tetano
Editor, Senior Moderator
J Biol Chem
. 2022 Feb 11;101724.
doi: 10.1016/j.jbc.2022.101724. Online ahead of print.
SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer
Kazuhiro Matsuoka[SUP] 1 [/SUP], Nobuhiko Imahashi[SUP] 2 [/SUP], Miki Ohno[SUP] 1 [/SUP], Hirotaka Ode[SUP] 1 [/SUP], Yoshihiro Nakata[SUP] 3 [/SUP], Mai Kubota[SUP] 1 [/SUP], Atsuko Sugimoto[SUP] 1 [/SUP], Mayumi Imahashi[SUP] 1 [/SUP], Yoshiyuki Yokomaku[SUP] 1 [/SUP], Yasumasa Iwatani[SUP] 4 [/SUP]
Affiliations
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 was 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 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.
Keywords: COVID-19; ORF8; SARS-CoV-2; glycosylation; secretory protein.
. 2022 Feb 11;101724.
doi: 10.1016/j.jbc.2022.101724. Online ahead of print.
SARS-CoV-2 accessory protein ORF8 is secreted extracellularly as a glycoprotein homodimer
Kazuhiro Matsuoka[SUP] 1 [/SUP], Nobuhiko Imahashi[SUP] 2 [/SUP], Miki Ohno[SUP] 1 [/SUP], Hirotaka Ode[SUP] 1 [/SUP], Yoshihiro Nakata[SUP] 3 [/SUP], Mai Kubota[SUP] 1 [/SUP], Atsuko Sugimoto[SUP] 1 [/SUP], Mayumi Imahashi[SUP] 1 [/SUP], Yoshiyuki Yokomaku[SUP] 1 [/SUP], Yasumasa Iwatani[SUP] 4 [/SUP]
Affiliations
- PMID: 35157849
- DOI: 10.1016/j.jbc.2022.101724
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 was 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 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.
Keywords: COVID-19; ORF8; SARS-CoV-2; glycosylation; secretory protein.