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: 8170867
• 负责人: Jianming Hu
• 金额: $ 0.46万
• 依托单位: BOSTON UNIVERSITY MEDICAL CAMPUS
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-06-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8170867
• 关键词: Atomic Force Microscopy; Biochemical; Capsid; Capsid Proteins; Cell Line; Cell physiology; Computer Retrieval of Information on Scientific Projects Database; Duck Hepatitis B Virus; Ducks; Fourier transform ion cyclotron resonance; Funding; Grant; Hepadnaviridae; Hepatitis B Virus; Institution; Link; Location; Manuscripts; Modification; Morphogenesis; Nature; Nucleocapsid; Peptides; Phosphopeptides; Phosphorylation; Phosphorylation Site; Protein Dephosphorylation; Proteins; Regulation; Reporting; Research; Research Personnel; Resources; Reverse Transcription; Role; Signal Transduction; Site; Source; Spottings; Staging; Structure; Ultracentrifugation; United States National Institutes of Health; Viral; Virion; Virus; 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. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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). After installation of the LTQ-Orbitrap MS, nanoLC/MS/MS analysis of the capsid digests were performed and allowed location of further phosphosites and other PTMs and to identhe identification of low-abundance proteins. Two manuscripts will be submitted shortly.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此可以在其他清晰的条目中代表。列出的机构是 对于中心，这不一定是调查员的机构。 丙型肝炎病毒（HBV）是一个主要的全球健康问题，目前在全球范围内有超过3亿个慢性感染。它的感染力极高，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的调节以及正常的细胞过程至关重要，尽管由于磷酸盐修饰的瞬时和不稳定性，它给MS研究带来了许多挑战。 HBV衣壳蛋白的磷酸化可能会调节其在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体的包裹和分泌。 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 损失。鸭HBV（DHBV）来自三个极端的病毒成熟（未成熟，含RNA的；成熟，含DNA的含DNA；和病毒粒子衍生的核素）的核蛋白酶是从表达病毒细胞系中分离出来的，并通过梯度超级肠系分纯化为同质性。将完整的核蛋白质直接发现到AFM靶标，并通过AFM可视化。 我们先前曾报道过，使用MALDI-TOF MS，ESI Nanospray和LC/MSN和VC Maldi-FTICR MS，我们检测到了一种新型的DHBV CAPSID磷酸肽，并通过对其进行连续的SORI-CAD实验（MS2，MS3）进行了磷酸化定位（TO S230）。我们还使用Esi-Q-otof MS/MS获得了新型DHBV CAPSID五磷酸化肽的完整测序和磷酸材料定位，从而确认了四个已知的衣壳磷酸化地点（T239，S245，S257和S259），并确定了第二种小说phosphosphorphoration（S257）。在安装LTQ-Orbitrap MS后，进行了capsid消化的纳米/MS/MS分析，并允许位置进一步的磷岩和其他PTM，并识别识别低丰度蛋白。两项手稿将很快提交。