ATPase-dependent duplex nucleic acid unwinding by SARS-CoV-2 nsP13 relies on facile binding and translocation along single-stranded nucleic acid.

Abstract

Nonstructural protein 13 (nsP13) of severe acute respiratory syndrome coronary virus (SARS-CoV-2) is a superfamily 1 helicase, which is essential for viral RNA replication. This protein can unwind dsRNA and DNA with a 5' single-stranded tail in the 5'-3' direction. Previous studies have demonstrated that nsP13 efficiently unwinds double-stranded nucleic acids with a single-stranded tail through a cooperative translocation fueled by ATP hydrolysis. However, the mechanism underlying the aforementioned unwinding remains unclear. In this study, we hypothesized that the differences in unwinding efficiency among duplex nucleic acids are driven by the ATP hydrolysis-induced changes in the binding affinity of nsP13 to a single-stranded tail. When nsP13 unwinds dsDNA with a 5' single-stranded tail, a long 5' single-stranded tail enhances ATP hydrolysis and promotes DNA unwinding efficiency. When the slowly hydrolyzable ATP analog adenosine-5'-O-3-thiotriphosphate was used for dsDNA unwinding by nsP13, duplex DNA unwinding was largely diminished, whereas the binding affinity onto the single-stranded DNA was more enhanced compared with ATP. Thus, unhindered ATP hydrolysis may allow nsP13 to bind and translocate along the single-stranded nucleic acid, resulting in the efficient unwinding of duplex nucleic acids.