Protein Primary Structure of the Vaccinia Virion at Increased Resolution.
Protein Primary Structure of the Vaccinia Virion at Increased Resolution.
复制标题
痘苗病毒粒子的蛋白质一级结构的高分辨率。
DOI：
10.1128/jvi.01042-16
复制
发表时间：
2016
影响因子：
5.4
通讯作者：
Gershon,PaulDavid
中科院分区：
文献类型：
--
作者：
Ngo,Tuan;Mirzakhanyan,Yeva;Moussatche,Nissin;Gershon,PaulDavid
在这里，我们研究了牛痘病毒病毒粒子的蛋白质共价结构。在两种病毒粒子制备中，bbbb88 %的理论牛苗病毒编码蛋白质组被高可信度地检测到，包括首次检测到的27个开放阅读框（orf）的产物，这些产物先前被指定为“预测的”、“未表征的”、“推断的”或“假设的”多肽，其中含有少至39个氨基酸（aa）和6个需要非色氨酸蛋白水解的蛋白质。我们还检测到四个短ORF的表达，每个短ORF都位于一个ORF内（“ORF- in-ORF”），包括一个以前未被识别或已知表达的ORF。使用定量质谱法（MS），确定了58至74种蛋白质被包装。总共有63种宿主蛋白也被确定为包装的候选蛋白。有证据表明，病毒粒子蛋白的某些部分通过内蛋白水解和协同外蛋白水解的组合被“切割”，其方式和位置与病毒来源或实验室程序无关。表征病毒粒子磷酸化蛋白质组的大小从189个（J. Matson， W. Chou， T. Ngo，和P. D. Gershon, Virology 452-453:310-323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012）增加到396个自信，独特的磷酸化位点，其中268个位于包装的蛋白质组内。这包括对病毒粒子蛋白磷酸化“热点”的明确识别。利用同位素富集的ATP，在9个病毒粒子蛋白中检测到23个病毒粒子内激酶磷酸化位点，这些位点全部位于病毒粒子制剂中已经部分占据的位点。RAP94和RP19蛋白的明显磷酸化与这些蛋白在病毒粒子内早期基因转录中的作用一致。在对蛋白质修饰的盲目研究中，半胱氨酸谷胱甘肽化和o -链糖基化具有显著的特点。我们为痘苗病毒蛋白的磷酸糖基化提供了证据。痘病毒是最复杂和不规则的病毒粒子之一，其内部结构鲜为人知。为了更好地了解痘病毒的病毒粒子结构，成像应辅以其他工具。在这里，我们提供了一个深入的研究牛痘病毒病毒粒子的共价结构使用当代质谱的各种工具。
Here we examine the protein covalent structure of the vaccinia virus virion. Within two virion preparations, >88% of the theoretical vaccinia virus-encoded proteome was detected with high confidence, including the first detection of products from 27 open reading frames (ORFs) previously designated “predicted,” “uncharacterized,” “inferred,” or “hypothetical” polypeptides containing as few as 39 amino acids (aa) and six proteins whose detection required nontryptic proteolysis. We also detected the expression of four short ORFs, each of which was located within an ORF (“ORF-within-ORF”), including one not previously recognized or known to be expressed. Using quantitative mass spectrometry (MS), between 58 and 74 proteins were determined to be packaged. A total of 63 host proteins were also identified as candidates for packaging. Evidence is provided that some portion of virion proteins are “nicked” via a combination of endoproteolysis and concerted exoproteolysis in a manner, and at sites, independent of virus origin or laboratory procedures. The size of the characterized virion phosphoproteome was doubled from 189 (J. Matson, W. Chou, T. Ngo, and P. D. Gershon, Virology 452-453:310–323, 2014, doi:http://dx.doi.org/10.1016/j.virol.2014.01.012) to 396 confident, unique phosphorylation sites, 268 of which were within the packaged proteome. This included the unambiguous identification of phosphorylation “hot spots” within virion proteins. Using isotopically enriched ATP, 23 sites of intravirion kinase phosphorylation were detected within nine virion proteins, all at sites already partially occupied within the virion preparations. The clear phosphorylation of proteins RAP94 and RP19 was consistent with the roles of these proteins in intravirion early gene transcription. In a blind search for protein modifications, cysteine glutathionylation and O-linked glycosylation featured prominently. We provide evidence for the phosphoglycosylation of vaccinia virus proteins.IMPORTANCEPoxviruses are among the most complex and irregular virions, about whose internal structure little is known. To better understand poxvirus virion structure, imaging should be supplemented with other tools. Here, we provide a deep study of the covalent structure of the vaccinia virus virion using the various tools of contemporary mass spectrometry.