The nonstructural protein 13 of porcine deltacoronavirus coordinates ATP-driven duplex unwinding and ATP-independent strand annealing for nucleic acid remodeling.

Abstract

BACKGROUND: Swine diarrheal death caused by intestinal coronavirus is a key problem that needs to be solved urgently in pig production. Porcine deltacoronavirus (PDCoV) is a novel enteropathogenic coronavirus that causes acute diarrhea in suckling piglets, resulting in serious economic losses to the cultivation industry. PDCoV helicase NSP13 plays a pivotal role in virus replication and is regarded as an ideal drug target, but its enzymatic characteristics remain poorly characterized. RESULTS: In this study, we systematically investigated the biochemical properties of PDCoV NSP13 through recombinant expression and functional assays. Our findings revealed that PDCoV NSP13 exhibited a 5'-to-3' directional unwinding activity powered by broad-spectrum nucleoside triphosphate hydrolysis, with the enhanced unwinding efficiency for dsDNA over dsRNA. Furthermore, we found that 4 nt was the minimum length of 5'-overhang required for NSP13 to unwind substrates, and the unwinding efficiency was inversely correlated with both 5'-overhang length and duplex region size. And NSP13 displayed concentration-dependent activity modulation, whereas excess NSP13 levels suppressed the unwinding reaction. Notably, we uncovered a previously unrecognized strand-annealing capability of NSP13, enabling efficient hybridization of complementary ssDNA into dsDNA. And a series of experiments with protein mutants revealed the effect of deletion of different domains on the unwinding and annealing activities of PDCoV NSP13. CONCLUSIONS: These mechanistic insights advanced our understanding of coronavirus replication machinery and established a biochemical foundation for the development of antiviral drugs targeting helicase NSP13.