Allosteric regulation of Senecavirus A 3Cpro proteolytic activity by an endogenous phospholipid.
Allosteric regulation of Senecavirus A 3Cpro proteolytic activity by an endogenous phospholipid.
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DOI：
10.1371/journal.ppat.1011411
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发表时间：
2023-05
期刊：
影响因子：
6.7
通讯作者：
中科院分区：
文献类型：
--
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塞内卡病毒A（SVA）是一种新出现的小核糖核酸病毒，最近已被确定为多个国家的许多猪水泡病病例的病原体。除了病毒多聚蛋白的切割之外，病毒3C蛋白酶（3Cpro）通过切割关键的细胞蛋白在参与细胞抗病毒应答的几个生理过程的调节中起重要作用。通过晶体学、非靶向脂质组学和免疫印迹的组合，我们鉴定了SVA 3Cpro与内源性磷脂分子的结合，所述内源性磷脂分子结合至邻近SVA 3Cpro的蛋白水解位点的独特区域。我们的脂质结合试验表明，SVA 3Cpro显示优先结合心磷脂（CL），其次是磷酸肌醇-4-磷酸（PI 4P）和硫苷脂。重要的是，我们发现，SVA 3Cpro的蛋白水解活性在磷脂的存在下被激活，并且当磷脂结合能力降低时，酶活性被抑制。有趣的是，在野生型SVA 3Cpro-底物肽结构中，切割残基不能与催化性半胱氨酸残基形成共价结合，从而形成在几种小核糖核酸病毒3Cpro结构中观察到的酰基酶中间体。我们观察到SVA突变体的感染性滴度降低，这些突变损害了3Cpro的脂质结合能力，表明磷脂介导的SVA感染能力的正调控。我们的研究结果揭示了SVA 3Cpro的蛋白水解活性和磷脂结合能力之间的相互调节，表明内源性磷脂可能作为一种变构激活剂，在感染过程中调节酶的蛋白水解活性。塞内卡病毒A（Seneca virus A，SVA）是一种新型的小核糖核酸病毒，近年来在一些国家爆发，对养猪业造成了极大的威胁。蛋白酶3Cpro负责切割病毒多聚蛋白，并且它还可以切割几种宿主蛋白，使得SVA能够逃避抗病毒先天免疫应答。我们通过晶体学、非靶向脂质组学和免疫印迹相结合，鉴定了一种与SVA 3Cpro中一个独特区域结合的内源性磷脂分子。优选的磷脂类型包括心磷脂、磷酸肌醇-4-磷酸和硫苷脂。我们发现当磷脂结合能力降低时，蛋白酶活性会受到显著抑制。同时，观察到含有损害3Cpro的脂质结合能力的突变的SVA突变体的感染性滴度降低。我们的研究结果表明，磷脂可能作为一种变构激活剂，调节与病毒复制和感染相关的SVA 3Cpro蛋白水解活性。
Seneca virus A (SVA) is an emerging novel picornavirus that has recently been identified as the causative agent of many cases of porcine vesicular diseases in multiple countries. In addition to cleavage of viral polyprotein, the viral 3C protease (3Cpro) plays an important role in the regulation of several physiological processes involved in cellular antiviral responses by cleaving critical cellular proteins. Through a combination of crystallography, untargeted lipidomics, and immunoblotting, we identified the association of SVA 3Cpro with an endogenous phospholipid molecule, which binds to a unique region neighboring the proteolytic site of SVA 3Cpro. Our lipid-binding assays showed that SVA 3Cpro displayed preferred binding to cardiolipin (CL), followed by phosphoinositol-4-phosphate (PI4P) and sulfatide. Importantly, we found that the proteolytic activity of SVA 3Cpro was activated in the presence of the phospholipid, and the enzymatic activity is inhibited when the phospholipid-binding capacity decreased. Interestingly, in the wild-type SVA 3Cpro-substrate peptide structure, the cleavage residue cannot form a covalent binding to the catalytic cysteine residue to form the acyl-enzyme intermediate observed in several picornaviral 3Cpro structures. We observed a decrease in infectivity titers of SVA mutants harboring mutations that impaired the lipid-binding ability of 3Cpro, indicating a positive regulation of SVA infection capacity mediated by phospholipids. Our findings reveal a mutual regulation between the proteolytic activity and phospholipid-binding capacity in SVA 3Cpro, suggesting that endogenous phospholipid may function as an allosteric activator that regulate the enzyme’s proteolytic activity during infection. Seneca virus A (SVA) is a novel picornavirus and there have been several recent outbreaks that caused a great threat to the swine industry in some countries. The protease 3Cpro is responsible for the cleavage of viral polyprotein and it can also cleave several host proteins enabling SVA to escape antiviral innate immune responses. We identified an endogenous phospholipid molecule that binds to a unique region in SVA 3Cpro by a combination of crystallography, untargeted lipidomics, and immunoblotting. The preferred phospholipid types include cardiolipin, phosphoinositol-4-phosphate and sulfatide. We found the protease activity can be significantly inhibited when the phospholipid-binding capacity decreases. Meanwhile, a decrease in infectivity titers of SVA mutants harboring mutations that impair the lipid-binding ability of 3Cpro was observed. Our findings suggest the phospholipid may function as an allosteric activator to regulate SVA 3Cpro proteolytic activity associated with the viral replication and infection.
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影响因子：
32.4
作者：
Iyer SS;He Q;Janczy JR;Elliott EI;Zhong Z;Olivier AK;Sadler JJ;Knepper-Adrian V;Han R;Qiao L;Eisenbarth SC;Nauseef WM;Cassel SL;Sutterwala FS
通讯作者：
Sutterwala FS
高尔基体蛋白 ACBD3 通过与 3A 相互作用促进肠道病毒 71 复制。
影响因子：
4.6
作者：
Lei X;Xiao X;Zhang Z;Ma Y;Qi J;Wu C;Xiao Y;Zhou Z;He B;Wang J
通讯作者：
Wang J
影响因子：
64.8
作者：
ALLAIRE, M;CHERNAIA, MM;JAMES, MNG
通讯作者：
JAMES, MNG
DOI：
10.1107/s0907444904019158
发表时间：
2004-12-01
影响因子：
2.2
作者：
Emsley, P;Cowtan, K
通讯作者：
Cowtan, K
影响因子：
4.3
作者：
Leme, R. A.;Zotti, E.;Alfieri, A. A.
通讯作者：
Alfieri, A. A.