The nonstructural protein 13 of porcine deltacoronavirus exhibits an ATP- independent and nondirectional annealing activity that is inhibited by Mg2.

Lethal diarrhea in suckling piglets caused by porcine deltacoronavirus (PDCoV) poses a significant threat to the global swine industry. The nonstructural protein 13 (NSP13) encoded by the PDCoV genome plays a crucial role in viral replication as a helicase and is considered an ideal target for antiviral drug development. However, the biochemical characteristics of PDCoV NSP13 remain unclear. In this study, we purified and characterized the PDCoV helicase NSP13. We found that NSP13 not only possessed the canonical ATP-dependent unwinding activity but also exhibited an opposite annealing activity, promoting the hybridization of complementary single-stranded DNA (ssDNA) into double-stranded DNA (dsDNA) without ATP involvement. Our results showed that Mg²⁺ addition suppressed dsDNA formation, with higher Mg2+ concentrations leading to lower annealing efficiency. In contrast, the dsDNA yield was enhanced by increasing the NSP13 concentrations. Moreover, NSP13 facilitated the formation of dsDNA with different terminals and duplex regions in a nondirectional manner, and longer 5'-overhangs and extended duplex regions increased the dsDNA yield. We also tested the effects of ATP and multiple substitutes on the annealing reaction. Among these, only ADP enhanced dsDNA generation in a concentration-dependent manner, whereas ATP and other substitutes inhibited annealing. This study elucidates the annealing function of NSP13 and identifies factors that influence this activity, thereby contributing to a deeper understanding of NSP13's role in viral replication and the development of antiviral drugs.