Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication
Mapping of Functional Domains of the Lipid Kinase Phosphatidylinositol 4-Kinase Type III Alpha Involved in Enzymatic Activity and Hepatitis C Virus Replication
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DOI：
10.1128/jvi.01063-14
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发表时间：
2014-09-01
影响因子：
5.4
通讯作者：
Lohmann, Volker
中科院分区：
文献类型：
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作者：
Harak, Christian;Radujkovic, Danijela;Lohmann, Volker
磷脂酰肌醇4-激酶III α (PI4KIII α)是一种内质网（ER）驻留酶，可合成磷脂酰肌醇4-磷酸（PI4P）。PI4KIII α是丙型肝炎病毒（HCV）复制的重要宿主因子。与HCV非结构蛋白5A （NS5A）的相互作用导致PI4P在细胞膜内的激酶激活和积累。在这项研究中，我们研究了PI4KIII α在HCV复制和酶活性中的结构要求。因此，我们分析了PI4KIII α突变体的亚细胞定位、PI4KIII α敲低细胞系中HCV复制的重建、HCV阳性细胞中PI4P的诱导以及体外脂质激酶活性。所有突变体仍然与NS5A相互作用并以与全长酶相似的方式定位，这表明PI4KIII α的多个区域参与了NS5A相互作用和亚细胞定位。有趣的是，n端1152个氨基酸对于HCV复制、PI4P诱导和酶功能是必不可少的，而进一步的n端或c端缺失是有害的，从而定义了最小的PI4KIII α核心酶，大小约为108 kDa。在PI4KIII α的c端半段内，除了提出的核定位信号外，预测的功能基序的额外缺失也对酶活性和PI4KIII α挽救HCV复制的能力有害，这表明PI4KIII α的整个c端半段参与了最小酶核的形成。PI4KIII α - C末端的结构模型支持了这一观点，表明催化中心由N-和C-末端叶和犰狳褶皱基序组成，其前面有三个不同的α -螺旋结构域可能参与酶活性的调节。脂质激酶PI4KIII α对细胞磷脂酰肌醇代谢至关重要，是丙型肝炎病毒复制的关键宿主细胞因子。然而，到目前为止，人们对这种240-kDa蛋白的结构以及特定亚结构域在脂质激酶活性和病毒复制方面的功能重要性知之甚少。这项工作的重点是不同的PI4KIII α突变体的表型分析在不同的生化和细胞为基础的分析，并建立了c端酶核的结构模型。这些结果揭示了酶活性的结构和功能要求以及HCV复制所需的决定因素。
The lipid kinase phosphatidylinositol 4-kinase III alpha (PI4KIII alpha) is an endoplasmic reticulum (ER)-resident enzyme that synthesizes phosphatidylinositol 4-phosphate (PI4P). PI4KIII alpha is an essential host factor for hepatitis C virus (HCV) replication. Interaction with HCV nonstructural protein 5A (NS5A) leads to kinase activation and accumulation of PI4P at intracellular membranes. In this study, we investigated the structural requirements of PI4KIII alpha in HCV replication and enzymatic activity. Therefore, we analyzed PI4KIII alpha mutants for subcellular localization, reconstitution of HCV replication in PI4KIII alpha knockdown cell lines, PI4P induction in HCV-positive cells, and lipid kinase activity in vitro. All mutants still interacted with NS5A and localized in a manner similar to that of the full-length enzyme, suggesting multiple regions of PI4KIII alpha are involved in NS5A interaction and subcellular localization. Interestingly, the N-terminal 1,152 amino acids were dispensable for HCV replication, PI4P induction, and enzymatic function, whereas further N-terminal or C-terminal deletions were deleterious, thereby defining the minimal PI4KIII alpha core enzyme at a size of ca. 108 kDa. Additional deletion of predicted functional motifs within the C-terminal half of PI4KIII alpha also were detrimental for enzymatic activity and for the ability of PI4KIII alpha to rescue HCV replication, with the exception of a proposed nuclear localization signal, suggesting that the entire C-terminal half of PI4KIII alpha is involved in the formation of a minimal enzymatic core. This view was supported by structural modeling of the PI4KIII alpha C terminus, suggesting a catalytic center formed by an N- and C-terminal lobe and an armadillo-fold motif, which is preceded by three distinct alpha-helical domains probably involved in regulation of enzymatic activity.IMPORTANCEThe lipid kinase PI4KIII alpha is of central importance for cellular phosphatidylinositol metabolism and is a key host cell factor of hepatitis C virus replication. However, little is known so far about the structure of this 240-kDa protein and the functional importance of specific subdomains regarding lipid kinase activity and viral replication. This work focuses on the phenotypic analysis of distinct PI4KIII alpha mutants in different biochemical and cell-based assays and develops a structural model of the C-terminal enzymatic core. The results shed light on the structural and functional requirements of enzymatic activity and the determinants required for HCV replication.