The ORF8 Protein of SARS-CoV-2 Modulates the Spike Protein and Its Implications in Viral Transmission

Abstract

COVID-19 is currently global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Accompanying the rapid spread of the error-prone RNA-based genome, several dominant SARS-CoV-2 variants have been genetically identified. The mutations in the spike protein, which are essential for receptor binding and fusion, have been intensively investigated for their contributions to viral transmission. Nevertheless, the importance of other viral proteins and their mutations in SARS-CoV-2 lifecycle and transmission remains fairly understood. Here, we report the strong potency of an accessory protein ORF8 in modulating the level and processing of the spike protein. The expression of ORF8 protein does not affect propagation but expression of spike protein, which may lead to pseudovirions with less spike protein on the surface, therefore less infection potential. At the protein level, ORF8 expression led to downregulation and insufficient S1/S2 cleavage of the spike protein in a dose-dependent manner. ORF8 exhibits a strong interaction with the spike protein mainly at S1 domains and mediates its degradation through multiple pathways. The dominant clinical isolated ORF8 variants with the reduced protein stability exhibited the increased capacity of viral transmission without compromising their inhibitory effects on HLA-A2. Although the increase in spike protein level and Spike pseudovirus production observed by using highly transmissible clinical spike variants, there was no significant compromise in ORF8-mediated downregulation. Because ORF8 is important for immune surveillance and might be required for viral fitness in vivo, the alteration of the spike protein might be an optional strategy used by SARS-CoV-2 to promote viral transmission by escaping the inhibitory effects of ORF8. Therefore, our report emphasized the importance of ORF8 in SARS-CoV-2 spike protein production, maturation, and possible evolution.

SARS-CoV-2 ORF8 limits expression levels of Spike antigen

“…The Δ382 strain replicates faster in vitro [20], but there is no significant change in the transcriptome of lung organoids infected with Δ382 [21]. ORF8 inhibits production of a viral component [22]. Consistently, we found that ORF8 restricts Spike incorporation into viral particles (Fig 7B ), and in turn, the virions were less infectious (Fig 7C).…”

Ganoderma microsporum immunomodulatory protein acts as a multifunctional broad-spectrum antiviral against SARS-CoV-2 by interfering virus binding to the host cells and spike-mediated cell fusion

“…An expression plasmid pcDNA3.1-SARS-CoV-2-Spike contains the wildtype (Wuhan strain) SARS-CoV-2 spike protein with a C-terminal C9 tag (gift from Fang Li; Addgene#145032). D614G or D614G plus N501Y (D614G/N501Y) mutation at Spike gene were performed by polymerase chain reaction (PCR) cloning [36] . The expression plasmid carrying spike gene derived from Beta strain (Academia Sinica, Taiwan), Delta strain (Addgene#179905) or Omicron strain (Addgene#179907) were used [37] .…”

“…Spike pseudovirions were produced as previously reported [36] . Different volumes of pseudovirions were inoculated with 3,000 HEK293T/hACE2 cells per well in 96-well plates.…”

SARS-CoV-2 evolution and evasion from multiple antibody treatments in a cancer patient

“…Recent studies revealed a myriad of functions facilitated by ORF8, with implications for transmission dynamics, host immunity and male fertility. However, whether or not these functions depend on the dimeric form of the protein is yet to be determined 23–25 .…”

“…Recent studies revealed a myriad of functions facilitated by ORF8, with implications for transmission dynamics, host immunity and male fertility. However, whether or not these functions depend on the dimeric form of the protein is yet to be determined [23][24][25] . NSP1 is a non-structural protein that causes a reduction in translation of cellular transcripts, promotes degradation of host mRNAs and inhibits export of nuclear mRNA, inhibiting host IFN-I response 26 .…”