CoV-2-S1 (319-541)

What is the CoV-2-S1 (319-541) domain and what is its significance in SARS-CoV-2 infection?

The CoV-2-S1 (319-541) domain refers to amino acid residues 319-541 of the S1 subunit of the SARS-CoV-2 spike protein. This region contains the receptor-binding domain (RBD) that recognizes and binds to the host receptor angiotensin-converting enzyme 2 (ACE2) . The S1 subunit contains this RBD that recognizes and binds to ACE2, while the S2 subunit mediates viral cell membrane fusion . This interaction between the RBD and ACE2 is the initial and critical step for SARS-CoV-2 entry into host cells, making it a key target for developing neutralizing antibodies and therapeutics.

The significance of this domain is highlighted by phylogenetic and structural analyses showing that the 319-541 residue region within the S1 domain is 100% similar among spike proteins from various geographical regions . This high conservation suggests evolutionary pressure to maintain this structure, likely due to its essential role in viral infection.

How does the conservation pattern of the CoV-2-S1 (319-541) region compare to other regions of the spike protein?

• The S1 subunit shows higher conservation (99.70%) compared to the S2 subunit (99.66%)

• Within the S1 domain, the RBD region (amino acids 319-541) demonstrates perfect conservation with 100% similarity among analyzed sequences

• The S1 subunit from amino acids 1 to 289 of domain 1 is also highly conserved without changes in the ligand interaction site

This pattern of conservation suggests that the RBD region (319-541) is under stronger selective pressure than other regions of the spike protein, likely due to its essential role in receptor binding and viral entry. The perfect conservation of this region makes it an ideal target for stable vaccine development and therapeutic interventions.

How was the RBD region (319-541) of SARS-CoV-2 initially identified?

The RBD region of SARS-CoV-2 was identified at residues 319-541 of the spike protein using homology search methods with the previously characterized RBD of SARS-CoV . This approach leveraged the genetic relationship between SARS-CoV-2 and the earlier SARS-CoV, which caused the 2003 SARS outbreak.

The identification process involved:

• Sequence alignment of the SARS-CoV-2 spike protein with that of SARS-CoV

• Determination of homologous regions based on sequence similarity

• Functional domain prediction based on structural properties

• Validation through experimental approaches

For more detailed analysis, researchers used tools such as ClustalW BioEdit software for pairwise amino-acid sequence alignments and PHYRE2 (Protein Homology/analogY Recognition Engine V 2.0) for structural modeling . Additional tools like Protein Peeling 3D were used to determine protein domains with specific parameters including R-value-95, minimal size of secondary structure-8, minimal size of protein unit-16, and other cutoff values .

What are the recommended protocols for expressing and purifying recombinant CoV-2-S1 (319-541) protein for research purposes?

Efficient expression and purification of recombinant CoV-2-S1 (319-541) protein involves several critical steps:

• Signal peptide identification: For efficient expression and purification, the signal peptide of the spike protein should be identified and included in expression constructs. This approach has been validated for generating functional recombinant RBD proteins .

• Expression construct design: Two main approaches have shown success:

  • RBD protein fused with the Fc domain of human IgG (SARS2-RBD-Fc)

  • RBD protein with a histidine tag (SARS2-RBD-His)

  Of these, the Fc-fusion approach has demonstrated superior expression and antiviral efficacy .

• Expression system: Mammalian cell expression systems are preferred for proper folding and post-translational modifications of the spike protein.

• Purification strategy: For Fc-fusion proteins, protein A/G affinity chromatography followed by size exclusion chromatography has proven effective. For His-tagged proteins, immobilized metal affinity chromatography (IMAC) is the primary purification method.

• Quality control: Verify protein integrity through SDS-PAGE, Western blotting with specific antibodies (such as 28991-1-AP at dilutions of 1:1000-1:4000) , and functional assays such as ACE2 binding tests.

How can researchers effectively evaluate the antiviral efficacy of CoV-2-S1 (319-541) proteins in vitro?

To evaluate the antiviral efficacy of recombinant CoV-2-S1 (319-541) proteins, researchers can follow this validated protocol:

• Cell culture preparation: Seed appropriate cells (e.g., Vero cells) in 96-well plates one day before infection, typically at a density of 2 × 10^4 cells per well .

• Protein treatment: Add serially diluted recombinant RBD proteins (typically ranging from 100 μg/mL to 0.16 μg/mL) to the cells 1 hour prior to viral infection .

• Viral infection: Infect the cells with SARS-CoV-2 at an appropriate multiplicity of infection (MOI), such as MOI of 3, in a BSL3 facility .

