Recombinant Human SARS coronavirus ORF8a protein (8a)

What are the structural differences between SARS-CoV ORF8a/b and SARS-CoV-2 ORF8?

SARS-CoV and SARS-CoV-2 ORF8 proteins exhibit significant structural and sequence divergence despite their functional similarities. The ORF8 protein of SARS-CoV-2 is the most divergent accessory protein from SARS-CoV, with the proteins possessing only 40% amino acid identity . A key structural difference is that SARS-CoV ORF8 contains a 29 base pair deletion that divides the gene into two separate ORFs, known as ORF8a and ORF8b . In contrast, SARS-CoV-2 ORF8 is a continuous 121-amino acid protein with an N-terminal signal sequence followed by an Ig-like fold .

Crystal structure analysis at 2.04 Å resolution reveals that SARS-CoV-2 ORF8 can form a covalent dimer with three sets of intramolecular disulfide bonds per monomer and a single intermolecular disulfide bond formed by Cys20 of each monomer . The core of each ORF8 monomer consists of two antiparallel β-sheets, with the smaller sheet consisting of β2, β5, and β6, while the larger is formed from β3, β4, β7, and β8 .

How have ORF8 proteins evolved across coronavirus lineages?

ORF8 is one of the most rapidly evolving betacoronavirus proteins, exhibiting atypical divergence patterns. Sequence analysis confirms that ORF8 likely originated from ORF7a, another gene in the coronavirus lineage that appears more conserved by comparison . While ORF7a and ORF8 share the same immunoglobulin-like fold, their sequence divergence is remarkable.

The evolution rates between closely related strains show striking differences. For instance, there is approximately 35% identity between SARS-CoV-1 and its closest bat coronavirus homologs, compared to approximately 88% identity between SARS-CoV-2 and its pangolin coronavirus homologs . This suggests different evolutionary pressures acting on ORF8 across different coronavirus lineages. Recent SARS-CoV-2 variants since B.1.1.7 (Alpha) in late 2020 all possess one or more ORF8 mutations, indicating ongoing selective pressure on this accessory protein .

What functional roles have been attributed to ORF8 in coronavirus pathogenesis?

While ORF8 is not strictly essential for viral replication and growth in vitro, it plays critical roles in modulating virus-host interactions during infection . Several functions have been attributed to SARS-CoV-2 ORF8, including:

• Down-regulation of MHC class I on the cell surface, potentially facilitating immune evasion by targeting MHC-I for lysosomal degradation

• Disruption of IFN-I signaling when exogenously overexpressed in cells

• Modulation of spike incorporation into virions

• Agonism of IL-17 receptor signaling

• Interaction with numerous host proteins, particularly factors involved in ER-associated degradation (ERAD)

For SARS-CoV specifically, the ORF8a and ORF8b proteins are hypothesized to facilitate the degradation of IRF3, further indicating roles in immune evasion .

What methodological approaches are most effective for expressing and purifying recombinant ORF8 proteins?

Successful expression and purification of ORF8 proteins require tailored approaches depending on the expression system and downstream applications. Two main methods have proven effective:

Bacterial Expression System:
SARS-CoV-2 ORF8 protein can be generated through expression in Escherichia coli followed by oxidative refolding . This approach is suitable for structural studies and has been used successfully to obtain crystals for X-ray crystallography.

Mammalian Cell Expression:
When expressing ORF8 in mammalian cells, researchers have encountered unexpected mRNA splicing at cryptic splice sites, resulting in truncated proteins . To address this issue, a methodological solution involves:

• Codon optimization of the orf8 gene

• Removal of cryptic splice sites

• Insertion into appropriate expression vectors (e.g., retroviral vector pQCXIP)

This optimized system enables production of properly folded ORF8 protein at the appropriate size, with approximately 41% secreted extracellularly as a homodimeric glycoprotein with intramolecular disulfide bonds .

For research applications, recombinant ORF8 is available with or without carrier proteins such as BSA. Carrier-free versions are recommended for applications where BSA might interfere with experimental outcomes .

How do naturally occurring ORF8 mutations affect viral pathogenesis in animal models?

Importantly, mice infected with a SARS-CoV-2 virus lacking ORF8 exhibited increased weight loss and exacerbated macrophage infiltration into the lungs compared to those infected with a clinical isolate . This suggests that ORF8 may normally function to moderate inflammatory responses during infection.

What are the implications of ORF8 secretion for its biological functions?

The discovery that approximately 41% of SARS-CoV-2 ORF8 is secreted extracellularly as a homodimeric glycoprotein has significant implications for understanding its biological functions . Several key aspects of ORF8 secretion merit consideration:

Understanding the trafficking and secretion mechanisms of ORF8 may provide insights into potential therapeutic targets that could modulate the protein's extracellular activities without affecting vital viral replication processes.

