Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1
Model of OSBP-Mediated Cholesterol Supply to Aichi Virus RNA Replication Sites Involving Protein-Protein Interactions among Viral Proteins, ACBD3, OSBP, VAP-A/B, and SAC1
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DOI：
10.1128/jvi.01952-17
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发表时间：
2018-04-01
影响因子：
5.4
通讯作者：
Sasaki, Jun
中科院分区：
文献类型：
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作者：
Ishikawa-Sasaki, Kumiko;Nagashima, Shigeo;Sasaki, Jun
正链RNA病毒，包括小核糖核酸病毒，利用细胞机制进行基因组复制。以前，我们报道了爱知病毒（AiV）的2B，2BC，2C，3A和3AB蛋白中的每一个，在病毒RNA复制位点（复制细胞器[RO]）与高尔基体蛋白ACBD 3和磷脂酰肌醇4-激酶III β（PI 4KB）形成复合物，增强PI 4KB依赖性磷脂酰肌醇4-磷酸（PI 4P）的产生。在这里，我们证明了AIV劫持的细胞胆固醇转运系统，涉及氧固醇结合蛋白（OSBP），一个PI 4P结合胆固醇转移蛋白。AiV RNA复制通过沉默已知是该途径组分的细胞蛋白质、OSBP、ER膜蛋白VAPA和VAP B（VAP-A/B）、PI 4 P-磷酸酶SAC 1和PI转移蛋白β来抑制。OSBP、VAP-A/B和SAC 1存在于RNA复制位点。我们还发现了AiV蛋白（2B、2BC、2C、3A和3AB）、ACBD 3、OSBP、VAP-A/B和SAC 1之间的各种先前未知的相互作用，并且这些相互作用被认为参与了将组分蛋白募集到AiV RO中。重要的是，OSBP-2B相互作用使得OSBP能够不依赖于PI 4P地募集到AiV RO，表明OSBP在PI 4P结合蛋白中的优先募集。其他小核糖核酸病毒尚未报道基于蛋白质-蛋白质相互作用的OSBP募集。胆固醇在AiV RO处积累，并且抑制OSBP介导的胆固醇转移损害胆固醇积累和AiV RNA复制。电子显微镜观察显示，AIV诱导的囊泡样结构靠近ER膜。总而言之，我们得出结论，AiV直接招募胆固醇运输机械通过蛋白质-蛋白质相互作用，导致在ER和AiV RO和胆固醇供应ROs.Importance之间的膜接触位点的形成正链RNA病毒利用宿主途径来调节复制的病毒RNA复制位点的脂质组成。以前，我们证明了爱知病毒（AiV），一种小核糖核酸病毒，形成一个复合物，包括某些蛋白质的AiV，高尔基体蛋白ACBD 3，和脂质激酶PI 4KB合成PI 4P脂质在网站AiV RNA复制。在这里，我们证实了胆固醇在AiV RNA复制位点的积累，这是通过劫持由PI 4P结合胆固醇转移蛋白OSBP介导的宿主胆固醇转移机制建立的。我们发现，OSBP，VAP，SAC 1和PITPNB的机器的组成蛋白，都是AiV复制的必要宿主因子。重要的是，该机制通过VAP/OSBP/SAC 1与AiV蛋白和ACBD 3的先前未知的相互作用直接募集到RNA复制位点。因此，我们提出了一种特定的策略，采用AiV通过蛋白质-蛋白质相互作用在RNA复制位点有效地积累胆固醇。
Positive-strand RNA viruses, including picornaviruses, utilize cellular machinery for genome replication. Previously, we reported that each of the 2B, 2BC, 2C, 3A, and 3AB proteins of Aichi virus (AiV), a picornavirus, forms a complex with the Golgi apparatus protein ACBD3 and phosphatidylinositol 4-kinase III beta (PI4KB) at viral RNA replication sites (replication organelles [ROs]), enhancing PI4KB-dependent phosphatidylinositol 4-phosphate (PI4P) production. Here, we demonstrate AiV hijacking of the cellular cholesterol transport system involving oxysterol-binding protein (OSBP), a PI4P-binding cholesterol transfer protein. AiV RNA replication was inhibited by silencing cellular proteins known to be components of this pathway, OSBP, the ER membrane proteins VAPA and VAPB (VAP-A/B), the PI4P-phosphatase SAC1, and PItransfer protein beta. OSBP, VAP-A/B, and SAC1 were present at RNA replication sites. We also found various previously unknown interactions among the AiV proteins (2B, 2BC, 2C, 3A, and 3AB), ACBD3, OSBP, VAP-A/B, and SAC1, and the interactions were suggested to be involved in recruiting the component proteins to AiV ROs. Importantly, the OSBP-2B interaction enabled PI4P-independent recruitment of OSBP to AiV ROs, indicating preferential recruitment of OSBP among PI4P-binding proteins. Protein-protein interaction-based OSBP recruitment has not been reported for other picornaviruses. Cholesterol was accumulated at AiV ROs, and inhibition of OSBP-mediated cholesterol transfer impaired cholesterol accumulation and AiV RNA replication. Electron microscopy showed that AiV-induced vesicle-like structures were close to ER membranes. Altogether, we conclude that AiV directly recruits the cholesterol transport machinery through protein-protein interactions, resulting in formation of membrane contact sites between the ER and AiV ROs and cholesterol supply to the ROs.IMPORTANCE Positive-strand RNA viruses utilize host pathways to modulate the lipid composition of viral RNA replication sites for replication. Previously, we demonstrated that Aichi virus (AiV), a picornavirus, forms a complex comprising certain proteins of AiV, the Golgi apparatus protein ACBD3, and the lipid kinase PI4KB to synthesize PI4P lipid at the sites for AiV RNA replication. Here, we confirmed cholesterol accumulation at the AiV RNA replication sites, which are established by hijacking the host cholesterol transfer machinery mediated by a PI4P-binding cholesterol transfer protein, OSBP. We showed that the component proteins of the machinery, OSBP, VAP, SAC1, and PITPNB, are all essential host factors for AiV replication. Importantly, the machinery is directly recruited to the RNA replication sites through previously unknown interactions of VAP/OSBP/SAC1 with the AiV proteins and with ACBD3. Consequently, we propose a specific strategy employed by AiV to efficiently accumulate cholesterol at the RNA replication sites via protein-protein interactions.