• Fixation and staining: At 1 day post-infection:

  • Fix cells with 4% paraformaldehyde

  • Permeabilize with 0.1% Triton X-100 in PBS

  • Immunostain with anti-dsRNA antibody and fluorescently-conjugated secondary antibody

  • Counterstain nuclei with Hoechst 33342 or DAPI

• Image acquisition and analysis: Capture fluorescence images using appropriate microscopy and quantify viral infection by dividing the number of cells stained with anti-dsRNA antibody by the total number of cells (determined by counting nuclei) .

• Determination of IC50 and selectivity index: Calculate the half-maximal inhibitory concentration (IC50) and compare to cytotoxicity concentration (CC50) to determine the selectivity index (SI = CC50/IC50). For reference, SARS2-RBD-Fc protein has demonstrated a high selectivity index, with a CC50 value approximately 3500-fold higher than the IC50 value .

What antibodies are available for detecting CoV-2-S1 (319-541) in various experimental applications?

Several antibodies targeting the CoV-2-S1 (319-541) region are available for research applications. One well-characterized example is:

Rabbit Polyclonal SARS-CoV-2 S protein (319-541 aa) antibody (28991-1-AP):

This antibody has been validated for:

• Western blot detection of recombinant protein

• ELISA with recombinant protein

• Reactivity with viral samples

Technical specifications:

• Host/Isotype: Rabbit/IgG

• Class: Polyclonal

• Immunogen: SARS-CoV-2 S protein (319-541 aa) fusion protein

• Storage: Liquid form in PBS with 0.02% sodium azide

• Purification method: Antigen affinity purification

For optimal results, titration of the antibody in each specific testing system is recommended, as optimal dilutions may be sample-dependent.

How does the expression of ACE2 influence the binding and entry of SARS-CoV-2 via the S1 (319-541) region?

The expression pattern of ACE2, the primary receptor for SARS-CoV-2, plays a critical role in determining tissue tropism and susceptibility to infection. The interaction between the viral S1 (319-541) RBD region and ACE2 is the initial and essential step for viral entry. Several important observations regarding ACE2 expression have been made:

These observations highlight the complex relationship between ACE2 expression, viral infection, and disease pathogenesis, suggesting that therapeutic approaches targeting the S1 (319-541) interaction with ACE2 must consider these dynamics.

What role do co-receptors play in facilitating SARS-CoV-2 entry in conjunction with the S1 (319-541) domain?

While ACE2 is the primary receptor for SARS-CoV-2, emerging evidence suggests that co-receptors, particularly neuropilins (NRPs), enhance viral entry in conjunction with the S1 (319-541) domain:

• Neuropilin-1 (NRP1) as co-receptor: NRP1 has been identified as a co-receptor that enhances SARS-CoV-2 infectivity:

  • Incubation of VSV-SARS-S2 or patient-isolated SARS-CoV-2 with monoclonal anti-NRP1 decreased infection efficiency, although to a lesser extent than anti-ACE2 antibodies

  • NRP1 and NRP2 are expressed in the respiratory epithelium at levels similar to ACE2 in AT2 cells

  • Cellular staining of post-mortem olfactory tissue sections showed higher NRP1 detection in infected epithelial cells of COVID-19 patients compared to control individuals

• Mechanism of enhancement: Current evidence suggests that NRP1 potentiates the attachment of SARS-CoV-2 to facilitate ACE2-mediated entry, rather than serving as an independent entry receptor .

• Significance for low ACE2 expression sites: The use of neuropilins as co-receptors may partially explain how SARS-CoV-2 efficiently infects tissues with relatively low ACE2 expression levels in the respiratory epithelium .

These findings highlight the complexity of SARS-CoV-2 entry mechanisms and suggest that therapeutic strategies targeting viral entry may need to consider multiple receptor interactions beyond the primary S1 (319-541)/ACE2 interaction.

How does the CoV-2-S1 (319-541) region function as a potential therapeutic agent against SARS-CoV-2 infection?

The CoV-2-S1 (319-541) region, which contains the receptor-binding domain (RBD), has demonstrated significant potential as a therapeutic agent against SARS-CoV-2 infection through several mechanisms:

• Competitive inhibition of viral entry: Recombinant RBD proteins can bind to ACE2 receptors on host cells, thereby preventing viral attachment and subsequent entry. This competitive inhibition mechanism has shown potent antiviral efficacy in experimental models .

