SARS-CoV-2 rORF3, a novel microprotein, impairs interferon-β production by targeting RanBP2 to inhibit p65 nuclear import.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the coronavirus disease 2019 (COVID-19). SARS-CoV-2 encodes various proteins to evade host innate immunity. rORF3 is a novel microprotein encoded by the negative-strand RNA (-RNA) of SARS-CoV-2 that antagonizes type I interferon (IFN-I) production. However, the molecular mechanism by which SARS-CoV-2 rORF3 suppresses IFN-I production remains elusive. Here we report rORF3 interacts with a nucleoporin Ran-Binding Protein 2 (RanBP2), and suppresses IFN-β production and promotes viral infection in a RanBP2-dependent manner. Specifically, we found RanBP2 inhibits the RIG-I-like receptor (RLR)-mediated signaling by targeting p65 and suppressing the nuclear translocation of p65. rORF3 targets RanBP2 and impedes the nuclear translocation of p65 upon tumor necrosis factor-alpha (TNF-α) stimulation, thereby antagonizing IFN-β production. Our findings not only reveal a novel function of RanBP2 in regulating RLR signaling but also provide insights into a new mechanism of the innate immune evasion by SARS-CoV-2.