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）：人类和猪之间共有的免疫学、生理学和遗传学使得猪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，已被证明可以感染并裂解培养的人类细胞。 PCV2几乎感染宿主的所有组织，因此阻碍了从猪到人类的异种移植。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问题的一种可行的解决方案。最近出现的对商业 PCV2 疫苗具有抗药性的菌株证明了 PCV2 能够快速突变并在其环境中茁壮成长的能力。因此，有很多原因说明为什么了解 PCV2 感染机制是当务之急。该项目的目标是了解 PCV2 实现几乎无差别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，因为无包膜动物病毒表现出它们可以与之相互作用并进入的一系列细胞类型。这种限制似乎取决于细胞表面上病毒衣壳可以与之相互作用的受体和/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成分以及导致广泛且几乎不加区别的细胞识别和进入的衣壳决定因素的机会。正在研究的三个独立但重叠的目标是： 1. 定义 PCV2 识别其受体并与其相互作用的机制。利用 PCV2 的晶体结构，我们假设实验鉴定的硫酸乙酰肝素和硫酸软骨素 B 受体结合的位置。我们将结合生物化学、生物物理和结构生物学技术来检验这一假设。 2. 定义PCV2 逃离内涵体的机制。 PCV2 已被证明可以利用网格蛋白依赖性和独立的内吞作用内化到不同的细胞类型中。我们假设 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 用于逃离内体并进入细胞。富含精氨酸的多肽已被证明具有这种活性。我们将结合生物化学和生物物理研究来检验这一假设。 3.研究PCV2进入细胞的机制。分别在目标 1 和 2 中，将研究为抑制受体识别和内体逃逸而产生的突变体感染通过网格蛋白依赖性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视角重新阐述我们的假设，以描述 PCV2 识别和进入多种不同细胞类型的机制。这些实验还将使我们能够研究 PCV2 是否使用单一或多种机制进入不同的细胞类型。