The greasy finger region of DTMUV NS1 plays an essential role in NS1 secretion and viral proliferation

Hantai Tan; Senzhao Zhang; Zhen Wu; Yu He; Tao Wang; Wangyang Tan; Xuedan Tang; Wei Li; Mingshu Wang; Renyong Jia; Dekang Zhu; Mafeng Liu; Xinxin Zhao; Qiao Yang; Ying Wu; Shaqiu Zhang; Juan Huang; Xumin Ou; Di Sun; Bin Tian; Anchun Cheng; Shun Chen

doi:10.1016/j.psj.2024.104322

The greasy finger region of DTMUV NS1 plays an essential role in NS1 secretion and viral proliferation

Poult Sci. 2024 Sep 12;103(12):104322. doi: 10.1016/j.psj.2024.104322. Online ahead of print.

Authors

Hantai Tan¹, Senzhao Zhang¹, Zhen Wu², Yu He², Tao Wang², Wangyang Tan¹, Xuedan Tang¹, Wei Li¹, Mingshu Wang², Renyong Jia², Dekang Zhu², Mafeng Liu², Xinxin Zhao², Qiao Yang², Ying Wu², Shaqiu Zhang², Juan Huang², Xumin Ou³, Di Sun², Bin Tian², Anchun Cheng², Shun Chen⁴

Affiliations

¹ Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China.
² Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China, Chengdu 611130, China.
³ Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China, Chengdu 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education of the People's Republic of China, Chengdu 611130, China.
⁴ Research Center of Avian Disease, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Key Laboratory of Animal Disease and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu, Sichuan 611130, China; Engineering Research Center of Southwest Animal Disease Prevention and Control Technology, Ministry of Education of the People's Republic of China, Chengdu 611130, China; Key Laboratory of Agricultural Bioinformatics, Ministry of Education of the People's Republic of China, Chengdu 611130, China. Electronic address: shunchen@sicau.edu.cn.

PMID: 39316982
DOI: 10.1016/j.psj.2024.104322

Abstract

Duck Tembusu virus (DTMUV) of the Orthoflavivirus genus poses a significant threat to waterfowl aquaculture. Nonstructural protein 1 (NS1), a multifunctional glycoprotein, exists in various oligomeric forms and performs diverse functions. The greasy finger (GF) region within NS1 of other flaviviruses has been shown to be a crucial component of the hydrophobic protrusion aiding in anchoring NS1 to the endoplasmic reticulum (ER). However, detailed studies on the role of the GF region in viral proliferation in vitro and the biological properties of NS1 remain scarce. A series of recombinant DTMUV (rDTMUV) with mutations in the GF region, including NS1-F158A, G159A, F160A, G161A, V162A, L163A, F160R, multipoint mutations (GF-4M), or regional deletions (ΔGF), were rescued using a DNA-based reverse genetics system. Only 5 rDTMUV variants (G159A, F160A, G161A, V162A, and L163A) could be rescued successfully, and these mutations were found to impair replication, reduce virulence, and decrease plaque size, as shown by growth kinetics, duck embryo virulence, and plaque assays, respectively. Upon examining NS1 expression by western blot, we discovered that secreted NS1 (sNS1) presented in large quantities in the supernatant of cells infected with rDTMUV-NS1-G159A, whereas intracellular NS1 was less abundant. These mutations also impacted the primary forms and secretion rates of NS1 in cases of overexpression by western blot and indirect ELISA. Exception for F160A and G161A, which showed decreased secretion rates, all other mutations increased sNS1 expression, with the most pronounced increase observed in F158A and ΔGF, and rDTMUV with these mutations can't be rescued. Co-localization studies of NS1 with the ER demonstrated that the ΔGF mutation attenuated NS1 anchoring to the ER, thereby inhibiting its intracellular residence and promoting secretion. Although these effects vary between flaviviruses, our data reveal that the GF region of NS1 is crucial for viral proliferation and NS1 secretion.

Keywords: Duck Tembusu virus; NS1 secretion; greasy finger; nonstructural protein 1; viral proliferation.