The ORF7a protein of SARS-CoV-2 initiates autophagy and limits autophagosome-lysosome fusion via degradation of SNAP29 to promote virus replication.

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is closely related to various cellular aspects associated with autophagy. However, how SARS-CoV-2 mediates the subversion of the macroautophagy/autophagy pathway remains largely unclear. In this study, we demonstrate that overexpression of the SARS-CoV-2 ORF7a protein activates LC3-II and leads to the accumulation of autophagosomes in multiple cell lines, while knockdown of the viral ORF7a gene via shRNAs targeting ORF7a sgRNA during SARS-CoV-2 infection decreased autophagy levels. Mechanistically, the ORF7a protein initiates autophagy via the AKT-MTOR-ULK1-mediated pathway, but ORF7a limits the progression of autophagic flux by activating CASP3 (caspase 3) to cleave the SNAP29 protein at aspartic acid residue 30 (D30), ultimately impairing complete autophagy. Importantly, SARS-CoV-2 infection-induced accumulated autophagosomes promote progeny virus production, whereby ORF7a downregulates SNAP29, ultimately resulting in failure of autophagosome fusion with lysosomes to promote viral replication. Taken together, our study reveals a mechanism by which SARS-CoV-2 utilizes the autophagic machinery to facilitate its own propagation via ORF7a.Abbreviations: 3-MA: 3-methyladenine; ACE2: angiotensin converting enzyme 2; ACTB/β-actin: actin beta; ATG7: autophagy related 7; Baf A1: bafilomycin A1; BECN1: beclin 1; CASP3: caspase 3; COVID-19: coronavirus disease 2019; GFP: green fluorescent protein; hpi: hour post-infection; hpt: hour post-transfection; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MERS: Middle East respiratory syndrome; MTOR: mechanistic target of rapamycin kinase; ORF: open reading frame; PARP: poly(ADP-ribose) polymerase; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; shRNAs: short hairpin RNAs; siRNA: small interfering RNA; SNAP29: synaptosome associated protein 29; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TCID50: tissue culture infectious dose; TEM: transmission electron microscopy; TUBB, tubulin, beta; ULK1: unc-51 like autophagy activating kinase 1.

• Authors: Peili Hou, Xuefeng Wang, Hongmei Wang, Tiecheng Wang, Zhangping Yu, Chunqing Xu, Yudong Zhao, Wenqi Wang, Yong Zhao, Fengyun Chu, Huasong Chang, Hongchao Zhu, Jiahui Lu, Fuzhen Zhang, Xue Liang, Xingyu Li, Song Wang, Yuwei Gao, Hongbin He
• Journal: Autophagy (Autophagy) Vol. 19 Issue 2 Pg. 551-569 (02 2023) ISSN: 1554-8635 [Electronic] United States
• PMID: 35670302 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Caspase 3
• Qb-SNARE Proteins
• Qc-SNARE Proteins
• SNAP29 protein, human
• TOR Serine-Threonine Kinases
• ORF7a protein, SARS-CoV-2

Topics

• Humans
• Autophagosomes (metabolism)
• Autophagy (genetics)
• Caspase 3 (metabolism)
• COVID-19
• Lysosomes (metabolism)
• Macroautophagy
• Qb-SNARE Proteins (metabolism)
• Qc-SNARE Proteins
• SARS-CoV-2
• TOR Serine-Threonine Kinases (metabolism)
• Virus Replication