Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.
Metabolism of phosphatidylinositol 4-kinase IIIα-dependent PI4P Is subverted by HCV and is targeted by a 4-anilino quinazoline with antiviral activity.
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DOI：
10.1371/journal.ppat.1002576
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发表时间：
2012
期刊：
影响因子：
6.7
通讯作者：
De Francesco R
中科院分区：
文献类型：
--
作者：
Bianco A;Reghellin V;Donnici L;Fenu S;Alvarez R;Baruffa C;Peri F;Pagani M;Abrignani S;Neddermann P;De Francesco R
4-苯胺基喹唑类化合物已被确定为丙型肝炎病毒复制的抑制剂。这类化合物的靶标被认为是病毒蛋白NS5A，尽管从未提出过明确的证据。在激酶抑制剂中经常发现4-苯胺基喹唑啉部分，这导致我们提出这样的假设，即这些化合物显示的抗丙型肝炎病毒活性可能是由于抑制了一种细胞激酶。新近发现，III型磷脂酰肌醇4-激酶α(PI4KIIIα)是丙型肝炎病毒复制的宿主因子。因此，我们评估了AL-9，一种4-苯胺基喹唑啉类化合物的原型，在选定的磷脂酰肌醇激酶上。Al-9对纯化的PI4KIIIα有抑制作用，对PI4KIIIβ的抑制作用较小。在Huh7.5细胞中，PI4KIIIα负责质膜中存在的磷脂酰肌醇-4磷酸(PI4P)池。因此，我们观察到与AL-9孵育后质膜上的PI4P逐渐减少，表明该药也抑制活细胞中的PI4KIIIα。相反，AL-9不影响高尔基体膜上PI4P的水平，这表明在我们的实验条件下，PI4KIIIβ亚型没有受到明显的抑制。表达丙型肝炎病毒蛋白的细胞与AL-9孵育产生异常大的NS5A簇，这是以前通过RNAi沉默PI4KIIIα时观察到的现象。根据我们的发现，我们认为4-苯胺基喹唑啉类化合物的抗病毒作用是通过抑制PI4KIIIα，从而耗尽丙型肝炎病毒膜网络所需的PI4P来实现的。此外，我们注意到，丙型肝炎病毒对细胞内PI4P的分布有深远的影响，导致PI4P在丙型肝炎病毒膜网络中显著丰富，并伴随着质膜中PI4P的减少。这一观察表明，丙型肝炎病毒通过在RNA复制复合体中招募PI4KIIIα来劫持PI4P的新陈代谢，最终导致PI4KIIIα产物的亚细胞分布显著改变。据估计，全球有3%的人口长期感染丙型肝炎病毒(HCV)。大多数感染成为慢性感染，最终演变为肝硬变和肝细胞癌。宿主因子是抗丙型肝炎病毒治疗的有趣靶点，因为它们固有的高耐药性遗传屏障。近年来，磷脂酰肌醇4-激酶α(PI4KIIIα)被认为是丙型肝炎病毒复制的重要宿主因子。许多不同的病原体，包括丙型肝炎病毒，颠覆了磷脂酰肌醇-4磷酸(PI4P)途径的组成部分，使其发挥有利于自身生命周期的作用。在本文中，我们发现，丙型肝炎病毒显著改变了细胞内PI4P的代谢和分布，导致病毒复制所需的膜网中PI4P的丰富，同时质膜中PI4P的减少。此外，我们证明了先前被认为针对HCVNS5A的抗病毒药物4-苯胺基喹唑啉确实通过抑制PI4KIIIα来抑制PI4P的形成。这类化合物为基于PI4KIIIα抑制的新型抗病毒疗法的开发提供了一个有前景的先导。特定的PI4KIIIα抑制剂也将是深入了解PI4P的功能和调节所需的重要研究工具。
4-anilino quinazolines have been identified as inhibitors of HCV replication. The target of this class of compounds was proposed to be the viral protein NS5A, although unequivocal proof has never been presented. A 4-anilino quinazoline moiety is often found in kinase inhibitors, leading us to formulate the hypothesis that the anti-HCV activity displayed by these compounds might be due to inhibition of a cellular kinase. Type III phosphatidylinositol 4-kinase α (PI4KIIIα) has recently been identified as a host factor for HCV replication. We therefore evaluated AL-9, a compound prototypical of the 4-anilino quinazoline class, on selected phosphatidylinositol kinases. AL-9 inhibited purified PI4KIIIα and, to a lesser extent, PI4KIIIβ. In Huh7.5 cells, PI4KIIIα is responsible for the phosphatidylinositol-4 phosphate (PI4P) pool present in the plasma membrane. Accordingly, we observed a gradual decrease of PI4P in the plasma membrane upon incubation with AL-9, indicating that this agent inhibits PI4KIIIα also in living cells. Conversely, AL-9 did not affect the level of PI4P in the Golgi membrane, suggesting