Mechanism of cellular recognition and entry by a circovirus

圆环病毒识别和进入细胞的机制

• 批准号: 8667019
• 负责人: Reza Khayat
• 金额: $ 34.33万
• 依托单位: CITY COLLEGE OF NEW YORK
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8667019
• 关键词: Affinity; Animal Viruses; Arginine; Binding; Biochemical; Biological Assay; Breathing; Calorimetry; Capsid; Capsid Proteins; Cells; Charge; Circovirus; Clathrin; Cleaved cell; Complement; Cryoelectron Microscopy; Cytolysis; Cytoplasm; Dermatan Sulfate; Development; Donor person; Endocytosis; Endosomes; Environment; Exhibits; Family suidae; Fluorescence; Generations; Genes; Genetic; Goals; Health; Heparitin Sulfate; Human; Human Cell Line; Immunology; Immunosuppressive Agents; Infection; Inorganic Sulfates; Kidney; Liposomes; Mass Spectrum Analysis; Membrane; Modeling; Modification; Molecular; Mutate; Nucleic Acids; Organ; Phenotype; Physiology; Population; Porcine circovirus; Proteoglycan; Proteolysis; Reporter; Resolution; Site-Directed Mutagenesis; Solutions; Structure; Surface; Techniques; Testing; Therapeutic; Time; Tissues; Titrations; Unspecified or Sulfate Ion Sulfates; Vaccines; Viral; Virus; Virus-like particle; Work; X-Ray Crystallography; Xenograft procedure; Zoonoses; base; cell type; design; endosome membrane; image reconstruction; insight; lens; mutant; particle; pathogen; polypeptide; public health relevance; receptor; receptor binding; research study; resistant strain; structural biology; therapeutic development

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.

描述（由申请人提供）：人类和猪之间共享的免疫学，生理和遗传学使猪病原体成为对人类健康的即将来临的威胁。猪圆环病毒2（PCV2）是一种全球分布的非发育的免疫抑制病毒，可在培养中感染和裂解人类细胞。 PCV2感染了其宿主中的几乎所有组织，因此是从猪到人类的异种移植的威慑作用。从猪到人类的异种移植正在成为人类捐助者的组织和器官短缺的实践解决方案。最近，对商业PCV2疫苗具有抗性菌株，最近证明了PCV2在其环境中快速突变而繁殖的能力。因此，了解PCV2感染机制是紧急优先级的许多原因。该项目的目的是了解PCV2几乎不加选择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极限。这种病毒识别和进入如此多细胞类型的能力令人惊讶，因为非发育的动物病毒表现出可以与之相互作用并进入的细胞类型的重点范围。该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。该项目很重要，因为它提供了理解自主复制病毒所需的最小成分的机会，以及导致广泛而几乎不加区分的细胞识别和进入的衣壳决定因素。研究的三个独立但重叠的目的是：1。定义PCV2识别并与其受体相互作用的机制。使用我们的PCV2的晶体结构，我们假设实验鉴定出硫酸乙酰肝素和硫酸软骨素B受体结合的位置。我们将使用生化，生物物理和结构生物学技术的结合来检验这一假设。 2。定义PCV2逃脱内体的机制。 PCV2已被证明使用依赖性和独立的内吞作用将其内部化为不同的细胞类型。我们假设PCV2衣壳蛋白的精氨酸N-末端具有膜不稳定活性，并由PCV2用于逃避内体并进入细胞。精氨酸富含多肽具有这种活性。我们将使用生化和生物物理研究的组合来检验这一假设。 3。研究PCV2进入细胞的机制。将研究以抑制受体识别和内体逃逸生成的突变体，分别在目标1和2中，研究其感染细胞类型的能力，这些细胞类型通过网格蛋白依赖性和独立的内吞作用将PCV2内化。这些研究将使我们能够通过细胞晶状体重新制定假设，以描述PCV2识别并进入许多不同细胞类型的机制。该实验还将使我们能够研究PCV2是使用单个或多种机制进入不同细胞类型的实验。