REVERSE TRANSCRIPTION-ASSOCIATED DEPHOSPHORYLATION OF HEPADNAVIRUS NUCLEOCAPSID

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 乙肝病毒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。其极高的传染性在一定程度上与其组装和形态形成的性质有关。蛋白质的动态磷酸化对于病毒以及正常的细胞过程的调节是至关重要的，尽管由于磷酸修饰的瞬时性和不稳定性，它给MS的研究带来了许多挑战。乙肝病毒衣壳蛋白的磷酸化可能调节其在乙肝病毒生命周期中的众多功能，包括在形态形成过程中传递病毒成熟信号，触发成熟感染性病毒粒子的包膜和分泌。为了确定病毒成熟的生物物理和生化相关性，我们用原子力显微镜(AFM)表征了病毒成熟三个阶段的纯化衣壳的大体结构，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，从而将磷酸盐损失降至最低。从表达病毒的细胞系中分离出鸭乙肝病毒(DHBV)核衣壳，并用梯度超速离心法将其纯化为均一。完整的核衣壳直接被发现在AFM靶标上，并由AFM可视化。我们先前报道，利用MALDI-TOF MS、ESI纳米光谱和LC/MSN和VC MALDI-FTICR MS，我们检测到了一种新的DHBV衣壳磷酸肽，并通过对其进行连续的Sori-CAD实验(MS2、MS3)获得了亚磷酸盐定位(TO S230)。我们还利用ESI-Q-OTOF MS/MS对一个新的DHBV衣壳五磷酸化肽进行了全序列测定和亚磷酸定位，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)。在LTQ-Orbitrap MS安装后，我们对衣壳消化进行了NanoLC/MS/MS分析，并能够定位更多的磷酸盐和其他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.