A high ATP concentration enhances the cooperative translocation of the SARS coronavirus helicase nsP13 in the unwinding of duplex RNA

Dong Young Kang,Nipin Sp,Dong-Eun Kim,Young Mok Yang,Seonghwan Jeong,Kyoung-Jin Jang

doi:10.1038/s41598-020-61432-1

Abstract

Severe acute respiratory syndrome coronavirus nonstructural protein 13 (SCV nsP13), a superfamily 1 helicase, plays a central role in viral RNA replication through the unwinding of duplex RNA and DNA with a 5′ single-stranded tail in a 5′ to 3′ direction. Despite its putative role in viral RNA replication, nsP13 readily unwinds duplex DNA by cooperative translocation. Herein, nsP13 exhibited different characteristics in duplex RNA unwinding than that in duplex DNA. nsP13 showed very poor processivity on duplex RNA compared with that on duplex DNA. More importantly, nsP13 inefficiently unwinds duplex RNA by increasing the 5′-ss tail length. As the concentration of nsP13 increased, the amount of unwound duplex DNA increased and that of unwound duplex RNA decreased. The accumulation of duplex RNA/nsP13 complexes increased as the concentration of nsP13 increased. An increased ATP concentration in the unwinding of duplex RNA relieved the decrease in duplex RNA unwinding. Thus, nsP13 has a strong affinity for duplex RNA as a substrate for the unwinding reaction, which requires increased ATPs to processively unwind duplex RNA. Our results suggest that duplex RNA is a preferred substrate for the helicase activity of nsP13 than duplex DNA at high ATP concentrations.

Highlights

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease caused by a novel coronavirus (SARS-CoV or SCV) that has claimed almost 800 deaths in early 20031
A representative reaction of duplex RNA unwinding by nsP13 is shown in Fig. 1B, in which the nsP13 could be bound to the 5′-ss tail in the presence of ATP without ATP hydrolysis
0.5 μM trap RNA, 0.5 μM nsP13, and 2 mM ATP were used in the duplex RNA standard assay, and the assay was designed to monitor the unwinding kinetics of nsP13 as described in the Materials and Methods section

Summary

Introduction

Severe acute respiratory syndrome (SARS) is an acute respiratory infectious disease caused by a novel coronavirus (SARS-CoV or SCV) that has claimed almost 800 deaths in early 20031. The replicase genes comprising open reading frames (OFR1a and 1b) are translated into two large replicative polyproteins, pp1ab (~790 kDa) and pp1a (~490 kDa), which are involved with and without ribosomal frameshifting into the −1 frame[4,5] These two translational polyproteins are processed autoproteolytically by the major viral cysteine proteases MPRO or 3CLPRO to produce 16 non-structural proteins (nsPs), including RNA-dependent RNA polymerases (RdRp, nsP12) and NTPase/helicase (nsP13)[6,7,8]. We used partial duplex RNAs and DNAs as a model and control substrate, and the single-turnover kinetics of duplex RNA with a 5′-ss tail of different lengths was investigated according to various times and the concentration of ATP or nsP13. We observed that the cooperative translocation by nsP13 is related to the amount of ATP

Methods

Results

Conclusion