Abstract
ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we herein established an ORF8 expression system in 293T cells. Using this system, approximately 41% of the ORF8 expressed in 293T cells were secreted extracellularly as a glycoprotein homodimer with inter/intramolecular disulfide bonds. Intracellular ORF8 was sensitive to the glycosidase Endo H, whereas the secreted portion was Endo-H-resistant, suggesting that secretion occurs via a conventional pathway. Additionally, immunoblotting analysis showed that the total amounts of the major histocompatibility complex class Ι (MHC-I), angiotensin-converting enzyme 2 (ACE2), and SARS-CoV-2 spike (CoV-2 S) proteins coexpressed in cells were not changed by the increased ORF8 expression, although FACS analysis revealed that the expression of the cell surface MHC-I protein, but not that of ACE2 and CoV-2 S proteins, was reduced by ORF8 expression. Finally, we demonstrate by RNA-seq analysis that ORF8 had no significant stimulatory effects in human primary monocyte-derived macrophages (MDMs). Taken together, our results provide fundamental evidence that the ORF8 glycoprotein acts as a secreted homodimer, and its functions are likely associated with the intracellular transport and/or extracellular signaling in SARS-CoV-2 infection.
Highlights
open reading frame 8 (ORF8) is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)
Previous studies showed that in cells, the ORF8 proteins of both SARS-CoV-1 and SARS-CoV-2 localize to the endoplasmic reticulum (ER) and that the SARS-CoV-2 ORF8 protein interacts with a variety of host proteins in the ER lumen, including the factors involved in ER-associated degradation and immunity [23–25]
Based on the crystal structure obtained by oxidative refolding of the recombinant ORF8 protein from E. coli, ORF8 forms a homodimer with three pairs of intramolecular disulfide bonds per protomer and one pair of intermolecular disulfide bonds via cysteine 20 (C20) (Fig. 1A) [32]
Summary
ORF8 is an accessory protein encoded by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Consensus regarding the biological functions of ORF8 is lacking, largely because the fundamental characteristics of this protein in cells have not been determined. To clarify these features, we established an ORF8 expression system in 293T cells. The viral genes of severe acute respiratory syndrome coronavirus (referred to as SARS-CoV-1 in this study) and SARS-CoV-2 are relatively conserved, many genetic mutations and deletions/insertions are observed in the locus near the open reading frame 8 (orf8) genes among betacoronaviruses [1–10]. The ORF8 protein was found to promote the expression of inflammatory factors by interacting with host interleukin(IL) receptor A (IL17RA) [31] Consensus on these phenomena is limited yet, and the fundamental functions of the SARS-CoV-2 ORF8 protein must be elucidated [9]
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