might offer higher sensitivity but lower specificity compared to those targeting the C-terminal region.

• Alpha vs. beta coronavirus cross-reactivity patterns: Interestingly, cross-reactivity with SARS-CoV-2 was stronger for alpha-HCoV rather than beta-HCoV despite having less sequence identity . This reveals the importance of conformational recognition rather than just sequence homology, suggesting that the three-dimensional structure of the N-terminal domain influences antibody recognition.

• Potential protective effects: Higher pre-existing IgG to OC43 N correlated with lower IgG to SARS-CoV-2 N in RT-PCR negative individuals, indicating a potential protective association . This finding suggests that cross-reactive antibodies targeting epitopes in the N protein, potentially including those in the N-terminal region, might provide some level of protection against SARS-CoV-2 infection.

Understanding these aspects of the antigenic profile of SARS-CoV-2 Nucleocapsid (1-49) is crucial for developing more specific and sensitive diagnostic tests and for interpreting serological data in epidemiological studies.

What is the role of SARS-CoV-2 Nucleocapsid (1-49) in T-cell immunity and vaccine development strategies?

While the search results don't specifically address the role of SARS-CoV-2 Nucleocapsid (1-49) in T-cell immunity and vaccine development, several inferences can be made based on the general properties of the nucleocapsid protein and its immunogenicity:

Research into the specific contribution of residues 1-49 to T-cell immunity and their potential in vaccine development represents an important area for future investigation, potentially leading to more effective vaccines against SARS-CoV-2 and related coronaviruses.