Unwinding Mechanism of SARS-CoV Helicase (nsp13) by Biochemical and Single-molecular Studies

Abstract

The recent outbreak of coronavirus diseases has highlighted the urgent need for effective therapeutic agents and vaccines. One potential therapeutic target is the non-structural protein 13 (nsp13) helicase of Severe Acute Respiratory Syndrome (SARS) CoV, which plays an essential role in the viral replication process and has a conserved amino acid sequence between species. Although the replication of coronaviruses and other retroviruses occurs in the cytoplasm of infected cells, little is known about the detailed mechanism of nsp13 unwinding activity. To address this, I utilized biochemical approaches and biophysical single-molecule assays to investigate the molecular unwinding mechanism of nsp13. In the first part of the study, I demonstrated that nsp13 exhibits cooperative unwinding activity on single-stranded regions of DNA, with inhibition by re-zipping. Additionally, I found that nsp13 interacts with double-stranded regions under the influence of ADP, leading to transient destabilization of the double strands, which enhances cooperative unwinding activity. These findings suggest a unique model that nsp13 exhibits unwinding activity through different cooperative actions. In second part of study, I further demonstrated that nsp13 can efficiently unwind double-stranded DNA under physiological concentrations of Ca2+ found in the cytosolic DMVs. Importantly, nsp13 achieves this unwinding activity while performing ATP hydrolysis in the presence of Ca2+. These novel findings provide new insights into the properties of nsp13 in the range of calcium in cytosolic DMVs. Taken together, understanding the molecular mechanism of nsp13 unwinding activity may provide important approaches into the development of effective therapeutic strategies against the coronavirus diseases that continue to affect humanity.