Sofosbuvir and its tri-phosphate metabolite inhibit the RNA-dependent RNA polymerase activity of non-Structural protein 5 from the Kyasanur forest disease virus

Abstract

Kyasanur forest disease is a neglected zoonotic disease caused by a single stranded RNA based flavivirus, the incidence of which, was first recorded in 1957, in the Southern part of India. Kyasanur forest disease virus is transmitted to monkeys and humans, through the infected tick bite of Haemophysalis spinigera. Kyasanur forest disease is a febrile illness, which in severe cases, results in neurological manifestations leading to mortality. The current treatment regimens are symptomatic and supportive in nature. There are no targeted therapies available for this disease. In this study, we evaluated the ability of the FDA approved drug, Sofosbuvir to inhibit the RNA dependent RNA polymerase activity of NS5 protein from Kyasanur forest disease virus. NS5 protein containing the N-terminal methyl transferase domain and C-terminal RNA dependent RNA polymerase domain was expressed in Escherichia coli and RNA dependent RNA polymerase activity, demonstrated with the purified protein. The RNA dependent RNA polymerase assay conditions were optimized, followed by determination of apparent Km,ATP. IC50 of Sofosbuvir against RNA dependent RNA polymerase activity was determined under the optimized conditions with the purified NS5 protein. Drug repurposing strategies can accelerate the development of targeted therapies for Kyasanur forest disease. This is the first demonstration of inhibition of RNA dependent RNA polymerase activity of Kyasanur forest disease virus with a small molecule inhibitor.