SARS-CoV-2 ORF8 Forms Intracellular Aggregates and Inhibits IFNγ-Induced Antiviral Gene Expression in Human Lung Epithelial Cells

Abstract

Infection with the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes COVID-19, a disease that involves significant lung tissue damage. How SARS-CoV-2 infection leads to lung injury remains elusive. The open reading frame 8 (ORF8) protein of SARS-CoV-2 (ORF8SARS-CoV-2) is a unique accessory protein, yet little is known about its cellular function. We examined the cellular distribution of ORF8SARS-CoV-2 and its role in the regulation of human lung epithelial cell proliferation and antiviral immunity. Using live imaging and immunofluorescent staining analyses, we found that ectopically expressed ORF8SARS-CoV-2 forms aggregates in the cytosol and nuclear compartments of lung epithelial cells. Using in silico bioinformatic analysis, we found that ORF8SARS-CoV-2 possesses an intrinsic aggregation characteristic at its N-terminal residues 1-18. Cell culture did not reveal any effects of ORF8SARS-CoV-2 expression on lung epithelial cell proliferation and cell cycle progression, suggesting that ORF8SARS-CoV-2 aggregates do not affect these cellular processes. Interestingly, ectopic expression of ORF8SARS-CoV-2 in lung epithelial cells suppressed basal expression of several antiviral molecules, including DHX58, ZBP1, MX1, and MX2. In addition, expression of ORF8SARS-CoV-2 attenuated the induction of antiviral molecules by IFNγ but not by IFNβ in lung epithelial cells. Taken together, ORF8SARS-CoV-2 is a unique viral accessory protein that forms aggregates when expressing in lung epithelial cells. It potently inhibits the expression of lung cellular anti-viral proteins at baseline and in response to IFNγ in lung epithelial cells, which may facilitate SARS-CoV-2 escape from the host antiviral innate immune response during early viral infection. In addition, it seems that formation of ORF8SARS-CoV-2 aggregate is independent from the viral infection. Thus, it would be interesting to examine whether any COVID-19 patients exhibit persistent ORF8 SARS-CoV-2 expression after recovering from SARS-CoV-2 infection. If so, the pathogenic effect of prolonged ORF8SARS-CoV-2 expression and its association with post-COVID symptoms warrant investigation in the future.

Defective ORF8 dimerization in SARS-CoV-2 delta variant leads to a better adaptive immune response due to abrogation of ORF8-MHC1 interaction

“…Deletion of ORF8 leads to decreased severity of infection as reported [27,46]. ORF8 is also involved in suppression antiviral proteins from INFγ [14]. SARS-CoV-2 drug or vaccines were using spike protein as an important target [20,37,38,41], but ORF8 can be also targeted for producing therapeutic approaches.…”

Epidemiological associations with genomic variation in SARS-CoV-2

“…We also identify mutational hotspots in the 3′-to-5′ exonuclease, endoRNAase, 2′-O-Ribose methyltransferase, Spike protein, ORF7a/b, and ORF8. Mutations in some regions have been linked to host functional alterations such as the role of ORF7a in host immune suppression 30 and ORF8’s role in intracellular stress pathways 31 , 32 . Functional roles of other viral mutations remain to be characterized.…”

SARS-CoV-2 and the Host Cell: A Tale of Interactions

“…Some in vitro evidence suggests that ORF8 may have more roles in immune evasion as previously described. Its ectopic expression has been correlated with a reduced expression of several IFNγ-induced antiviral molecules with different biochemical activities in lung epithelial cells (311). Interestingly, the same group observed that ectopic expression of ORF8 in HEK293 cells reduced the expression of different antiviral proteins, suggesting that the effects of ORF8 on ISGs may be cell type specific.…”