Investigating SARS-CoV-2 ORF3a binding to human TRAF2 and TRAF3

Abstract Severe COVID-19, caused by Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is characterized widespread inflammation and cytokine storm. A number of viral proteins are capable inducing inflammatory responses, one which open reading frame 3a (ORF3a). ORF3a from SARS-CoV shares 73% homology with SARS-CoV-2 was shown to stimulate NF-κB signaling inflammasome activation in a manner dependent on TNF receptor-associated factor 3 (TRAF3). TRAF family regulate thereby influence responses. Both homologs contain TRAF-binding consensus sequence, has been bind human TRAFs 2, 3, 6. We therefore investigated the binding as potential mechanism through may activate signaling. TRAF-C domains were purified co-crystallized peptide containing sequence (PIQAS). Fluorescence polarization (FP) used assess affinity between peptide, dual-luciferase assays measure cells transfected wildtype or mutated alanines. X-ray crystallography revealed that able domain FP indicated for these interactions low. When we compared HEK293T expressing WT vs mutant ORF3a, there no significant difference. Therefore, while TRAFs, this interaction does not significantly contribute our assay. This work supported NIH grants R01GM127609 P01AI141350.