Crosstalk zwischen nichtstrukturellen Proteinen von SARS-CoV-2 und der Autophagie-Signalmaschinerie
Immunologie
Zusammenfassung der Projektergebnisse
SARS-CoV-2 is the causative virus of the coronavirus disease 2019 (COVID-19). SARS- CoV-2 contains a single-stranded positive RNA genome of approximately 30 kb. The 5’-terminal two-thirds of the genome harbor two overlapping open reading frames (ORFs), ORF1a and 1b, which encode 16 nonstructural proteins (NSPs) involved in viral genome replication and subgenomic mRNA synthesis. In this project, we aimed at elucidating the crosstalk between selected NSPs of SARS-CoV-2 and the (macro-)autophagy signaling machinery. Autophagy is an intracellular recycling process responsible for the recycling of longlived or damaged proteins and organelles. Generally, autophagy might remove viral components during xenophagy/virophagy. However, there is an additional level of crosstalk between viral replication and autophagy. Positive-sense RNA viruses employ the host cell’s membranes to generate viral replication organelles, including double membrane vesicles (DMVs). DMVs are structures that are analogous to autophagosomes. Here, we expressed HA-tagged NSP3, NSP4, NSP5 and C-terminally 3xFLAG/MYC-tagged NSP6 (in the following abbreviated as NSP3-6) in HeLa and A549 cells and observed colocalization of NSP3, NSP6, and ubiquitin. We did not detect any co-localization of NSP3 or NSP6 with autophagosome markers. In NSP3-6-expressing cells, we observed extensive rearrangements of the cellular endomembrane system. We also investigated the crosstalk between NSPs or SARS-CoV-2 with the autophagy-inducing ULK1 and PIK3C3 complexes. We found that NSP3 inhibits ULK1 signaling via proteolytic cleavage, whereas expression of NSP5 and the nucleoprotein as well as the copy number of viral RNAs were reduced in Vero cells transfected with siRNA targeting the PIK3C3-complex subunit BECN1. Taken together, it appears that there exists mutual regulation of SARS-CoV-2 and the autophagy signaling machinery, i.e. NSP3-mediated regulation of ULK1 and PIK3C3-supported viral replication.
