Structural and Functional Analysis of Coronavirus Cysteine Protease Nsp5

Abstract

Coronaviruses (CoV) are positive-strand RNA viruses that encode large replicase polyproteins that are processed by two or three viral proteases to yield intermediate and mature nonstructural proteins (nsps). Nsp5 (3CLpro, Mpro) is a cysteine protease that is essential for virus replication and mediates processing at 11 cleavage sites, yielding nsps 4 through 16. Due to its critical role in replication, nsp5 is a key target for development of antivirals against coronaviruses. However, the intramolecular and intermolecular mechanisms that govern nsp5 protease structure and function remain unclear. These issues and the limited ability to culture many human coronaviruses make testing of inhibitors against nsp5 challenging during infection in culture. This dissertation discusses the role of newly identified intramolecular residue interactions on protease function, and the conservation of these interactions across coronaviruses. Using mutagenesis, I identified temperature-sensitive and second-site suppressor mutations in murine hepatitis virus (MHV) nsp5 that are distant from known functional determinants and have a profound impact on viral replication and nsp5-mediated polyprotein processing. To evaluate the role of these mutations and their associations in other viruses, I have engineered nsp5-substitution chimeras by introducing the nsp5 proteases of closely and distantly related coronaviruses into the background of MHV. These data show that coronavirus nsp5 protease activity is governed by complex long-distance residue interactions that span the protease structure and have tightly co-evolved within the context of the greater polyprotein and viral background. Collectively, these data define a new approach for the study of human coronavirus nsp5 proteases in an efficiently replicating non-human coronavirus, provide a platform for testing antivirals against the proteases from virus that are difficult to cultivate, and provide key new insights into the regulatory mechanisms directing nsp5 structure and function.