Files
Abstract
Coronaviruses are single-stranded, positive-sense RNA viruses from the family Coronaviridae, whose members have severe impact on human health and cause significant economic hardships. Pertinent examples of coronaviruses include porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and severe acute and Middle East respiratory syndrome coronavirus (SARS-CoV; MERS-CoV). Interestingly, these viruses encode a dual functioning protease, the papain like-protease (PLP), that aids in RNA replication via processing of the viral polyprotein as well as antagonizes the host antiviral innate immune response. The latter function is achieved through the removal of post-translational modifications of host and viral proteins by ubiquitin (Ub) or Ub-like interferon-stimulated-gene-product-15 (ISG15), processes known as deubiquitination and deISGylation. The overall coronavirus PLP deubiquitinase and deISGylase activities are not well characterized, especially among the different porcine coronaviruses. Ub is known to be almost completely conserved amongst eukaryotes; however, ISG15 is highly divergent with sequence identities as low as 35% among a broad range of animals. The implications of this sequence divergence on the ability of coronavirus PLPs to recognize and cleave certain species of ISG15 conjugates is poorly understood. In addition, the lack of coronavirus PLPs with altered deISGylase activity has been an obvious barrier in defining the roles of deubiquitinase and deISGylase activities on pathogenesis and viral host evasion. This study aims to address these three major knowledge gaps to provide insight into the deubiquitination and deISGylation roles of the coronavirus PLPs. First, the biochemical characterization of the PLPs from PEDV and PDCoV are described in detail. Second, a biochemical and structural look into the coronavirus PLP interface with ISG15 utilizing the first X-ray structures of SARS-CoV in complex with the principle binding domain of human and mouse ISG15. Third, utilizing structural information, from the first X-ray structure of MERS-CoV in complex with the C-terminal domain of human ISG15, disruptive mutants were generated that lacked deISGylase activity but retained wild-type deubiquitinase activity. These studies provide insight into the varied deubiquitinase and deISGylase activity of the coronavirus PLP and provides important new molecular tools to engineer coronavirus PLPs to further advance the understanding of CoV pathogenesis.