REVERSE TRANSCRIPTION-ASSOCIATED DEPHOSPHORYLATION OF HEPADNAVIRUS NUCLEOCAPSID

该子项目是利用NIH/NCRR资助的中心赠款提供的资源的许多研究子项目之一。子项目和研究者（PI）可能从另一个NIH来源获得主要资金，因此可以在其他CRISP条目中表示。所列机构为中心，不一定是研究者所在机构。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。动态蛋白磷酸化是至关重要的病毒，以及正常的，细胞过程的调节，虽然，它提出了许多挑战，MS研究由于磷酸修饰？的短暂性和不稳定的性质。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在AFM靶上，并使用Veeco Multimode？原子力显微镜连接到纳米镜？IIIa控制器。使变性的衣壳经受SDS-PAGE并在凝胶中用Lys-C或胰蛋白酶消化。通过ZipTip微色谱法纯化肽。用Bruker Reflex IV MALDI-TOF MS对来自病毒成熟的不同阶段的磷酸肽进行比较分析。用内部构建的VC MALDI-FTICR MS对磷酸肽进行MSn，或用MDS Sciex/ABI QStar ESI Q-oTOF MS对磷酸肽进行串联MS测序。使用VC MALDI-FTICR MS，我们能够检测新的DHBV衣壳磷酸肽，然后通过进行连续的SORI-CAD实验获得磷酸盐定位（至S230此外，我们使用ESI-Q-oTOF MS/MS获得了新的DHBV衣壳富含脯氨酸的五磷酸化肽的完整测序和磷酸化位点定位，确认了衣壳磷酸化的四个已知位点（T239、S245、S257和S259）和鉴定第二新磷酸化位点（S232）。从病毒成熟的三个不同阶段的完整的纯化衣壳的AFM分析表明病毒成熟和逐渐增加的衣壳大小之间的相关性。比较MALDI-TOF MS分析的肽来源于这些相同的三个衣壳人口揭示，衣壳成熟过程是相关的衣壳蛋白在所有六个磷酸化位点的显着去磷酸化。这些结果，连同已知的在病毒生命周期的早期阶段衣壳磷酸化的要求，表明嗜肝DNA病毒衣壳经历磷酸化的动态变化，以满足其在病毒生命周期的不同阶段的多种作用。成熟衣壳的去磷酸化可能提供了触发其释放和分泌的信号，这可能是由我们通过AFM检测到的衣壳结构变化所促进的。这些成熟相关的差异，衣壳的扩展能力，以及显着的脱磷酸化的衣壳蛋白，反映了以前不受重视的衣壳动力学，我们推测这可能是重要的衣壳稳定性，附着，和/或拆卸感染期间。

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 phosphate modification?s transience and labile nature. 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 with a Veeco Multimode? AFM attached to a Nanoscope? IIIa controller. Denatured capsids were subjected to SDS-PAGE and digested with Lys-C or trypsin in-gel. Peptides were purified by ZipTip microchromatography. Comparative analysis of phosphopeptides from different stages of viral maturation was conducted with a Bruker Reflex IV MALDI-TOF MS. Phosphopeptides were subjected to MSn with a VC MALDI-FTICR MS, constructed in-house, or were subjected to tandem MS sequencing with an MDS Sciex/ABI QStar ESI Q-oTOF MS. Using VC MALDI-FTICR MS, we were able to detect a novel DHBV capsid phosphopeptide, then obtain phosphosite localization (to S230) by conducting successive SORI-CAD experiments (MS2, MS3) upon it. Furthermore, we obtained complete sequencing and phosphosite localization for a novel DHBV capsid proline-rich 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). AFM analysis of the intact purified capsids from the three different stages of viral maturation demonstrated a correlation between viral maturation and a progressively increased capsid size. Comparative MALDI-TOF MS analysis of peptides derived from these same three capsid populations revealed that the capsid maturation process is correlated to a dramatic dephosphorylation of the capsid protein at all six phosphosites. These results, together with the known requirement of capsid phosphorylation at earlier stages in the viral lifecycle, suggest that the hepadnaviral capsid undergoes a dynamic change in phosphorylation in order to fulfill its multiple roles at different stages in the viral lifecycle. Dephosphorylation of the maturing capsids may provide the signal that triggers their envelopment and secretion, perhaps facilitated by the capsid structural changes that we detected by AFM. These maturation-associated differences in the capsids' capacity for expansion, as well as the dramatic dephosphorylation of the capsid protein, reflect previously unappreciated capsid dynamics, which we speculate may be important for capsid stability, envelopment, and/or disassembly during infection.