The SARS-CoV-2 protein ORF3a inhibits fusion of autophagosomes with lysosomes

Yabin Zhang,Yong Lin,Binli Mao,Rongjuan Pei,Huihui Li,Kefeng Lu,Hao Sun,Zhenyu Zhao

doi:10.1038/s41421-021-00268-z

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the ongoing coronavirus disease 2019 pandemic. How SARS-CoV-2 regulates cellular responses to escape clearance by host cells is unknown. Autophagy is an intracellular lysosomal degradation pathway for the clearance of various cargoes, including viruses. Here, we systematically screened 28 viral proteins of SARS-CoV-2 and identified that ORF3a strongly inhibited autophagic flux by blocking the fusion of autophagosomes with lysosomes. ORF3a colocalized with lysosomes and interacted with VPS39, a component of the homotypic fusion and protein sorting (HOPS) complex. The ORF3a–VPS39 interaction prohibited the binding of HOPS with RAB7, which prevented the assembly of fusion machinery, leading to the accumulation of unfused autophagosomes. These results indicated the potential mechanism by which SARS-CoV-2 escapes degradation; that is, the virus interferes with autophagosome–lysosome fusion. Furthermore, our findings will facilitate strategies targeting autophagy for conferring potential protection against the spread of SARS-CoV-2.

Highlights

The pandemic of coronavirus disease 2019 (COVID-19), caused by the most-recently emergent member of the coronavirus family, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has transitioned this relatively understudied group of viruses to a worldwide public health priority in a matter of months[1,2,3,4]
We expressed these proteins in HEK293T cells to determine their effect on autophagy by detecting the protein levels of SQSTM1/p62 and LC3-II
We constructed four chimeras: TM from SARS-CoV-2 ORF3a combined with C-terminus from SARS-CoV or MERS and the C-terminus from SARS-CoV-2 ORF3a combined with TM from SARS-CoV or MERS (Fig. 4f). These chimeras and TM + C-truncated SARS-CoV-2 ORF3a were tested to determine their effect on autophagy, and the results showed that none of these chimeras influenced the protein levels of p62 or LC3-II (Fig. 4f, g)

Summary

Introduction

The pandemic of coronavirus disease 2019 (COVID-19), caused by the most-recently emergent member of the coronavirus family, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has transitioned this relatively understudied group of viruses to a worldwide public health priority in a matter of months[1,2,3,4]. Autophagy is a highly conserved cellular pathway involving the formation of autophagosomes to deliver cargoes, including long-lived proteins, protein aggregates, organelles, and infected viruses or bacteria, to lysosomes for degradation[13,14,15]. The mature autophagosomes move to, dock onto, and subsequently fuse with lysosomes. This fusion process is mediated by the N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins syntaxin-17, SNAP29, and VAMP8, as well as the homotypic fusion and protein sorting (HOPS) complex[24,25,26,27].

Methods

Results

Conclusion