CellRep: New Crown Virus Family ORF6 Protein Inhibits Antiviral Interferon Signaling Activity

The ORF6 gene is the most important difference between the sarbecoviruses virus family (e.g., SARS-CoV and SARS-CoV-2) and other beta coronaviruses. According to a recent study published in the journal CellReports, Professor Kei Sato and colleagues from the University of Tokyo in Japan revealed that ORF6-encoded proteins inhibit the activity of innate immune signaling, such as inhibiting the up-regulation of type I and type III IFN signaling after viral infection. In addition, it was found that SARS-CoV-2-derived ORF6 protein was more effective in inhibiting host cell innate immune activity than its homologous protein from SARS-CoV. Mutational analysis indicated that E46 and Q56 are important determinants of the antagonistic activity of SARS-CoV-2ORF6. It has been shown that the anti-innate immune activity of ORF6 depends on its C-terminal region, which is able to inhibit the nuclear transport activity of IRF3. Finally, studies reveal frameshift/nonsense mutations that occur under natural conditions and these mutations cause about 0.2% of the SARS-CoV-2 strains to inactivate ORF6.

First, the authors assessed the phylogenetic relationships of beta coronaviruses, including SARS-CoV, SARS-CoV-2, MERS-CoV, OC43 and HKU1. The virus strains were classified according to the phylogenetic tree of the full-length viral genome and the genetic relationship of the five viral core genes encoding ORF1ab, spike protein (S), envelope protein (E), and membrane protein. The analysis showed topological difference characteristics in the phylogeny of different viral genes within the subgenus sarbecoviruses, which is consistent with previous studies. However, certain viral genes, for example, E (composed of 75 amino acids in SARS-CoV-2) are relatively short, making it difficult to reliably deduce their phylogenetic relationships. Two viruses belonging to Sarbecovirus, BtKY72 and BM48, were isolated in the systematic tree of the E gene. In contrast, the other six phylogenetic trees showed almost identical relationships between the five subgenera of beta coronaviruses. These results suggest that although recombination events can occur between sarbecoviruses, no viral recombination phenomenon occurs between the β-coronaviruses analyzed.

The authors then compared the genome organization of the different subgenera. The results showed that the arrangement of the core gene (ORF1ab-S-E-M-N) was conserved. A variable open reading frame (ORF) was detected between ORF1ab and S in Hibecovirus and Embecovirus members, whereas in all beta coronaviruses, a variable ORF was detected between S and E. However, the phenomenon of ORF insertion between M and N was observed only in members of the Sarbecovirus and Hibecovirus subgenera. When the sequences of these ORFs were compared, the genes in Sarbecovirus did not match those in Hibecovirus, indicating that these ORFs emerged independently after the divergence of these subgenera. Notably, ORF6 is highly conserved in sarbecovirus viruses including SARS-CoV and SARS-CoV-2, but not in other beta coronaviruses.

Since previous reports indicated that the ORF6 protein of SARS-CoV has the ability to inhibit IFN-I activation as well as inhibit ISG activity, the authors compared the phenotypic properties of representative SarbecovirusORF6 proteins. The results showed that the phylogenetic topological characteristics of the SarbecovirusORF6 gene were similar to those of the full-length viral genome, which indicated that recombination events involving the ORF6 gene occurred in Sarbecovirus virus. For phenotypic analysis, the authors cloned an expression plasmid for ORF6 from SARS-CoV-2 (Wuhan-Hu-1) and derived relevant viral genes from SARS-CoV-2 produced from bats (RmYN02, RaTG13 and ZXC21) and pangolin (P4L). Western Blot results showed that the expression level of ORF6 protein of SARS-CoV-2 lineage was lower than that of SARS-CoV lineage and two outgroup viruses. The results of luciferase reporter assay showed that ORF6 was able to inhibit the expression of a series of IFN-related genes, including IFN-B1, IFN-L1, IFI-44L, and so on. These results indicate that Sarbecovirus-derived ORF6 has the ability to inhibit IFN innate immune signaling.

To investigate the intrinsic mechanism by which ORF6 inhibits IFN signaling activity, the authors performed fragment deletion mutagenesis with point mutation analysis. The results showed that the peptide at the C-terminus had a key effect on the activity of ORF6. Further, the authors found that two of these amino acid residues, E46 with Q56, are essential for this activity.

Finally, the authors analyzed the ORF6 protein evolutionary features in the currently circulating SARS-CoV-2. The results showed that 0.2% (124/66741) of the pathogenic strains lost their C-terminal activity due to frameshift or nonsense mutations during evolution, suggesting that these mutated strains may lead to IFN signaling after infecting the human body.