The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

Hazel Stewart; Yongxu Lu; Sarah O'Keefe; Anusha Valpadashi; Luis Daniel Cruz-Zaragoza; Hendrik A Michel; Samantha K Nguyen; George W Carnell; Nina Lukhovitskaya; Rachel Milligan; Yasmin Adewusi; Irwin Jungreis; Valeria Lulla; David A Matthews; Stephen High; Peter Rehling; Edward Emmott; Jonathan L Heeney; Andrew D Davidson; James R Edgar; Geoffrey L Smith; Andrew E Firth

doi:10.1016/j.isci.2023.108080

The SARS-CoV-2 protein ORF3c is a mitochondrial modulator of innate immunity

iScience. 2023 Sep 28;26(11):108080. doi: 10.1016/j.isci.2023.108080. eCollection 2023 Nov 17.

Authors

Hazel Stewart¹, Yongxu Lu¹, Sarah O'Keefe², Anusha Valpadashi³, Luis Daniel Cruz-Zaragoza³, Hendrik A Michel¹, Samantha K Nguyen¹, George W Carnell⁴, Nina Lukhovitskaya¹, Rachel Milligan⁵, Yasmin Adewusi¹, Irwin Jungreis^{6

7}, Valeria Lulla¹, David A Matthews⁵, Stephen High², Peter Rehling³, Edward Emmott⁸, Jonathan L Heeney⁴, Andrew D Davidson⁵, James R Edgar¹, Geoffrey L Smith¹, Andrew E Firth¹

Affiliations

¹ Department of Pathology, University of Cambridge, Cambridge, UK.
² Faculty of Biology, Medicine and Health, School of Biological Sciences, University of Manchester, Manchester, UK.
³ Department of Cellular Biochemistry, University Medical Center Göttingen, Göttingen, Germany.
⁴ Department of Veterinary Medicine, University of Cambridge, Cambridge, UK.
⁵ School of Cellular and Molecular Medicine, Faculty of Life Sciences, University of Bristol, Bristol, UK.
⁶ Broad Institute of MIT and Harvard, Cambridge, MA, USA.
⁷ MIT Computer Science and Artificial Intelligence Laboratory, Cambridge, MA, USA.
⁸ Centre for Proteome Research, Department of Biochemistry & Systems Biology, Institute of Systems, Molecular and Integrative Biology, University of Liverpool, Liverpool, UK.

Abstract

The SARS-CoV-2 genome encodes a multitude of accessory proteins. Using comparative genomic approaches, an additional accessory protein, ORF3c, has been predicted to be encoded within the ORF3a sgmRNA. Expression of ORF3c during infection has been confirmed independently by ribosome profiling. Despite ORF3c also being present in the 2002-2003 SARS-CoV, its function has remained unexplored. Here we show that ORF3c localizes to mitochondria, where it inhibits innate immunity by restricting IFN-β production, but not NF-κB activation or JAK-STAT signaling downstream of type I IFN stimulation. We find that ORF3c is inhibitory after stimulation with cytoplasmic RNA helicases RIG-I or MDA5 or adaptor protein MAVS, but not after TRIF, TBK1 or phospho-IRF3 stimulation. ORF3c co-immunoprecipitates with the antiviral proteins MAVS and PGAM5 and induces MAVS cleavage by caspase-3. Together, these data provide insight into an uncharacterized mechanism of innate immune evasion by this important human pathogen.

Keywords: Immunology; Microbiology; Molecular biology; Proteomics.

Abstract

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