*12.2. nsp3*

As noted previously, nsp3 is a large nonstructural protein containing multiple domains, and it participates in a wide array of functions. Its PLpro domains have deubiquitinating and deISGylating activities and are speculated to participate in immunomodulation. Indeed, SARS and the HCoV-NL63 PL2pro domains can interfere with IRF-3 phosphorylation without affecting respective kinases and stimulation of NFκB dependent genes [329–331]. Interestingly, the enzymatic activities of nsp3 are not solely responsible for IFN-β suppression. Deleting the catalytic residue, C1678 in SARS-CoV-1, and H1836 in HCoV-NL63

PL2pro, to eliminate proteolytic and deubiquitinating activities of the protein, only slightly decreased IFN suppression [331]. Treating PL2pro transfected cells with protease inhibitor GRL-0617S had no effect on IFN suppression but did abrogate NFκB stimulated gene suppression [331]. Since nsp3 is a membrane-spanning protein, the authors of this study also examined if TM forms of the PL2pro domain could inhibit IFN expression. Truncating nsp3 to only include the PL2pro domain attached to a TMS was shown to be a potent IFN antagonist, and it could suppress N-RIG stimulated IFN-β production [331].

To investigate the possible role deubiquitination has on IFN suppression, IRF-3(5D), a phosphomimetic of IRF3, was shown to be deubiquitinated by PL2pro. Despite being deubiquitinated, IRF-3(5D) was still able to dimerize, translocate to the nucleus and bind to DNA, but it could not induce IFN expression. The authors proposed that its interaction with other transcriptional machinery is altered so that IFN expression cannot be achieved [332]. These results sugges<sup>t</sup> that nsp3 is a potent inhibitor of IFN expression both upstream and downstream of IRF-3 phosphorylation. It has ye<sup>t</sup> to be detailed how nsp3 can prevent the phosphorylation of IRF-3. Enzymatic activity may also be required for NFκB stimulated gene suppression [331].

#### **13. Accessory Protein Interference with IFN Expression** *13.1.ORF6*

The SARS-CoV-1 ORF6 protein has been shown to have IFN-inhibiting abilities, suppressing both upstream and downstream effectors of IFN pathways. Expression of the protein suppressed Sendai virus-induced IFN expression by inhibiting phosphorylation and subsequent translocation of IRF3 to the nucleus [308,333]. ORF6 was also shown to inhibit STAT1 nuclear translocation, despite not preventing STAT1 phosphorylation [334]. Similarly, SARS-CoV-2 ORF6 is able to broadly suppress type-I IFN expression in vitro. Clonal expression of SARS-CoV-2 ORF6 and C-terminally truncated ORF6 inhibited multiple stages of IFN activation as well as downstream pathways of IFN signaling. Specifically, residues 53–61 of the protein overexpressed in HEK293T cells suppressed IRF3 activation by interfering with RIG-I, MDA5, and MAVS complex assembly [308]. Interestingly, the same region was also able to inhibit STAT1 nuclear translocation in IFN-stimulated HEK293T cells [308]. A possible explanation is that SARS-CoV-1 ORF6 localizes in the ER/Golgi membranes. Its C-terminus binds to nuclear import factors karyopherinα2 and karyopherin-β1, disrupting the formation of nuclear import complexes. Phosphorylated STAT1 is then unable to enter the nucleus. Deletion of ORF6, or removal of the C-terminus, restored STAT1 nuclear translocation [335]. It therefore seems that ORF6 can prevent the expression and secretion of IFNs, thus preventing the downstream upregulation of ISGs.
