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

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.