that the PI4KIIIβ isoform was not significantly inhibited under our experimental conditions. Incubation of cells expressing HCV proteins with AL-9 induced abnormally large clusters of NS5A, a phenomenon previously observed upon silencing PI4KIIIα by RNA interference. In light of our findings, we propose that the antiviral effect of 4-anilino quinazoline compounds is mediated by the inhibition of PI4KIIIα and the consequent depletion of PI4P required for the HCV membranous web. In addition, we noted that HCV has a profound effect on cellular PI4P distribution, causing significant enrichment of PI4P in the HCV-membranous web and a concomitant depletion of PI4P in the plasma membrane. This observation implies that HCV – by recruiting PI4KIIIα in the RNA replication complex – hijacks PI4P metabolism, ultimately resulting in a markedly altered subcellular distribution of the PI4KIIIα product. It is estimated that 3% of the world's population are chronically infected by the hepatitis C virus (HCV). Most infections become chronic and eventually evolve into cirrhosis and hepatocellular carcinoma. Host factors are interesting targets for anti-HCV therapies due to their inherent high genetic barrier to resistance. Recently, phosphatidylinositol 4-kinase α (PI4KIIIα) has been identified as a crucial host factor for HCV replication. Many different pathogens, including HCV, subvert components of the phosphatidylinositol-4 phosphate (PI4P) pathway to function in favor of their own life cycle. In this paper, we show that HCV dramatically alters cellular PI4P metabolism and distribution, resulting in the enrichment of PI4P in the membranous web required for viral replication with a concomitant decrease of PI4P in the plasma-membrane. Moreover, we demonstrate that 4-anilino quinazolines, antiviral agents previously believed to target HCV NS5A, do in fact inhibit PI4P formation by inhibition of PI4KIIIα. This compound class is a promising lead for the development of a novel antiviral therapy based on PI4KIIIα inhibition. Specific PI4KIIIα inhibitors would also be important research tools required for a deeper understanding of the functions and regulation of PI4P.
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影响因子：
5.4
作者：
Amako, Yutaka;Sarkeshik, Ali;Siddiqui, Aleem
通讯作者：
Siddiqui, Aleem
DOI：
10.1042/bj20090428
发表时间：
2009-07-29
期刊：
The Biochemical journal
影响因子：
--
作者：
Hammond GR;Schiavo G;Irvine RF
通讯作者：
Irvine RF
影响因子：
2.9
作者：
Liang, Yu;Ye, Hong;Yoon, Ho Sup
通讯作者：
Yoon, Ho Sup
影响因子：
64.5
作者：
Knight, Zachary A.;Gonzalez, Beatriz;Shokat, Kevan M.
通讯作者：
Shokat, Kevan M.
DOI：
10.1073/pnas.0902693106
发表时间：
2009-05-05
影响因子：
11.1
作者：
Berger, Kristi L.;Cooper, Jacob D.;Randall, Glenn
通讯作者：
Randall, Glenn