SARS-CoV-2 ORF6 protein does not antagonize interferon signaling in respiratory epithelial Calu-3 cells during infection

Minghua Li, Kasirajan Ayyanathan, Mark Dittmar, Jesse Miller, Iulia Tapescu, Jae Seung Lee, Marisa E. McGrath, Yong Xue, Sanjay Vashee, David C. Schultz, Matthew B. Frieman, Sara Cherry

Research output: Contribution to journalArticlepeer-review


Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused millions of deaths, posing a substantial threat to global public health. Viruses evolve differentstrategies to antagonize or evade host immune responses. While ectopic expression of SARS-CoV-2 accessory protein ORF6 blocks interferon (IFN) production and downstream IFN signaling, the role of ORF6 in IFN signaling during bona fideviral infection of respiratory cells is unclear. By comparing wild-type (WT) and ORF6-deleted (ΔORF6) SARS-CoV-2 infection and IFN signaling in respiratory cells, we found that ΔORF6 SARS-CoV-2 replicates more efficientlythan WT virus and, thus, stimulates more robust immune signaling. Loss of ORF6 does not alter innate signaling in infected cells: both WT and ΔORF6 virus induce delayed IFN responses only in bystander cells. Moreover, expression of ORF6 in the context of SARS-CoV-2 infection has no effecton Sendai virus-stimulated IFN induction: robust translocation of IRF3 is observed in both SARS-CoV-2 infected and bystander cells. Furthermore, IFN pretreatment potently blocks WT and ΔORF6 virus replication similarly, and both viruses fail to suppress the induction of interferon-stimulated genes (ISGs) upon IFN-β treatment. However, upon treatment with IFN-β, only bystander cells induce STAT1 translocation during infection with WT virus, whereas ΔORF6 virus-infected cells now show translocation. This suggests that under conditions of high IFN activation, ORF6 can attenuate STAT1 activation. These data provide evidence that ORF6 is not sufficientto antagonize IFN production or IFN signaling in SARS-CoV-2-infected respiratory cells but may impact the efficacyof therapeutics that stimulate innate immune pathways.

Original languageEnglish (US)
Issue number4
StatePublished - Jul 2023
Externally publishedYes


  • ORF6
  • SARS-CoV-2
  • antagonism
  • interferon

ASJC Scopus subject areas

  • Microbiology
  • Virology


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