Regulation of virion production by the ORF8 signal peptide across SARS-CoV-2 variants

The open reading frame 8 (ORF8), an accessory protein of SARS-CoV-2, is prone to deletions and mutations across different viral variants, which was first described in several Singapore variants. The reason why viral evolution favors loss or inactivation of ORF8 is not fully understood, although the effects of ORF8 on inflammation, immune evasion, and disease severity have been described. Here we show -using clinical ORF8-deficient viral isolates, virus-like particles (VLPs) and viral replicons- that ORF8 expression dampens viral particle production. ORF8 physically interacts with the viral Spike protein and induces Golgi fragmentation, overall contributing to less virus particle production. Using systematic ORF8 deletions, we mapped the particle-reducing function to its N-terminal signal peptide. Interestingly, this part of ORF8 is severely truncated in the recent XBB.1.5 variant, and when restored, suppresses viral particle production in the context of the entire viral genome. Collectively, our data supports the model that evolutionary pressure exists to delete ORF8 sequence and expression across SARS-CoV-2 variants to fully enable viral particle production. ImportanceSARS-CoV-2 variants continue to emerge worldwide with advantages in replication and immune evasion. Many variants have acquired distinct mutations in independent lineages to abolish ORF8 expression. To understand the molecular mechanisms behind this evolutionary trend, we utilized reverse genetics, molecular virology, and confocal microscopy to show that ORF8 has antiviral functions by dampening viral particle production and inducing Golgi stress during infection. Our data demonstrate that SARS-CoV-2 is continuing its adaptation to optimize viral particle production and other unknown aspects of viral infection.