SARS Spike (14-667)

What is the significance of the SARS Spike (14-667) region in viral infection?

The 14-667 amino acid region of the SARS spike protein encompasses critical components of the S1 subunit, including the receptor binding domain (RBD), which is the primary target for host immune defenses . This region is responsible for initial attachment to host cell receptors. Methodologically, researchers can verify this significance through binding assays using recombinant proteins of this specific region compared to full-length spike proteins. Surface plasmon resonance (SPR) measurements using BIAcore systems have been effectively employed to quantify binding kinetics, where CM5 sensor chips are activated with EDC/NHS chemistry, followed by coupling of recombinant proteins in acetate buffer at specified pH values .

How do researchers accurately express and purify the SARS Spike (14-667) construct for experimental studies?

For high-yield expression of the SARS Spike (14-667) construct, insect cell expression systems are commonly employed as demonstrated in recent studies with SARS-CoV-2 spike proteins . The typical methodology involves:

• Cloning the gene sequence encoding amino acids 14-667 into expression vectors

• Transfecting insect cells (typically Sf9 or High Five)

• Expressing the protein with appropriate tags for purification

• Purifying via affinity chromatography, followed by size exclusion chromatography

• Confirming structural integrity through negative-stain electron microscopy

For stability enhancement, researchers often introduce modifications such as proline substitutions in key positions, similar to the K986P and V987P modifications used in full-length SARS-CoV-2 spike protein studies .

What glycan binding properties have been observed with SARS Spike proteins, and how can they be systematically investigated?

SARS spike proteins demonstrate binding affinity to heparan sulfate (HS) in a sulfation-dependent manner, while no significant binding to sialic acid residues has been detected . For systematic investigation of these interactions, researchers should:

• Prepare glycan microarrays containing diverse sulfated HS oligosaccharides

• Express and purify spike protein constructs, including the 14-667 region

• Conduct binding assays using surface plasmon resonance (SPR)

• Compare binding affinities across different coronaviruses (SARS-CoV, SARS-CoV-2, MERS-CoV)

• Analyze the effect of sulfation patterns on binding affinity

The sulfation pattern significantly influences binding, suggesting that HS binding represents a general mechanism for coronavirus attachment to host cells and could be exploited for developing antiviral agents .

How do naturally occurring mutations in the 14-667 region impact spike protein function and viral fitness?

Mutations in the spike protein, such as D614G (which falls just outside the 14-667 region but affects its function), have demonstrated significant impacts on viral fitness . To investigate mutations within the 14-667 region:

• Generate a panel of spike protein variants using site-directed mutagenesis

• Incorporate these mutations into pseudotyped virus systems

• Assess infectivity through in vitro assays measuring viral entry

• Analyze structural changes using cryo-electron microscopy

• Correlate structural changes with functional differences

Research has shown that certain mutations can significantly enhance viral infectivity. For example, the G614 variant grows to higher titers as pseudotyped virions compared to D614 counterparts and is associated with higher viral loads in patients without increasing disease severity . This pattern of enhanced fitness has been observed consistently across multiple geographic regions, suggesting a significant competitive advantage .

What methodologies are most effective for monitoring structural conformational changes in the SARS Spike (14-667) region?

For monitoring conformational changes in the SARS Spike (14-667) region, researchers should employ complementary structural biology techniques:

• Cryo-electron microscopy (cryo-EM) for visualizing spike protein conformations at near-atomic resolution

• Hydrogen-deuterium exchange mass spectrometry (HDX-MS) to map dynamic regions

• Single-molecule Förster resonance energy transfer (smFRET) to track conformational dynamics in real-time

• Molecular dynamics simulations to model conformational transitions

• X-ray crystallography of stabilized constructs to obtain high-resolution static structures

These techniques have revealed that modifications like proline substitutions can stabilize the prefusion conformation of spike proteins, which is crucial for maintaining immunogenic epitopes in vaccine formulations .

How can researchers develop experimental systems to study the interaction between SARS Spike (14-667) and potential therapeutic agents?

To develop robust systems for screening potential therapeutic agents targeting the SARS Spike (14-667) region:

• Establish cell-based assays using ACE2/TMPRSS2-expressing cell lines (e.g., modified Caco-2 cells)

• Develop pseudotyped virus systems incorporating the spike protein

• Implement high-throughput screening platforms based on viral entry inhibition

• Design competitive binding assays using purified spike protein constructs and ACE2 receptors

• Validate hits through plaque reduction neutralization tests with live virus in appropriate biosafety facilities

For quantitative analysis, researchers should titrate virus stocks by incubating ACE2/TMPRSS2-expressing cells with serial dilutions of virus, overlay with medium containing 1:1 mixture of 2X DMEM/4% FBS and 1.2% Avicel, and visualize plaques after three days using paraformaldehyde fixation and crystal violet staining .

What are the optimal conditions for expressing recombinant SARS Spike (14-667) for structural studies?

