Mechanism of cellular recognition and entry by a circovirus

描述（由申请人提供）：人类和猪之间共有的免疫学、生理学和遗传学使猪病原体对人类健康构成迫在眉睫的威胁。猪圆环病毒2型（PCV 2）是一种全球分布的无包膜免疫抑制病毒，可感染并裂解培养中的人类细胞。PCV 2几乎感染宿主的每一个组织，因此对从猪到人的异种移植是一种威慑。从猪到人的异种移植正在成为解决人类供体组织和器官短缺的一种实用方法。PCV 2在其环境中快速突变以茁壮成长的能力最近被对商业PCV 2疫苗具有抗性的新菌株所证明。因此，了解PCV 2感染机制是当务之急的原因有很多。该项目的目标是了解PCV 2实现几乎无差别的细胞识别和进入的详细分子机制。PCV 2是最小的自主复制病毒，因此可以定义病毒的限制。这种病毒识别和进入如此多的细胞类型的能力是令人惊讶的，因为无包膜动物病毒表现出它们可以相互作用并进入的集中范围的细胞类型。这种限制似乎取决于病毒衣壳可以与之相互作用的细胞表面上的受体和/或共受体的存在。该项目意义重大，因为它提供了了解自主复制病毒所需的最小组件以及导致广泛和几乎不加选择的细胞识别和进入的衣壳决定因素的机会。研究中的三个独立但重叠的目标是：1。定义PCV 2识别并与其受体相互作用的机制。使用我们的PCV 2晶体结构，我们假设实验鉴定的硫酸乙酰肝素和硫酸软骨素B受体结合在哪里。我们将使用生物化学、生物物理学和结构生物学技术相结合的方法来检验这一假设。2.定义PCV 2逃逸内体的机制。已显示PCV 2使用网格蛋白依赖性和非依赖性内吞作用内化到不同的细胞类型中。我们假设PCV 2衣壳蛋白的富含精氨酸的N-末端具有膜去稳定活性，并被PCV 2用于逃离内体并进入细胞。富含精氨酸的多肽已显示具有这种活性。我们将结合生物化学和生物物理学研究来验证这一假设。3.研究PCV 2进入细胞的机制。分别在目的1和2中，将研究产生的抑制受体识别和内体逃逸的突变体感染通过网格蛋白依赖性和非依赖性内吞作用内化PCV 2的细胞类型的能力。这些研究将使我们能够通过细胞透镜重新制定我们的假设，以描述PCV 2识别和进入如此多不同细胞类型的机制。这些实验还将使我们能够研究PCV 2是否使用单一或多种机制进入不同的细胞类型。

DESCRIPTION (provided by applicant): The immunology, physiology, and genetics shared between humans and swine make swine pathogens an impending threat to human health. Porcine circovirus 2 (PCV2) is a globally distributed non-enveloped immunosuppressive virus demonstrated to infect and lyse human cells in culture. PCV2 infects nearly every tissue in its host and is thus a deterrent to xenotransplantation from swine to humans. Xenotransplantation from swine to human is becoming a practiced solution to the shortage of tissue and organs from human donors. PCV2's ability to rapidly mutate to thrive in its environment was recently demonstrated by emergent strains resistant to commercial PCV2 vaccines. Thus there are a number of reasons why understanding the PCV2 infection mechanism is of urgent priority. The goal of this project is to understand the detailed molecular mechanism by which PCV2 achieves nearly indiscriminate cellular recognition and entry. PCV2 is the smallest autonomously replicating virus, and may therefore define a limit for viruses. The ability of this virus to recognize and enter so many cell types is surprising as non-enveloped animal viruses exhibit a focused range of cell types that they can interact with and enter. This limitation appears to be dependent on the presence of a receptor and/or co-receptor on the cellular surface that the virus capsid can interact with. The project is significant as it provides the opportunity to understand the minimal components necessary to be an autonomously replicating virus, and the capsid determinants that result in broad and nearly indiscriminate cellular recognition and entry. The three independent yet overlapping aims under study are: 1. Define the mechanism by which PCV2 recognizes and interacts with its receptor. Using our crystal structure of PCV2 we hypothesize where the experimentally identified heparan sulfate and chondroitin sulfate B receptors bind. We will test this hypothesis using a combination of biochemical, biophysical and structural biology techniques. 2. Define the mechanism by which PCV2 escapes the endosome. PCV2 has been shown to internalize into different cell types using clathrin-dependent and independent endocytosis. We hypothesize that the arginine rich N-terminus of the PCV2 capsid protein possesses membrane destabilizing activity and used by PCV2 to escape the endosome and enter the cell. Arginine rich polypeptides have been shown to possess such activity. We will use a combination of biochemical and biophysical studies to test this hypothesis. 3. Study the mechanism by which PCV2 enters the cell. The mutants generated to inhibit receptor recognition and endosome escape, in aims 1 and 2 respectively, will be studied for their ability to infect cell types that internalize PCV2 via clathrin-dependent and independent endocytosis. These studies will allow us to reformulate our hypotheses through a cellular lens to describe the mechanism by which PCV2 recognizes and enters so many different cell types. The experiments will also allow us to study whether PCV2 uses a single or multiple mechanisms to enter into different cell types.