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.

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