SARS-CoV-2 enhances lysosomal exocytosis and deacidifies lysosomes to facilitate viral release.

The mechanism of SARS-CoV-2 egress predominantly governs the quantity and quality of progeny viruses, thereby significantly contributing to viral pathogenicity. However, the key factors influencing viral egress remain largely unclear. In this study, using transcription- and replication-competent SARS-CoV-2 virus-like-particle (SARS-CoV-2 trVLP), electron microscopy, drug inhibition assays, and cellular pH-sensitive fluorescent probes, we demonstrate that increased lysosomal exocytosis efficiency and lysosome deacidification play a pivotal role in facilitating SARS-CoV-2 egress. Specifically, SARS-CoV-2 may use multiple egress pathways, with lysosomal exocytosis as the primary mechanism and the biosynthetic secretory pathway as a less efficient route. Viral infection enhances lysosomal exocytosis via the ORF3a protein, thus facilitating viral release. SARS-CoV-2 infection also induces lysosome deacidification; moreover, treatment with bafilomycin A1, which induces lysosome deacidification, further enhances viral egress. Furthermore, we systematically investigate how viral proteins affect lysosomal pH and enzymatic activities. Our findings reveal that ORF3a and E proteins induce lysosome deacidification and diminish lysosomal enzyme activities, probably protecting progeny viruses from premature cleavage and degradation. This study provides mechanistic insight into how SARS-CoV-2 promotes lysosomal exocytosis and triggers lysosome deacidification for viral release.