Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity
Mutations That Hamper Dimerization of Foot-and-Mouth Disease Virus 3A Protein Are Detrimental for Infectivity
复制标题
DOI：
10.1128/jvi.00580-12
复制
发表时间：
2012-10-01
影响因子：
5.4
通讯作者：
Sobrino, Francisco
中科院分区：
文献类型：
--
作者：
Gonzalez-Magaldi, Monica;Postigo, Raul;Sobrino, Francisco
口蹄疫病毒（FMDV）非结构蛋白3A在病毒复制、毒力和宿主范围中起重要作用。在其他小核糖核酸病毒中，3A的同二聚化已被证明与其生物活性有关。本研究通过原位蛋白荧光连接实验证实了FMDV 3A二聚体的存在。根据脊髓灰质炎病毒3A蛋白的核磁共振（NMR）结构建立的FMDV 3A蛋白分子模型预测，跨越残基25至44的疏水界面是3A二聚化的主要决定因素。替换L38E和L41E，涉及到预计有助于疏水界面的残基上的电荷获取，减少了蛋白质连接实验中的二聚体信号，并且在瞬时表达的3A蛋白和复制3A N端的合成肽中阻止了二聚体/多聚体的检测。这些替换也导致感染性病毒的产生，这些病毒取代了由非极性氨基酸引入的酸性残基(E)，这表明疏水界面的保存对病毒复制至关重要。替换有利于（Q44R）或损害（Q44D）残基Q44和D32之间预测的极性相互作用并没有消除瞬时表达3A二聚体的形成，这表明这些相互作用对3A二聚体的形成并不重要。然而，Q44R导致了保持突变的病毒的恢复，而Q44D导致了替代带有酸性电荷但结构特征与亲本病毒相似的D44E的感染病毒的选择，这表明Q44参与了3A二聚化以外的功能。
Foot-and-mouth disease virus (FMDV) nonstructural protein 3A plays important roles in virus replication, virulence, and host range. In other picornaviruses, homodimerization of 3A has been shown to be relevant for its biological activity. In this work, FMDV 3A homodimerization was evidenced by an in situ protein fluorescent ligation assay. A molecular model of the FMDV 3A protein, derived from the nuclear magnetic resonance (NMR) structure of the poliovirus 3A protein, predicted a hydrophobic interface spanning residues 25 to 44 as the main determinant for 3A dimerization. Replacements L38E and L41E, involving charge acquisition at residues predicted to contribute to the hydrophobic interface, reduced the dimerization signal in the protein ligation assay and prevented the detection of dimer/multimer species in both transiently expressed 3A proteins and in synthetic peptides reproducing the N terminus of 3A. These replacements also led to production of infective viruses that replaced the acidic residues introduced (E) by nonpolar amino acids, indicating that preservation of the hydrophobic interface is essential for virus replication. Replacements that favored (Q44R) or impaired (Q44D) the polar interactions predicted between residues Q44 and D32 did not abolish dimer formation of transiently expressed 3A, indicating that these interactions are not critical for 3A dimerization. Nevertheless, while Q44R led to recovery of viruses that maintained the mutation, Q44D resulted in selection of infective viruses with substitution D44E with acidic charge but with structural features similar to those of the parental virus, suggesting that Q44 is involved in functions other than 3A dimerization.