ABSTRACTLike many pathogenic viruses, SARS-CoV-2 must overcome interferon (IFN)-mediated host defenses for infection establishment. To achieve this, SARS-CoV-2 deploys overlapping mechanisms to antagonize IFN production and signaling. The strongest IFN antagonist is the accessory protein ORF6, which localizes to multiple membranous compartments, including the nuclear envelope, where it directly binds the nuclear pore components Nup98-Rae1 to inhibit nuclear translocation of activated STAT1/IRF3 transcription factors. However, a direct cause-and-effect relationship between ORF6 localization and IFN antagonism has yet to be explored experimentally. Here, we use extensive mutagenesis studies to define the structural determinants required for steady-state localization and demonstrate that mis-localized ORF6 variants can still potently inhibit nuclear trafficking and IFN signaling. Additionally, expression of a peptide that mimics the ORF6/Nup98 interaction domain robustly inhibited nuclear trafficking. Furthermore, pharmacologic and mutational approaches combined to suggest that ORF6 is likely a peripheral-membrane protein, opposed to being a transmembrane protein as previously speculated. Thus, ORF6 localization and IFN antagonism are independent activities, which raises the possibility that ORF6 may have additional functions within membrane networks to enhance virus replication.