Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.
Hepatitis B virus core protein allosteric modulators can distort and disrupt intact capsids.
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DOI：
10.7554/elife.31473
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发表时间：
2018-01-29
期刊：
影响因子：
7.7
通讯作者：
Zlotnick A
中科院分区：
文献类型：
--
作者：
Schlicksup CJ;Wang JC;Francis S;Venkatakrishnan B;Turner WW;VanNieuwenhze M;Zlotnick A
定义直接作用的抗病毒药物的机制有助于药物开发和我们对病毒功能的理解。杂芳基二氢嘧啶（HAP）不适当地激活B肝炎病毒（HBV）核心蛋白（Cp）的组装，抑制病毒体的形成。我们检测了荧光团标记的HAP，HAP-TAMRA。HAP-TAMRA诱导Cp组装并且还结合预组装的衣壳。动力学和光谱研究表明，HAP结合位点通常不可用，但结合合作。使用cryo-EM，我们观察到HAP-TAMRA不对称地变形衣壳，产生锐角、平坦区域和完全断裂的异质阵列。为了实现高分辨率重建（<4 μ m），我们引入了二硫键交联，拯救了粒子对称性。我们推断，HAP-TAMRA导致准六重顶点变得更平坦和五倍多的角度。这种转变导致了不对称刻面。无序的交联可以挽救对称性，这意味着衣壳具有张紧整体性质。衣壳扭曲和破坏是一种新的机制，通过这种机制，HAP等分子可以阻断HBV感染。病毒是由包裹在外壳内的遗传信息组成的简单结构。对于B型肝炎病毒来说，这个外壳看起来像个足球。它由240个相同蛋白质的拷贝组成，以五边形和六边形的模式排列。这些蛋白质为病毒的遗传信息形成了保护屏障：它们还在病毒生命周期的关键事件中与宿主细胞相互作用。当B型肝炎病毒感染细胞时，它会劫持细胞机器进行复制。新的壳蛋白在细胞内产生和组装。一种名为CpAM的潜在抗病毒药物会破坏这一过程：它会导致外壳过早且不准确地组装，从而损害病毒的生命周期。然而，CpAM可以结合到壳体上，即使在其已经组装之后。这种结合如何影响病毒仍不清楚。在这里，Schlicksup等人将荧光分子连接到CpAM上，并使用尖端的显微镜方法在原子水平上观察结构。这使得可以详细检查CpAM如何附着到正确形成的病毒外壳上。Schlicksup等人的研究表明，当CpAM与壳结合时，它会破坏，有时甚至打破壳的足球状图案：六边形变平，五边形弯曲。这些变形的外壳可以阻止病毒与感染所必需的细胞结构相互作用，或者阻止它释放病毒的遗传信息。这是CpAM的一种新的抗病毒机制。通过在外壳组装之前和之后起作用，CpAM在病毒生命周期的不同阶段靶向病毒。B型肝炎影响全世界超过2.4亿人。虽然有疫苗存在，但仍然没有治愈它的方法。更好地了解病毒外壳的物理特性和CpAM的作用方式可能会导致更好的药物对抗这种疾病。
Defining mechanisms of direct-acting antivirals facilitates drug development and our understanding of virus function. Heteroaryldihydropyrimidines (HAPs) inappropriately activate assembly of hepatitis B virus (HBV) core protein (Cp), suppressing formation of virions. We examined a fluorophore-labeled HAP, HAP-TAMRA. HAP-TAMRA induced Cp assembly and also bound pre-assembled capsids. Kinetic and spectroscopic studies imply that HAP-binding sites are usually not available but are bound cooperatively. Using cryo-EM, we observed that HAP-TAMRA asymmetrically deformed capsids, creating a heterogeneous array of sharp angles, flat regions, and outright breaks. To achieve high resolution reconstruction (<4 Å), we introduced a disulfide crosslink that rescued particle symmetry. We deduced that HAP-TAMRA caused quasi-sixfold vertices to become flatter and fivefold more angular. This transition led to asymmetric faceting. That a disordered crosslink could rescue symmetry implies that capsids have tensegrity properties. Capsid distortion and disruption is a new mechanism by which molecules like the HAPs can block HBV infection. Viruses are simple structures formed of genetic information wrapped inside a shell. For the hepatitis B virus, this casing looks like a soccer ball. It is composed of 240 copies of the same protein, arranged in a pattern of pentagons and hexagons. These proteins form a protective shield for the virus’ genetic information: they also interact with the cells of the host during key events of the virus’ life cycle. When the hepatitis B virus infects a cell, it hijacks the cellular machinery to replicate. New shell proteins are produced and assemble within the cell. A type of potential antiviral drug called a CpAM disrupts this process: it causes the shell to assemble too early and inaccurately, which impairs the life cycle of the virus. However, a CpAM can bind to the shell even after it has already assembled. How this binding affects the virus is still unclear. Here, Schlicksup et al. attach a fluorescent molecule to a CpAM, and use a cutting-edge microscopy method to look at the structures at the atomic level. This makes it possible to examine in detail how the CpAM attaches to a correctly formed virus shell. Schlicksup et al. show that when the CpAM binds to the shell, it disrupts and sometimes even breaks the soccer-like pattern of the shell: the hexagons flatten, and the pentagons buckle. These misshaped shells could prevent the virus from interacting with the cellular structures necessary for infection or prevent it from releasing the virus’ genetic information. This is a new antiviral mechanism for a CpAM. By acting both before and after the shell has assembled, the CpAM targets the virus at different points of its life cycle. Hepatitis B affects over 240 million people worldwide. While a vaccine exists, there is still no cure for it. A better understanding of the physics of the virus’ shell and the mode of action of CpAMs could lead to better drugs against the disease.