To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to co-express PLpro and a substrate, murine interferon stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition o... More
To combat emerging coronaviruses, developing safe and efficient platforms to evaluate viral protease activities and the efficacy of protease inhibitors is a high priority. Here we exploit a biosafety level 2 (BSL-2) chimeric Sindbis virus system to evaluate protease activities and the efficacy of inhibitors directed against the papain-like protease (PLpro) of Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), a biosafety level 3 (BSL-3) pathogen. We engineered Sindbis virus to co-express PLpro and a substrate, murine interferon stimulated gene 15 (ISG15), and found that PLpro mediates removal of ISG15 (deISGylation) from cellular proteins. Mutation of the catalytic cysteine residue of PLpro or addition of a PLpro inhibitor blocked deISGylation in virus-infected cells. Thus, deISGylation is a marker of PLpro activity. Infection of Interferon-alpha/beta receptor knockout (IFNAR-/-) mice with these chimeric viruses revealed that PLpro deISGylation activity removed the ISG15-mediated protection during viral infection. Importantly, administration of a PLpro inhibitor protected these mice from lethal infection demonstrating the efficacy of a coronavirus protease inhibitor in a mouse model. However, this PLpro inhibitor was not sufficient to protect mice from lethal infection with SARS-CoV MA15, suggesting that further optimization of the delivery and stability of PLpro inhibitors is needed. We extended the chimeric virus platform to evaluate papain-like protease/deISGylating activity of Middle East Respiratory Syndrome Coronavirus (MERS-CoV), to provide a small animal model to evaluate PLpro protease inhibitors to this recently emerged pathogen. This platform has the potential to be universally adaptable to other viral and cellular enzymes that have deISGylating activity.
