Abstract - Text

Abstract text (incl. references and figure legends)

Introduction

Autophagy is a primal part of the innate immune system. During autophagy, cytoplasmic cargo including viral components is degraded in double-membrane vesicles (=autophagosomes) upon fusion with lysosomes. However, successful viral pathogens have evolved ways to evade or even exploit autophagy to facilitate their replication. The novel, pandemic SARS-CoV-2 is likely to be no exception to this, however, its strategies to subvert autophagy are currently unclear.

Objectives

Our goal was to systematically identify SARS-CoV-2 proteins that counteract autophagy and analyze their molecular mechanism.

Materials & Methods

We screened all SARS-CoV-2 proteins for modulation of autophagy using a high-throughput flow cytometry-based system. Subsequently, we employed a variety of methods, including quantitative proteomics, western blotting, RT-qPCR, and immunofluorescence microscopy, to characterize prominent hits.

Results

Our approach identified the SARS-CoV-2 proteins Nsp15, Orf3a, Orf7a, and E as the major viral regulators of autophagy. Nsp15 inhibited the induction of autophagy but could be counteracted by rapamycin. This suggests the mTOR signaling cascade as a potential target. Presence of Orf3a, Orf7a, and E lead to an accumulation of autophagosomes accompanied by reduced autophagic flux. Blockage of autophagic turnover with bafilomycin A1 drastically decreased this effect, implying that the viral proteins interfere with late steps of autophagy. Proteome analysis showed that Orf3a and Orf7a achieve this by interfering with cellular trafficking at the late endosome, fragmenting the trans-Golgi network. Interestingly, SARS-CoV-2 M accumulated autophagosomal membranes in the perinuclear region. However, this did not affect autophagic flux, hinting at activity separate from classical autophagy. Coronaviruses require double-membrane vesicles, such as autophagosomes, for their replication factories, thus, M might play a role in their establishment.

Conclusion

In summary, we identified major SARS-CoV-2 antagonists of cellular autophagy in an unbiased approach and characterized their molecular mechanisms. Notably, our data hint that the M protein exploits autophagy to potentially help establish viral replication factories. These results advance our understanding of SARS-CoV-2 pathogenesis and interplay with cellular defense mechanisms, which may help us to identify novel targets for therapeutic intervention.