Adenovirus Composition, Proteolysis, and Disassembly Studied by In-depth Qualitative and Quantitative Proteomics
Adenovirus Composition, Proteolysis, and Disassembly Studied by In-depth Qualitative and Quantitative Proteomics
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DOI：
10.1074/jbc.m113.537498
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发表时间：
2014-04-18
影响因子：
4.8
通讯作者：
Heck, Albert J. R.
中科院分区：
文献类型：
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作者：
Benevento, Marco;Di Palma, Serena;Heck, Albert J. R.
背景：腺病毒（Adenoviruses，AdV）被广泛用作基因递送载体.结果：测定了AdV蛋白的拷贝数，并研究了热应激时蛋白的释放。结论：病毒蛋白酶在腺病毒蛋白的节段释放过程中起着重要作用。重要性：我们的质谱表征提供了新的见解在HAdV拆卸进入宿主cells.Using高分辨率MS为基础的蛋白质组学结合多蛋白酶消化，我们剖析，平均90%的序列覆盖率，所有13个病毒蛋白存在于人腺病毒（HAdV）载体。这种深入的概况提供了多种基于肽的证据，内在的蛋白酶活性影响几个HAdV蛋白。接下来，使用产生的肽文库开发靶向蛋白质组学方法，该方法使用选择反应监测（SRM），旨在定量分析HAdV中存在的所有13种蛋白质的化学计量。我们还使用这种方法来探测由热刺激引发的特定病毒蛋白的释放，模拟HAdV在进入宿主细胞期间解体的早期阶段。我们确认了最充分表征的病毒衣壳组分的拷贝数，并建立了迄今为止尚未准确定义其化学计量的蛋白质的拷贝数。我们还发现，加热HAdV诱导五邻体基底和纤维蛋白的完全释放以及蛋白VIII和VI的大量释放。对于这些后者的蛋白质，腺病毒蛋白酶的成熟蛋白水解导致片段的差异释放，其中某些肽被完全释放，而其他肽大部分保留在AdV颗粒中。这一信息可能有利于高分辨率cryoEM和X射线电子密度图的持续解释。
Background: Adenoviruses (AdV) are broadly employed as gene delivery vectors. Results: Copy numbers of all AdV proteins were measured, and the release of proteins upon heat stress investigated. Conclusion: The viral protease plays a distinct role in the segmented release of AdV proteins. Significance: Our characterization by mass spectrometry provides new insight in HAdV disassembly during entry into host cells.Using high-resolution MS-based proteomics in combination with multiple protease digestion, we profiled, with on average 90% sequence coverage, all 13 viral proteins present in an human adenovirus (HAdV) vector. This in-depth profile provided multiple peptide-based evidence on intrinsic protease activity affecting several HAdV proteins. Next, the generated peptide library was used to develop a targeted proteomics method using selected reaction monitoring (SRM) aimed at quantitative profiling of the stoichiometry of all 13 proteins present in the HAdV. We also used this method to probe the release of specific virus proteins initiated by thermal stimulation, mimicking the early stage of HAdV disassembly during entry into host cells. We confirmed the copy numbers of the most well characterized viral capsid components and established the copy numbers for proteins whose stoichiometry has so far not been accurately defined. We also found that heating HAdV induces the complete release of the penton base and fiber proteins as well as a substantial release of protein VIII and VI. For these latter proteins, maturational proteolysis by the adenoviral protease leads to the differential release of fragments with certain peptides being fully released and others largely retained in the AdV particles. This information is likely to be beneficial for the ongoing interpretation of high resolution cryoEM and x-ray electron density maps.