Effects of multiple Nsp proteins on SARS-CoV-2 polymerase activity
10.16303/j.cnki.1005-4545.2025.08.11
- VernacularTitle:SARS-CoV-2的多种Nsp对其聚合酶活性的影响
- Author:
Mingxin CHENG
1
;
Pengfei ZHU
;
Fang YAN
;
Fang SONG
;
Xuefeng WANG
;
Tiecheng WANG
;
Xianzhu XIA
;
Yuwei GAO
;
Fang YAN
Author Information
1. 山西农业大学动物医学学院,山西晋中 310029;中国农业科学院长春兽医研究所病原微生物生物安全全国重点实验室/吉林省人兽共患病预防控制重点实验室,吉林长春 130122
- Publication Type:Journal Article
- Keywords:
SARS-CoV-2;
polymerase activity reporting systerm;
capping;
DMV
- From:
Chinese Journal of Veterinary Science
2025;45(8):1665-1671
- CountryChina
- Language:Chinese
-
Abstract:
The novel coronavirus(severe acute respiratory syndrome coronavirus 2,SARS-CoV-2),classified as a single-stranded RNA virus,replicates and transcribes its genome through the action of an RNA-dependent RNA polymerase,which is itself comprised of numerous non-structural pro-teins(non-structural proteins,Nsps).The present study delineates the development of a detection system founded on a bicistronic reporter plasmid in conjunction with an array of Nsp plasmids,ai-ming to investigate the influence of SARS-CoV-2 Nsps on the virus's replication and expression profiles.Specifically,a bicistronic reporter gene plasmid along with twelve distinct Nsp plasmids(encompassing Nsp3C-Flag,Nsp4,Nsp6-Nsp10,Nsp12-Nsp16)were meticulously constructed via molecular cloning techniques.The successful expression of these Nsps was subsequently confirmed through Western blot analysis.Initially,the activity of the RNA-dependent RNA polymerase was assessed by co-transfection of the reporter plasmid with Nsp12,in the presence of its auxiliary fac-tors Nsp7 and Nsp8,with careful regulation of the co-transfection ratio,culturing temperature,and the timing of activity determination for the triad of Nsp plasmids.The normalized NLuc fluorescein value,in reference to the FLuc fluorescein value of the housekeeping gene,served as a metric for determining the polymerase activity.Building upon this foundation,the co-transfection concentra-tions of Nsp9-16 were fine-tuned,followed by the incremental addition of varying doses of Nsp3C,Nsp4,and Nsp6,to further elucidate the activity of RNA-dependent RNA polymerase(RdRp).The findings indicated that upon transfection with varying ratios of Nsp7,Nsp8,and Nsp12 at a propor-tion of 1∶8∶24,the polymerase activity was markedly elevated compared to the control group,with a statistical significance level(P＜0.001).Furthermore,in the absence of Nsp3,Nsp4,and Nsp6,the inclusion of Nsp10-16 substantially augmented the activity of the RdRp,particularly in scenarios where Nsp9 was not introduced,achieving statistical significance(P＜0.001).In the pres-ence of Nsp3 and Nsp4,the RdRp activity was augmented further upon the addition of Nsp9,reac-hing a level of significance(P＜0.05).The data imply that Nsp9 is capable of enhancing the RdRp activity of SARS-CoV-2 exclusively in the context of Nsp3 and Nsp4 coexistence,suggesting that the stimulatory influence of Nsp9 on viral replication may be contingent upon the formation of double-membrane vesicles.
