Abstract
ORF8 is a viral immunoglobulin-like (Ig-like) domain protein encoded by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA genome. It tends to evolve rapidly and interfere with immune responses. However, the structural characteristics of various coronavirus ORF8 proteins and their subsequent effects on biological functions remain unclear. Herein, we determined the crystal structures of SARS-CoV-2 ORF8 (S84) (one of the epidemic isoforms) and the bat coronavirus RaTG13 ORF8 variant at 1.62 Å and 1.76 Å resolution, respectively. Comparison of these ORF8 proteins demonstrates that the 62-77 residues in Ig-like domain of coronavirus ORF8 adopt different conformations. Combined with mutagenesis assays, the residue Cys20 of ORF8 is responsible for forming the covalent disulfide-linked dimer in crystal packing and in vitro biochemical conditions. Furthermore, immune cell-binding assays indicate that various ORF8 (SARS-CoV-2 ORF8 (L84), ORF8 (S84), and RaTG13 ORF8) proteins have different interaction capabilities with human CD14+ monocytes in human peripheral blood. These results provide new insights into the specific characteristics of various coronavirus ORF8 and suggest that ORF8 variants may influence disease-related immune responses.
Highlights
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed substantial global challenges to health and economic systems
The protruding b-hairpin exists between strands bC’ and bD, consisting of strands bI1 and bI2 (Figure 1D). It is the specific acquisition of a long insertion between strands C and D that distinguishes the ORF8 protein from other coronavirus Ig-like fold proteins, such as SARS-CoV-2 ORF7a (PDB ID: 7CI3) [12, 23]
The insertion region is highly dynamic compared with equivalent residues between SARS-CoV-2 ORF8 (S84) and RaTG13 ORF8, especially 62 to 77 amino acids, which can adopt different conformations
Summary
The coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has posed substantial global challenges to health and economic systems (https://covid19.who.int/). The rapid and persistent mutations of SARS-CoV-2 triggered a new phase of the pandemic outbreak [1–4]. The accessory proteins of SARS-CoV-2 appear not necessary for virus replication, several mutations have potentially contributed to increasing the pathogenesis and transmissibility of the virus [5] (https:// covariants.org/). SARS-CoV-2 ORF8 is confirmed as an Ig-like fold containing accessory protein, which may be involved in a potential evolutionary arms race between virus and host [11, 12]. The clinical effect of a 382-nucleotide deletion (D382) in SARS-CoV-2, an ORF8 deleted variant, appears to be a milder infection with the less systemic release of pro-inflammatory cytokines [13]. A deep understanding of ORF8 may provide new clues into the pathogenesis of SARS-CoV-2. The S84 variant in the ORF8 protein is considered related to mild disease among the clinical outcomes [17]
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