The Role of the Tyrosine-Based Sorting Signals of the ORF3a Protein of SARS-CoV-2 on Intracellular Trafficking, Autophagy, and Apoptosis

ABSTRACT The open reading frame 3a (ORF3a) is an accessory transmembrane protein that is important to the pathogenicity of SARS-CoV-2. The cytoplasmic domain of ORF3a has three canonical tyrosine-based sorting signals (YxxΦ; where x is any amino acid and Φ is a hydrophobic amino acid with a bulky -R group). They have been implicated in the trafficking of membrane proteins to the cell plasma membrane and to intracellular organelles. Previous studies have indicated that mutation of the 160 YSNV 163 motif abrogated plasma membrane expression and inhibited ORF3a-induced apoptosis. However, two additional canonical tyrosine-based sorting motifs ( 211 YYQL 213 , 233 YNKI 236 ) exist in the cytoplasmic domain of ORF3a that have not been assessed. We removed all three potential tyrosine-based motifs and systematically restored them to assess the importance of each motif or combination of motifs that restored efficient trafficking to the cell surface and lysosomes. Our results indicate that the YxxΦ motif at position 160 was insufficient for the trafficking of ORF3a to the cell surface. Our studies also showed that ORF3a proteins with an intact YxxΦ at position 211 or at 160 and 211 were most important. We found that ORF3a cell surface expression correlated with the co-localization of ORF3a with LAMP-1 near the cell surface. These results suggest that YxxΦ motifs within the cytoplasmic domain may act cooperatively in ORF3a transport to the plasma membrane and endocytosis to lysosomes. Further, our results indicate that certain tyrosine mutants failed to activate caspase 3 and did not correlate with autophagy functions associated with this protein. IMPORTANCE Open reading frame 3a (ORF3a) encodes for the largest of the SARS-CoV-2 accessory proteins. While deletion of the ORF3a gene from SARS-CoV-2 results in a virus that replicates slightly less efficiently in cell culture, deletion also results in a virus that is less pathogenic in mouse models of SARS-CoV-2 infections. The ORF3a has been reported to be a viroporin, induces apoptosis and incomplete autophagy in cells. Thus, determining the domains involved in these functions will further our understanding of how this protein influences virus assembly and pathogenesis. Here, we investigated the role of the three potential tyrosine-based sorting signals in the cytoplasmic domain of the ORF3a on intracellular protein trafficking, apoptosis, and in the initiation of autophagy. Our results indicate that more than one YxxΦ motif is required for efficient transport of ORF3a, ORF3a expression resulted in minimal apoptosis, and cell surface expression was not required for autophagy.