The Functional Classification of ORF8 in SARS-CoV-2 Replication, Immune Evasion, and Viral Pathogenesis Inferred through Phylogenetic Profiling

Abstract

ORF8 is a highly variable genomic region of SARS-CoV-2. Although non-essential and the precise functions are unknown, it has been suggested that this protein assists in SARS-CoV-2 replication in the early secretory pathway and in immune evasion. We utilized the binding partners of SARS-CoV-2 proteins in human HEK293T cells and performed genome-wide phylogenetic profiling and clustering analyses in 446 eukaryotic species to predict and discover ORF8 binding partners that share associated functional mechanisms based on co-evolution. Results classified 47 ORF8 binding partner proteins into 3 clusters (groups 1-3), which were conserved in vertebrates (group 1), metazoan (group 2), and eukaryotes (group 3). Gene ontology analysis indicated that group 1 had no significant associated biological processes, while groups 2 and 3 were associated with glycoprotein biosynthesis process and ubiquitin-dependent endoplasmic reticulum-associated degradation pathways, respectively. Collectively, our results classified potential genes that might be associated with SARS-CoV-2 viral pathogenesis, specifically related to acute respiratory distress syndrome, and the secretory pathway. Here, we discuss the possible role of ORF8 in viral pathogenesis and in assisting viral replication and immune evasion via secretory pathway, as well as the possible factors associated with the rapid evolution of ORF8.

In silico Analysis of SARS-CoV-2 ORF8-Binding Proteins Reveals the Involvement of ORF8 in Acquired-Immune and Innate-Immune Systems

“…In contrast, the percentage of proteins localized in the endoplasmic reticulum was lower (11.8%) in Class 1 compared to the other classes ( Figure 3 ). These results are consistent with the previously published results ( 9 ). In contrast, proteins from Classes 2 and 3 showed a tendency for ER localization compared to extracellular proteins, with a very slight difference for Class 2.…”

“…This enormous quantity of genomic data has made possible, through comparative genomics, a number of advances in the elucidation of gene function and crosstalk. One such approach is phylogenetic profiling, an independent technique to infer functionally related genes and protein-protein interactions via the correlation of the occurrence across a set of genomes or so-called profiles that show the degree of co-evolution between genes ( 9 ). Phylogenetic profiling relies on the hypothesis that genes that function together are associated with similar evolutionary pressures and are thus lost and gained together throughout evolution ( 20 ).…”

“…We later identified the associated function of each cluster and suggested that Class 1 might contribute to viral pathogenesis and Classes 2 and 3 might contribute to the immune evasion mechanism. Generally, we suggest that ORF8 is associated with SARS-CoV-2 immune evasion and pathogenicity ( 9 ). Experimentally, ORF8 was reported to bypass the host immune surveillance mechanism by downregulating HLA class I and inhibiting IFN-β production ( 8 , 10 ).…”

“…Although the ORF8 protein is likely to be involved in the progression of COVID-19 pathology, the precise function of ORF8 and its pathogenic mechanism remain elusive. Previously, we explored the function of ORF8 using bioinformatics and found that it is localized in the endoplasmic reticulum and the secretory/intercellular compartments, which affect the endoplasmic reticulum and immunity systems, respectively ( 9 ). It has been shown in laboratory experiments that SARS-CoV-2 ORF8 induces endoplasmic reticulum stress by causing the divergence of activating transcription factor 6 (ATF6) and inositol-requiring enzyme 1 (IRE1) in the endoplasmic reticulum stress pathway ( 10 ).…”

Genomic and transcriptomic characterization of Delta SARS-CoV-2 infection in free-ranging white-tailed deer (Odocoileus virginianus)

“…Additionally, an ORF8 inframe deletion of 6 amino acids at positions 61–66 and L60F mutation were observed in sample 4205 although with an allele fraction of 59% (262/442 observations of TGTGCGTGGATGAGGCTGG to T at nucleotide position 28,072). High variability and truncation of ORF8 arising from nonsense mutations and deletions has been observed previously, underscoring the potential role of ORF8 in SARS-CoV-2 adaptation 42,43 .…”