Viral proteases play critical roles in the host cell and immune remodeling that allows virus production. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) papain-like protease (PLpro) encoded in the large nonstructural protein 3 (Nsp3) also possesses isopeptidase activity with specificity for ubiquitin and ISG15 conjugates. Here, we interrogated the cellular interactome of the SARS-CoV-2 PLpro catalytic domain to gain insight into the putative substrates and cellular functions affected by the viral deubiquitinase. PLpro was detected in protein complexes that control multiple ubiquitin and ubiquitin-like (UbL) regulated signaling and effector pathways. By restricting the analysis to cytosolic and ... More
Viral proteases play critical roles in the host cell and immune remodeling that allows virus production. The severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) papain-like protease (PLpro) encoded in the large nonstructural protein 3 (Nsp3) also possesses isopeptidase activity with specificity for ubiquitin and ISG15 conjugates. Here, we interrogated the cellular interactome of the SARS-CoV-2 PLpro catalytic domain to gain insight into the putative substrates and cellular functions affected by the viral deubiquitinase. PLpro was detected in protein complexes that control multiple ubiquitin and ubiquitin-like (UbL) regulated signaling and effector pathways. By restricting the analysis to cytosolic and membrane-associated ubiquitin ligases, we found that PLpro interacts with N-recognin ubiquitin ligases and preferentially rescues type I N-degron substrates from proteasomal degradation. PLpro stabilized N-degron carrying HSPA5/BiP/GRP78, which is arginylated in the cytosol upon release from the endoplasmic reticulum (ER) during ER stress, and enhanced the Arg-HSPA5-driven oligomerization of the N-recognin SQSTM1/p62 that serves as a platform for phagophore assembly. However, while in addition to Arg-HSPA5 and SQSTM1/p62, ATG9A, WIPI2, and BECN1/Beclin 1 were detected in PLpro immunoprecipitates, other components of the autophagosome biogenesis machinery, such as the ATG12-ATG5-ATG16L1 complex and MAP1LC3/LC3 were absent, which correlated with proteolytic inactivation of ULK1, impaired production of lipidated LC3-II, and inhibition of reticulophagy. The findings highlight a novel mechanism by which, through the reprogramming of autophagy, the PLpro deubiquitinase may contribute to the remodeling of intracellular membranes in coronavirus-infected cells.
