Zhou Saisai, Liu Nankun, Tian Yang, Pan Hong, Han Yang, Li Zhen, Zhang Jinhua, Guan Shuaiyin, Chen Huanchun, Song Yunfeng
State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan, China.
College of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China.
Heliyon. 2024 Jul 30;10(15):e35449. doi: 10.1016/j.heliyon.2024.e35449. eCollection 2024 Aug 15.
Foot-and-mouth disease virus (FMDV) 2C protein is a conserved non-structural protein and crucial for replication of the virus. In this study, FMDV 2C protein was prepared and the enzymatic activities were investigated in detail. The protein could digest ssDNA or ssRNA into a small fragment at about 10 nt, indicating that the protein has nuclease activity. But it did not show digestion to blunt-end dsDNA or dsRNA. The nuclease activity of 2C protein could be inhibited in 2 mM Zn or Ca while enhanced by Mg or Mn. FMDV 2C protein exhibited unwinding activity to all the three kinds of dsDNA and dsRNA (5' protruded, 3' protruded, and blunt-end). The unwinding velocity to 5' protruded dsRNA was higher than to the blunt-end dsRNA. 2C protein only showed unwinding activity in high concentration of Mg, but no unwinding activity in physiological concentrations of Mg and Ca, as well as in cell lysate. The 2C protein could catalyze two structured ssRNA to form double strand, thus it was proved to have RNA chaperone activity. The Mg and ATP in different concentrations did not show promotion to the RNA chaperone activity. Finally, six mutant proteins (K116A, D160A, D170A, N207A, R226A, and F316A) were constructed and the enzymatic activities were analyzed. All the six mutations reduced the ATPase activity, D170A and F361A could inactivate the nuclease activity, while the N207A and F316A could inactivate the helicase activity. Our study provides a comprehensive understanding of the enzymatic activities of FMDV 2C protein.
口蹄疫病毒（FMDV）2C蛋白是一种保守的非结构蛋白，对病毒复制至关重要。在本研究中，制备了FMDV 2C蛋白并详细研究了其酶活性。该蛋白可将单链DNA或单链RNA消化成约10个核苷酸的小片段，表明该蛋白具有核酸酶活性。但它对平端双链DNA或双链RNA没有消化作用。2C蛋白的核酸酶活性在2 mM锌或钙中可被抑制，而在镁或锰中增强。FMDV 2C蛋白对三种双链DNA和双链RNA（5'突出端、3'突出端和平端）均表现出解旋活性。对5'突出端双链RNA的解旋速度高于平端双链RNA。2C蛋白仅在高浓度镁中表现出解旋活性，而在生理浓度的镁和钙以及细胞裂解物中没有解旋活性。2C蛋白可催化两条结构化单链RNA形成双链，因此被证明具有RNA伴侣活性。不同浓度的镁和ATP对RNA伴侣活性没有促进作用。最后，构建了六个突变蛋白（K116A、D160A、D170A、N207A、R226A和F316A）并分析了其酶活性。所有六个突变均降低了ATP酶活性，D170A和F361A可使核酸酶活性失活，而N207A和F316A可使解旋酶活性失活。我们的研究提供了对口蹄疫病毒2C蛋白酶活性的全面理解。
