REVERSE TRANSCRIPTION-ASSOCIATED DEPHOSPHORYLATION OF HEPADNAVIRUS NUCLEOCAPSID

反转录相关的肝炎病毒核衣壳去磷酸化

• 批准号: 8365503
• 负责人: Jianming Hu
• 金额: $ 0.23万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-06-01 至 2012-08-09
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8365503
• 关键词: Atomic Force Microscopy; Biochemical; Biology; Capsid; Capsid Proteins; Cell Line; Cell physiology; DNA; Duck Hepatitis B Virus; Ducks; Fourier transform ion cyclotron resonance; Funding; Grant; Hepadnaviridae; Hepatitis B Virus; Link; Location; Manuscripts; Mass Spectrum Analysis; Medicine; Modification; Morphogenesis; National Center for Research Resources; Nature; Nucleocapsid; Peptides; Phosphopeptides; Phosphorylation; Phosphorylation Site; Principal Investigator; Protein Dephosphorylation; Proteins; RNA; Regulation; Reporting; Research; Research Infrastructure; Resources; Reverse Transcription; Role; Signal Transduction; Site; Source; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; Spottings; Staging; Structure; Ultracentrifugation; United States National Institutes of Health; Viral; Virion; Virus; cost; global health; inorganic phosphate; novel; research study

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Hepatitis B Virus (HBV) is a major global health problem, with currently more than 300 million chronically infected worldwide. Its extremely high degree of infectivity has been in part linked to the nature of its assembly and morphogenesis. Dynamic protein phosphorylation is paramount to the regulation of viral, as well as normal, cellular processes, although it presents numerous challenges for MS study due to the transience and labile nature of the phosphate modification. Phosphorylation of the HBV capsid protein may regulate its numerous functional roles in the HBV lifecycle, including, during morphogenesis, transmitting the viral maturation signal that triggers envelopment and secretion of mature infectious virions. To identify biophysical and biochemical correlates of viral maturation, we have characterized the gross structure of purified capsids from three stages of viral maturation by atomic force microscopy (AFM), and the phosphorylation of the capsid protein at these stages by MALDI-TOF MS ESI-Q-oTOF MS/MS, and vibrational cooling (VC) MALDI-FT MS, which minimizes phosphate loss. Duck HBV (DHBV) nucleocapsids from three extremes of viral maturation (immature, RNA-containing; mature, DNA-containing; and virion-derived nucleocapsids) were isolated from virus-expressing cell lines and purified to homogeneity by gradient ultracentrifugation. Intact nucleocapsids were spotted directly onto AFM targets and visualized by AFM. We previously reported that, using MALDI-TOF MS, ESI nanospray and LC/MSn and VC MALDI-FTICR MS, we detected a novel DHBV capsid phosphopeptide, and obtained phosphosite localization (to S230) by conducting successive SORI-CAD experiments (MS2, MS3) upon it. We also obtained complete sequencing and phosphosite localization for a novel DHBV capsid pentaphosphorylated peptide using ESI-Q-oTOF MS/MS, confirming four known sites of capsid phosphorylation (T239, S245, S257, and S259) and identifying a second novel phosphorylation site (S232). NanoLC/MS/MS analysis of the capsid digests, carried out with the LTQ-Orbitrap MS allowed location of further phosphosites and other PTMs and identification of low-abundance proteins. Two manuscripts are being prepared.

该子项目是利用资源的众多研究子项目之一 由 NIH/NCRR 资助的中心拨款提供。子项目的主要支持 并且子项目的首席研究员可能是由其他来源提供的， 包括其他 NIH 来源。 子项目可能列出的总成本 代表子项目使用的中心基础设施的估计数量， NCRR 赠款不直接向子项目或子项目工作人员提供资金。 乙型肝炎病毒 (HBV) 是一个主要的全球健康问题，目前全球有超过 3 亿人慢性感染。其极高的传染性部分与其组装和形态发生的性质有关。动态蛋白质磷酸化对于病毒以及正常细胞过程的调节至关重要，尽管由于磷酸修饰的短暂性和不稳定性质，它给 MS 研究带来了许多挑战。 HBV 衣壳蛋白的磷酸化可以调节其在 HBV 生命周期中的众多功能作用，包括在形态发生过程中，传递病毒成熟信号，从而触发成熟感染性病毒颗粒的包封和分泌。为了确定病毒成熟的生物物理和生化相关性，我们通过原子力显微镜 (AFM) 表征了病毒成熟三个阶段的纯化衣壳的总体结构，并通过 MALDI-TOF MS ESI 表征了这些阶段衣壳蛋白的磷酸化。 Q-oTOF MS/MS 和振动冷却 (VC) MALDI-FT MS，可最大限度地减少磷酸盐损失。从病毒表达细胞系中分离出来自三种极端病毒成熟状态（未成熟、含 RNA；成熟、含 DNA；和病毒粒子衍生的核衣壳）的鸭 HBV (DHBV) 核衣壳，并通过梯度超速离心纯化至同质。完整的核衣壳直接发现到 AFM 目标上并通过 AFM 可视化。 我们之前报道过，使用 MALDI-TOF MS、ESI nanospray 和 LC/MSn 以及 VC MALDI-FTICR MS，我们检测到了一种新型 DHBV 衣壳磷酸肽，并通过连续的 SORI-CAD 实验（MS2、 MS3) 就可以了。我们还使用 ESI-Q-oTOF MS/MS 获得了新型 DHBV 衣壳五磷酸化肽的完整测序和磷酸位点定位，确认了四个已知的衣壳磷酸化位点（T239、S245、S257 和 S259），并确定了第二个新的磷酸化位点（ S232）。使用 LTQ-Orbitrap MS 对衣壳消化物进行 NanoLC/MS/MS 分析，可以进一步定位磷酸位点和其他 PTM，并鉴定低丰度蛋白质。两份手稿正在准备中。