For optimal expression of SARS Spike (14-667) for structural studies:

• Clone the construct into baculovirus expression vectors with a C-terminal purification tag

• Include a signal peptide for secretion into the culture medium

• Maintain insect cell cultures at 27°C with appropriate shaking (120-130 rpm)

• Harvest cell culture supernatant 72-96 hours post-infection

• Purify using Ni-NTA affinity chromatography followed by size-exclusion chromatography

For structural integrity verification, negative-stain electron microscopy has proven effective for assessing protein quality before proceeding to more resource-intensive techniques like cryo-EM . The protein should be formulated in suitable detergents like 0.01% (v/v) polysorbate 80 (PS 80) to maintain stability and prevent aggregation .

How can researchers generate recombinant SARS viruses to study spike protein mutations?

For generating recombinant SARS-CoV-2 incorporating specific spike mutations, the circular polymerase extension reaction (CPER) methodology has proven effective:

• Amplify the viral genome into overlapping fragments (typically 8-9 fragments)

• Use plasmids containing the spike gene region (F8) as templates for introducing desired mutations

• Include a UTR linker containing hepatitis delta virus ribozyme, BGH polyadenylation signal, and CMV promoter

• Perform CPER to assemble the complete viral genome

• Transfect the assembled genome into susceptible cells for virus production

This approach allows for systematic introduction of mutations in the spike protein to study their functional consequences while maintaining the remainder of the viral genome constant.

What methods effectively track SARS coronavirus spike protein evolution in real-time during outbreaks?

To effectively track SARS coronavirus spike protein evolution during outbreaks:

• Implement systematic genomic surveillance through regular sampling and sequencing

• Develop bioinformatics pipelines to identify emerging amino acid variants

• Monitor variant frequencies across geographic regions

• Apply statistical approaches to assess the significance of observed frequency shifts

• Correlate genetic changes with phenotypic properties (infectivity, immune evasion)

For statistical validity, ensure sampling meets three key requirements: (1) co-circulation of variants in the geographic area, (2) sampling over adequate duration to observe frequency changes, and (3) sufficient samples for statistical power . This approach successfully tracked the D614G mutation, revealing its consistent rise to dominance across multiple geographic regions .

How do modifications at the S1/S2 cleavage site affect the structure and function of SARS spike proteins?

The S1/S2 cleavage site plays a crucial role in spike protein activation. Modifications at this site, such as changing the polybasic cleavage sequence from RRAR to QQAQ (as in the NVAX-CoV2373 vaccine candidate), render the protein protease-resistant and enhance stability . To study these effects:

• Generate constructs with various cleavage site modifications

• Express and purify the modified proteins

• Assess structural integrity through negative-stain EM and cryo-EM

• Evaluate protease susceptibility through in vitro cleavage assays

• Determine functional impacts through cell-based fusion and entry assays

These modifications are particularly important for vaccine development, as they help maintain the spike protein in its prefusion conformation, which presents important neutralizing epitopes to the immune system .

What structural features of the SARS Spike (14-667) region are most critical for antibody neutralization?

Within the 14-667 region, several structural features are critical for antibody neutralization:

• The receptor binding domain (RBD), which can adopt "up" or "down" conformations

• The N-terminal domain (NTD), which contains several neutralizing epitopes

• Glycan shields that partially protect the protein from antibody recognition

• Conserved epitopes that are targets for broadly neutralizing antibodies

• Regions involved in conformational changes during receptor binding

To identify these features, researchers should employ structural vaccinology approaches, combining high-resolution structural studies with epitope mapping using monoclonal antibodies and mutational analysis . This information is crucial for designing improved vaccines and therapeutic antibodies.

How does glycan binding differ between SARS-CoV and SARS-CoV-2 spike proteins, and what are the implications for cross-species transmission?

Both SARS-CoV and SARS-CoV-2 spike proteins bind to heparan sulfate in a sulfation-dependent manner, but with potential differences in binding affinity and specificity . To investigate these differences:

• Perform comparative glycan microarray analyses with purified spike proteins from both viruses

• Quantify binding kinetics using surface plasmon resonance with various glycan structures

• Map binding sites through mutagenesis and structural studies

• Assess the impact of species-specific glycan variations on binding affinity

• Correlate glycan binding properties with cellular tropism

Understanding these differences may provide insights into the mechanisms of cross-species transmission and help identify potential intermediate hosts in the zoonotic transmission chain .

What evolutionary patterns have been observed in the SARS Spike (14-667) region across coronavirus outbreaks?

Evolutionary analysis of the spike protein 14-667 region reveals:

• Selective pressure on the receptor binding domain, particularly at residues involved in receptor interaction

• Conservation of structural elements required for protein folding and stability

• Variation in surface-exposed regions targeted by neutralizing antibodies

• Convergent evolution in different coronavirus lineages

To study these patterns, researchers should implement phylogenetic analysis of sequence data from multiple outbreaks, calculate selection pressures on individual residues, and correlate genetic changes with structural and functional consequences . The emergence and rapid dominance of mutations like D614G highlight the importance of continued surveillance to identify variants with potential fitness advantages .