Protein Interactions Involved in Orthopoxvirus Envelopment

参与正痘病毒包膜的蛋白质相互作用

• 批准号: 7265425
• 负责人: BRIAN M WARD
• 金额: $ 34.65万
• 依托单位: UNIVERSITY OF ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-02-15 至 2012-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7265425
• 关键词: Affect; Alanine; Animal Model; Antiviral Agents; Attenuated Vaccines; Biochemistry; Biological; Biological Assay; Capital; Cells; Charge; Co-Immunoprecipitations; Complex; Data; Defect; Discipline; Disease; Disruption; Drug Design; Energy Transfer; Event; Facility Construction Funding Category; Family; Fluorescence; GPA33 gene; Genes; Genetic; Genetic Transcription; Glycoprotein A33; Goals; Golgi Apparatus; Immunization; Infection; Letters; Life; Localized; Location; Maps; Membrane; Microscopy; Molecular; Molecular and Cellular Biology; Morphogenesis; Mus; Mutagenesis; Mutation; Numbers; Open Reading Frames; Orthopoxvirus; Phenotype; Play; Point Mutation; Positioning Attribute; Poxviridae; Process; Production; Proteins; Recombinant Vaccines; Recombinants; Relative (related person); Research Personnel; Role; Series; Site; Smallpox; Smallpox Vaccine; Smallpox Viruses; Staging; Structure; System; Systemic infection; Testing; Vaccines; Vaccinia virus; Viral; Viral Proteins; Virion; Virulence; Virus; Virus Diseases; Work; base; cellular targeting; design; expression vector; in vivo; innovation; interest; member; mutant; neutralizing antibody; novel; programs; protein function; protein protein interaction; recombinant virus; research study; size; trafficking; vector; virus envelope; ward

DESCRIPTION (provided by applicant): The Orthopoxvirus genus of the family Poxviridae includes both the causative agent of smallpox, variola virus, and the vaccine used in its eradication, vaccinia virus. Recent events have renewed interest in Orthopoxviruses because of concerns that variola virus or one of its close relatives could be used as a biological weapon. Orthopoxviruses produce both enveloped and unenveloped virions and remarkably both types are infectious. While non-enveloped virions make up the majority of progeny virions the enveloped form (EV) is required for cell-to-cell spread, systemic infection and virulence. The major objective of work is to determine the molecular mechanism employed by envelope virus specific proteins to coordinate the intracellular envelopment of Orthopoxviruses. Our hypothesis is that the lumenal domain of three EV specific proteins, A33, A34 and B5, interact and that interaction is required for exit from the ER and subsequently proper sub-cellular targeting of B5, which is the main target of neutralizing antibodies, to the site of intracellular envelopment and subsequently into infectious enveloped virions. The specific aims are: 1) Mapping residues of A33, A34 and B5 required for proper targeting and interaction. The studies proposed here will use an innovative live trans complementation microscopy assay and co-IP to map regions of interaction. 2) Characterization of the relationship between A33, A34 and B5 in EV production. Recombinant viruses will be created that express A3R, A3R and BR mutations defined in Aim1 to characterize the role the interaction has during viral morphogenesis. 3) Temporal and spatial characterization of A33, A34 and B5 interaction. The innovative fluorescent assays FRET and BiFC will be used to track the interaction in live cells. Mapping sites of interaction on these proteins will help define functional domains and provide for a better understanding of their structure and function. The B5R protein is required for the formation of enveloped poxviruses. We will investigate how this important protein is incorporated into and coordinates the formation of newly made viruses. A better understanding of this process will provide new targets for antivirals directed against poxviruses.

描述（由申请人提供）：痘病毒科的正痘病毒属包括天花的病原体天花病毒和用于根除天花的疫苗痘苗病毒。由于担心天花病毒或其近亲之一可能被用作生物武器，最近发生的事件重新引起了人们对正痘病毒的兴趣。正痘病毒产生有包膜和无包膜病毒粒子，并且值得注意的是这两种类型都具有传染性。虽然无包膜病毒粒子构成了子代病毒粒子的大部分，但有包膜病毒粒子 (EV) 是细胞间传播、全身感染和毒力所必需的。工作的主要目标是确定包膜病毒特异性蛋白用于协调正痘病毒的细胞内包膜的分子机制。我们的假设是，三种 EV 特异性蛋白 A33、A34 和 B5 的腔结构域相互作用，并且这种相互作用是从 ER 退出以及随后将 B5（中和抗体的主要靶标）正确亚细胞靶向至细胞内包膜位点并随后进入感染性包膜病毒体所需的。具体目标是： 1) 绘制正确靶向和相互作用所需的 A33、A34 和 B5 残基图谱。这里提出的研究将使用创新的活反式互补显微镜检测和 co-IP 来绘制相互作用区域。 2）电动汽车生产中A33、A34和B5之间关系的表征。将创建表达 Aim1 中定义的 A3R、A3R 和 BR 突变的重组病毒，以表征相互作用在病毒形态发生过程中的作用。 3) A33、A34 和 B5 相互作用的时间和空间特征。创新的荧光检测 FRET 和 BiFC 将用于追踪活细胞中的相互作用。绘制这些蛋白质上的相互作用位点将有助于定义功能域并提供对其结构和功能的更好理解。 B5R 蛋白是形成有包膜痘病毒所必需的。我们将研究这种重要的蛋白质如何融入并协调新病毒的形成。更好地了解这一过程将为针对痘病毒的抗病毒药物提供新的靶点。