• Superior efficacy of Fc-fusion proteins: Among recombinant RBD proteins, those fused with the Fc domain of human IgG (SARS2-RBD-Fc) have demonstrated superior antiviral activity compared to proteins with histidine tags (SARS2-RBD-His) . Key metrics include:

  • SARS2-RBD-Fc demonstrated a highly favorable selectivity index, with a CC50 value approximately 3500-fold higher than the IC50 value

  • This high therapeutic index suggests minimal cellular toxicity while maintaining potent antiviral activity

• Dose-dependent inhibition: Serially diluted recombinant RBD proteins (100 μg/mL to 0.16 μg/mL) have shown dose-dependent inhibition of SARS-CoV-2 infection in cell culture models .

The efficacy demonstrated by recombinant RBD proteins makes them promising candidates for therapeutic development, either as standalone agents or as part of combination approaches targeting different stages of the viral life cycle.

What are the key considerations in designing stable vaccines targeting the CoV-2-S1 (319-541) domain?

The CoV-2-S1 (319-541) domain presents an ideal target for vaccine development due to its perfect conservation (100% similarity) among analyzed SARS-CoV-2 variants. Key considerations for designing stable vaccines targeting this region include:

The development of vaccines targeting the highly conserved S1 (319-541) domain offers the potential for broad and durable protection against SARS-CoV-2 variants.

How do interferon responses modulate ACE2 expression and impact the interaction with the S1 (319-541) domain?

The relationship between interferon (IFN) responses, ACE2 expression, and SARS-CoV-2 S1 (319-541) domain interactions is complex and involves multiple regulatory mechanisms:

• IFN effects on ACE2 expression: Evidence regarding IFN-mediated regulation of ACE2 shows context-dependent effects:

  • Immunoblot analysis of ACE2 in fully differentiated HBEC derived from asthmatic patients stimulated with IFNγ showed a slight increase in only 1 out of 4 donors

  • Studies suggest that IFNs, particularly type I IFNs, may upregulate a truncated form of ACE2 (dACE2) that lacks the interaction domain for the SARS-CoV-2 spike protein, potentially as a host defense mechanism

• IFN dynamics during SARS-CoV-2 infection: The pattern of IFN production during infection may influence ACE2 expression:

  • In critically ill COVID-19 patients, IFN-α2 (but neither IFNβ nor IFNλ) was detected in plasma, with peak levels around 10 days after symptom onset

  • IFNα levels were lower in critically ill patients than in those with mild to moderate COVID-19, and decreased more rapidly in severe cases

  • This suggests that severely ill patients may have impaired or delayed IFN responses, potentially affecting ACE2 regulation

• Viral evasion of IFN responses: SARS-CoV-2 proteins can inhibit IFN responses, which may indirectly affect ACE2 expression:

  • Several viral proteins including Nsp1, Nsp13, and Orf6 have been shown to inhibit IFNβ promoter induction and IFN signaling

  • Orf6 specifically interferes with IRF3 and STAT1 nuclear translocation, potentially affecting downstream IFN-stimulated gene expression

  • A truncation variant of SARS-CoV-2 Orf3b may also be associated with inhibition of IFNβ production

These complex interactions between IFN responses and ACE2 expression highlight the importance of considering host immune responses when developing therapeutics targeting the S1 (319-541) domain.

What is the relationship between viral load, ACE2 expression, and inflammation in specific patient populations with respect to S1 (319-541) domain interactions?

The complex interplay between viral load, ACE2 expression, and inflammation varies across patient populations and may significantly impact SARS-CoV-2 S1 (319-541) domain interactions:

• Relationship in smokers and COPD patients:

  • Future studies should investigate the relationship between ACE2 levels in the lung, viral load, and inflammation in smokers versus non-smokers and in COPD patients versus healthy individuals

  • This relationship is critical as these populations may have altered baseline ACE2 expression, potentially affecting susceptibility to infection and disease severity

• Post-mortem observations:

  • Post-mortem examination of lungs from deceased COVID-19 patients (n = 7) compared to age-matched uninfected individuals (n = 10) showed significantly greater numbers of ACE2-positive alveolar epithelial cells in COVID-19 individuals

  • This increased ACE2 expression may reflect the virus-induced changes in receptor expression, potentially creating a feed-forward loop that enhances infection

• Pathophysiological implications:

  • During SARS-CoV-2 infection, reduced availability of ACE2 for its enzymatic functions (such as degradation of des-Arg9-bradykinin and Lys-des-Arg9-bradykinin peptides) may lead to overactivation of B1R and subsequently lung angioedema

  • This suggests that beyond viral entry, the interaction between the S1 (319-541) domain and ACE2 may have broader pathophysiological consequences by disrupting normal ACE2 function

These observations highlight the need for comprehensive studies examining the dynamic relationships between viral factors (including S1 domain interactions), host factors (ACE2 expression and regulation), and inflammatory responses across different patient populations to better understand disease pathogenesis and identify targeted therapeutic approaches.

