Mechanism of cellular recognition and entry by a circovirus

描述(申请人提供)：人类和猪之间共享的免疫学、生理学和遗传学使猪的病原体对人类健康构成迫在眉睫的威胁。猪圆环病毒2(PCV2)是一种全球分布的无包膜免疫抑制病毒，可在培养条件下感染和溶解人类细胞。PCV2几乎感染宿主的每一个组织，因此对从猪到人的异种移植具有威慑作用。从猪到人的异种移植正在成为解决人类捐赠者组织和器官短缺的实际解决方案。新近出现的对商业PCV2疫苗有抗药性的毒株证明了PCV2的S在其环境中快速变异并茁壮成长的能力。因此，了解PCV2感染机制是当务之急的原因有很多。这个项目的目标是了解PCV2实现几乎不分青红皂白的细胞识别和进入的详细分子机制。PCV2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细胞类型的能力令人惊讶，因为非包膜动物病毒展示了它们可以与之相互作用和进入的一系列集中的细胞类型。这种限制似乎依赖于细胞表面是否存在病毒衣壳可以相互作用的受体和/或辅助受体。该项目意义重大，因为它提供了机会，了解自主复制病毒所需的最低限度的成分，以及导致广泛和几乎不分青红皂白的细胞识别和进入的衣壳决定因素。正在研究的三个独立但相互重叠的目标是：1.确定PCV2识别其受体并与其相互作用的机制。利用我们的PCV2的晶体结构，我们假设实验确定的硫酸乙酰肝素和硫酸软骨素B受体结合的地方。我们将使用生化、生物物理和结构生物学技术的组合来检验这一假设。2.确定PCV2逃逸内体的机制。PCV2已被证明通过依赖和独立的内吞作用内化成不同类型的细胞。我们推测PCV2衣壳蛋白富含精氨酸的N末端具有膜不稳定活性，并被PCV2用来逃脱内体进入细胞。富含精氨酸的多肽已被证明具有这种活性。我们将结合生化和生物物理研究来验证这一假说。3.研究PCV2进入细胞的机制。分别在AIMS 1和AIMS 2中产生的抑制受体识别和内小体逃逸的突变体将被研究，以了解它们感染通过依赖和独立内吞作用内化PCV2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阐述我们的假设，以描述PCV2识别和进入如此多不同类型细胞的机制。这些实验还将使我们能够研究PCV2是使用一种还是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.