Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude. Author summary In the last 3 years, it has become clear that emerging coronaviruses still pose a significant threat to human health. Despite the vaccines that are now available in many countries against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccine scarcity or the inability to gain proper protection from immunization due to an immunocompromised status still leaves various people that will benefit from the administration of potent and safe antiviral agents. In this research, we have developed ubiquitin variants (UbVs), small proteins that resemble a natural component of human cells called ubiquitin, that are able to bind a part of the virus that is essential for its survival, with high affinity and selectivity. This way, these UbVs are able to inhibit the production of new viral particles after infection, thus preventing the virus from spreading from cell to cell and wreaking havoc on the body.

Automated summary

In this study, researchers have identified ubiquitin variants (UbVs) that can specifically and effectively block the activity of the papain-like protease (PLpro) in SARS-CoV-2. These UbVs inhibit the essential cleavage activities of the protease, reducing viral replication and providing a potential therapeutic approach for combating coronaviruses.