What are the optimal experimental conditions for evaluating the binding kinetics between the CoV-2-S1 (319-541) domain and ACE2?

To accurately evaluate the binding kinetics between the CoV-2-S1 (319-541) domain and ACE2, researchers should consider the following methodological approaches:

• Surface Plasmon Resonance (SPR):

  • Immobilize purified ACE2 or RBD protein on a sensor chip

  • Flow the complementary protein at varying concentrations

  • Measure association (kon) and dissociation (koff) rates

  • Calculate equilibrium dissociation constant (KD = koff/kon)

  • Control for non-specific binding using irrelevant proteins

  • Consider multiple buffer conditions to assess pH and salt dependencies

• Bio-Layer Interferometry (BLI):

  • Similar to SPR but uses optical interference patterns to detect binding

  • Particularly useful for real-time kinetic measurements with minimal sample consumption

  • Use purified recombinant RBD proteins such as those developed with Fc or His tags

• Isothermal Titration Calorimetry (ITC):

  • Provides direct measurement of binding thermodynamics

  • Determines enthalpy (ΔH), entropy (ΔS), and Gibbs free energy (ΔG)

  • No protein immobilization required, measuring interactions in solution

  • Requires larger protein quantities than surface-based methods

• Enzyme-Linked Immunosorbent Assay (ELISA)-based approaches:

  • Use validated antibodies such as Rabbit Polyclonal SARS-CoV-2 S protein (319-541 aa) antibody at appropriate dilutions (1:10-1:100 for ELISA)

  • Establish standard curves using recombinant proteins

  • Implement competitive binding assays to determine relative affinities

• Quality control considerations:

  • Ensure protein quality through SDS-PAGE and Western blot analysis

  • Verify proper folding using circular dichroism or other structural techniques

  • Test multiple batches of proteins to account for preparation variability

  • Include positive and negative controls in all binding assays

These methodological approaches provide complementary data on binding kinetics, affinity, and thermodynamics, allowing for comprehensive characterization of the interaction between the CoV-2-S1 (319-541) domain and ACE2.

How can researchers effectively compare and reconcile conflicting data regarding ACE2 regulation during SARS-CoV-2 infection?

Conflicting data regarding ACE2 regulation during SARS-CoV-2 infection presents significant challenges for researchers. To effectively compare and reconcile these contradictions, consider the following methodological approaches:

• Standardize experimental systems:

  • Use consistent cell types and culture conditions across studies

  • Clearly define multiplicity of infection (MOI) and infection time course

  • Control for virus preparation quality, as defective viral particles can trigger interferon production that may affect ACE2 regulation

  • Implement systematic analysis of infection parameters to resolve discrepancies

• Distinguish between mRNA and protein measurements:

  • Separately analyze ACE2 mRNA and protein levels, as post-transcriptional regulation appears significant

  • For example, some studies show increased ACE2 mRNA but decreased protein levels during infection

  • Use multiple antibodies targeting different ACE2 epitopes to confirm protein measurements

  • Implement techniques like polysome profiling to assess translational efficiency

• Consider isoform-specific effects:

  • Distinguish between full-length ACE2 (flACE2) and truncated forms like dACE2

  • Design primers and antibodies that can specifically detect different ACE2 variants

  • Assess functional differences between ACE2 isoforms in binding studies

• Account for contextual factors:

  • Analyze the impact of interferons and inflammatory mediators on ACE2 expression

  • Consider tissue-specific regulation patterns, as effects may differ between respiratory and non-respiratory tissues

  • Evaluate the influence of patient-specific factors (age, comorbidities, genetics)

• Meta-analysis approach:

  • Systematically compare methodologies across conflicting studies

  • Identify patterns in experimental conditions that correlate with specific outcomes

  • Develop integrated models that incorporate multiple regulatory mechanisms

  • Design experiments specifically to test hypotheses that could explain apparent contradictions

By implementing these methodological approaches, researchers can better understand the complex and context-dependent regulation of ACE2 during SARS-CoV-2 infection, potentially reconciling seemingly contradictory observations and developing a more unified model of host-pathogen interactions.