REVERSE TRANSCRIPTION-ASSOCIATED DEPHOSPHORYLATION OF HEPADNAVIRUS NUCLEOCAPSID

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 中心，但不一定是研究者所在的机构。 B型肝炎病毒（HBV）是一个主要的全球性健康问题，目前全世界有超过3亿的慢性感染者。其极高的感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瞬时性和不稳定性，它对MS研究提出了许多挑战。HBV衣壳蛋白的磷酸化可以调节其在HBV生命周期中的许多功能作用，包括在形态发生期间，传递触发成熟感染性病毒体的增殖和分泌的病毒成熟信号。为了鉴定病毒成熟的生物物理和生物化学相关性，我们通过原子力显微镜（AFM）表征了来自病毒成熟的三个阶段的纯化衣壳的总体结构，并通过MALDI-TOF MS ESI-Q-oTOF MS/MS和振动冷却（VC）MALDI-FT MS表征了这些阶段衣壳蛋白的磷酸化，其使磷酸盐损失最小化。从病毒表达细胞系中分离出三种极端病毒成熟（未成熟，含RNA;成熟，含DNA;和病毒体衍生的核衣壳）的鸭HBV（DHBV）核衣壳，并通过梯度离心纯化至均一。将完整的核衣壳直接点样到AFM靶上并通过AFM可视化。 我们以前报道过，使用MALDI-TOF MS，ESI nanospray和LC/MSn和VC MALDI-FTICR MS，我们检测到一个新的DHBV衣壳磷酸肽，并通过进行连续的SORI-CAD实验获得磷酸位点定位（至S230我们还使用ESI-Q-oTOF MS/MS获得了新的DHBV衣壳五磷酸化肽的完整测序和磷酸化位点定位，确认衣壳磷酸化的四个已知位点（T239、S245、S257和S259）并鉴定第二个新的磷酸化位点（S232）。安装LTQ-Orbitrap MS后，我们对衣壳蛋白进行了nanoLC/MS/MS分析，并能够定位进一步的磷酸化位点和其他PTM，并鉴定低丰度蛋白。一份手稿即将完成。

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 the installation of the LTQ-Orbitrap MS, we carried out nanoLC/MS/MS analysis of the capsid digests, and have been able to locate further phosphosites and other PTMs and to identify low-abundance proteins. A manuscript is nearing completion.