Multifunctional Coronavirus Papain-Like Proteases as Targets for Antiviral Therapeutics and Vaccines

Severe Acute Respiratory Syndrome coronavirus (SARS-CoV) and Middle Ease Respiratory Syndrome coronavirus (MERS-CoV) pose a severe threat to humans because of high mortality. Despite the risk of coronavirus (CoV) emerging in the human population there are no antiviral drugs or vaccines to combat coronavirus infection. The focus of my dissertation was to study the multifunctionality of papain-like proteases (PLPs) encoded within coronavirus genomes to facilitate the development of antiviral drugs and vaccines. The viral PLPs are critical for processing the amino-terminal end of the replicase during virus replication and are attractive targets for antiviral therapies. In my research, I analyzed the activities of multiple PLPs to determine if those proteases can be targets for broad spectrum therapeutics. I determined that SARS-CoV PLpro is an effective target of small molecule inhibitors by evaluating their efficacy and ability to inhibit SARS-CoV replication in cell culture. Further, I determined that the predicted PLpro domain from MERS-CoV is a multifunctional enzyme with protease, deubiquitinase, interferon antagonism and deISGylating activities. Despite low sequence identity, this multifunctionality of PLPs seems to be conserved among many coronaviruses. I demonstrated protease and deubiquitinase activity of PLP2s from HCoV-OC43, HCoV-229E, HCoV-HKU1, MHV and FIPV. To evaluate the role of PLP during virus replication and pathogenesis I utilized two approaches and a mouse model system, mouse hepatitis virus (MHV). First, I performed deletion analysis and mutagenesis of the PLP2 ubiquitin-like (UBL) domain and showed, for the first time, that the UBL domain is important for PLP2 stability and virus pathogenesis. The UBL mutant virus is attenuated in mice and protects the mice from the disease upon challenge with wild-type virus making it a possible vaccine candidate. Secondly, based on the MHV PLP2 crystal structure and modeling of the PLP2 with an ubiquitin moiety, I identified residues on PLP2 that likely interact with ubiquitin. I tested mutants of predicted PLP2-ubiquitin interaction sites and found residues that are important for both protease and deubiquitinase function. Overall, my data suggest that PLP multifunctionality is conserved among coronaviruses and is a valuable target for vaccines and antiviral drugs development for existing and emerging coronaviruses