# The Stalk and 1B Domains Are Required for Porcine Deltacoronavirus Helicase NSP13 to Separate the Double-Stranded Nucleic Acids, and the Deletion of the ZBD Impairs This Activity

## TL;DR

This study identifies key domains in the PDCoV helicase NSP13 that are essential for unwinding double-stranded nucleic acids, which is crucial for viral replication and drug development.

## Contribution

The first study to determine the specific roles of NSP13 domains in PDCoV helicase activity and unwinding efficiency.

## Key findings

- The Stalk and 1B domains are required for NSP13 to unwind double-stranded nucleic acids.
- Deleting the ZBD reduces unwinding efficiency but does not eliminate ATPase activity.
- NSP13ΔZBD-Stalk, NSP13Δ1B, and NSP13ΔZBD-Stalk-1B mutants lose unwinding activity entirely.

## Abstract

The helicase NSP13, encoded by porcine deltacoronavirus (PDCoV), regulates viral replication and is considered to bean ideal target for antiviral drugs. In our work, the wild-type NSP13WT and various deletion mutants were generated through expression and purification, and we tested the activities of these proteins using multiple methods. The results revealed that both the Stalk and 1B domains were necessary for NSP13WT to separate the double-stranded nucleic acids, and the deletion of the ZBD impaired this unwinding activity. Our study is the first to explore the role of each domain in the activities of PDCoV NSP13, providing mechanistic insights into helicase-regulated viral replication and laying the foundation for the development of antiviral drugs.

The nonstructural protein 13 (NSP13) of PDCoV is a highly conservative helicase and plays key roles in viral replication. NSP13 contains a zinc-binding domain (ZBD), a helical Stalk domain, a beta-barrel 1B domain, and a core helicase domain. However, the specific functions of these domains of PDCoV NSP13 remain largely unknown. Here, we expressed and purified the wild-type NSP13WT and various mutants with domain deletions, and the activities of these proteins were analyzed using multiple methods. We found that NSP13ΔZBD possessed the abilities to hydrolyze ATP and unwind double-stranded nucleic acids, but the unwinding efficiency was lower than that of NSP13WT. In contrast, NSP13ΔZBD-Stalk, NSP13Δ1B, and NSP13ΔZBD-Stalk-1B all lost their unwinding activity, but not their ATPase activity. These results revealed that the deletion of the ZBD impaired the unwinding activity of PDCoV helicase NSP13, and the Stalk and 1B domains were critical for NSP13 to separate the duplexes. The identification of the roles of each domain in this study was helpful to gain an in-depth understanding of the overall functions of helicase NSP13, providing a theoretical basis for the development of antiviral drugs targeting helicase.