How do clinical isolates with ORF8 deletions correlate with disease outcomes?

Deletions in ORF8 have been identified in clinical isolates from multiple geographic regions, including Singapore and Taiwan, demonstrating the high level of selective pressure on this accessory protein . These natural deletion variants have provided valuable insights into ORF8's role in pathogenesis:

• A 29-nucleotide deletion (Δ29) that occurred early in human-to-human transmission of SARS-CoV (splitting ORF8 into ORF8a and ORF8b) correlated with milder disease .

• A 382-nucleotide deletion (Δ382) in SARS-CoV-2 was found to correlate with milder disease and a lower incidence of hypoxia in infected patients .

These clinical observations align with laboratory findings in mouse models where ORF8 deletion viruses caused increased lung inflammation compared to clinical isolates . This apparent paradox—where deletion increases inflammation yet decreases disease severity—suggests complex relationships between inflammation, immune response, and clinical outcomes.

Researchers investigating these phenomena should consider that ORF8 may play different roles at different stages of infection, potentially promoting viral replication or immune evasion early while contributing to pathological inflammation later.

What strategies can be employed to study ORF8-host protein interactions?

Understanding ORF8's interactions with host proteins is crucial for elucidating its functions in immune evasion and pathogenesis. Several methodological approaches can be employed:

• Affinity Purification-Mass Spectrometry (AP-MS): This approach has identified that within the lumen of the ER, SARS-CoV-2 ORF8 interacts with various host proteins, including many factors involved in ER-associated degradation (ERAD) .

• Co-Immunoprecipitation Assays: These can be used to confirm specific protein-protein interactions identified through screening approaches.

• Proximity Labeling: Methods such as BioID or APEX2 can identify transient or weak interactions occurring in specific cellular compartments.

• Functional Validation Assays: For example, studying the ORF8-dependent downregulation of MHC-I requires appropriate cell surface protein detection methods and trafficking studies .

• Structural Biology Approaches: X-ray crystallography has been successfully used to determine the structure of ORF8 , and could potentially be applied to ORF8-host protein complexes.

When designing interaction studies, researchers should consider ORF8's localization in the ER and its partial secretion, as these factors will influence which host proteins it can physiologically encounter.

What are the key considerations for designing ORF8 mutation studies in recombinant viruses?

When designing studies to investigate ORF8 mutations in recombinant viruses, researchers should consider several methodological aspects:

• Reference Strain Selection: The choice of backbone strain (e.g., WA-1) can impact results. Studies have successfully used recombinant WA-1 virus containing variant ORF8 genes from B.1.1.7, B.1.351, and P.1 to characterize the functional consequences of mutations .

• Mutation Types to Investigate:

  • Naturally occurring point mutations (e.g., S84L, E92K)

  • Truncations (e.g., premature stop codons as seen in B.1.1.7 and XBB lineages)

  • Complete deletions (ΔORF8)

  • Chimeric constructs between different coronavirus ORF8 variants

• Readouts for Functional Assessment:

  • In vivo: Weight loss, lung inflammation, macrophage infiltration

  • In vitro: MHC-I downregulation, interferon signaling, cytokine responses

• Control Considerations: Include both wildtype virus and, when possible, viruses with mutations in other accessory proteins to distinguish ORF8-specific effects from general perturbations to viral fitness.

• Biosafety Considerations: Studies with recombinant SARS-CoV-2 require appropriate biosafety level facilities (typically BSL-3).

How can researchers effectively compare the functions of SARS-CoV ORF8a/b with SARS-CoV-2 ORF8?

Comparative studies between SARS-CoV ORF8a/b and SARS-CoV-2 ORF8 require careful methodological approaches:

• Expression Systems: Due to the different structures and properties of these proteins, optimization may be required for each. For SARS-CoV ORF8a/b, separate expression constructs should be created for each fragment .

• Functional Assays: Select assays that can detect potential functional differences:

  • MHC-I downregulation (observed with SARS-CoV-2 ORF8 but not SARS-CoV ORF8a/b)

  • IRF3 degradation (hypothesized for SARS-CoV ORF8a/b)

  • IL-17 receptor signaling (for SARS-CoV-2 ORF8)

• Chimeric Constructs: Creating chimeric proteins between SARS-CoV ORF8a/b and SARS-CoV-2 ORF8 can help identify which domains are responsible for functional differences.

• Structural Comparisons: Comparing the solved structure of SARS-CoV-2 ORF8 with models of SARS-CoV ORF8a/b can provide insights into mechanistic differences.