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Comparative Analysis of Rhinovirus 2A Protease Cleavage of the Nuclear Pore Complex.

机译：鼻孔复合体鼻病毒2A蛋白酶切割的比较分析。

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摘要

Several active nucleocytoplasmic transport pathways are disrupted in picornavirus infected cells. Recent studies have shown that rhinoviruses (RV) use encoded 2A protease, 2Apro, to cleave nucleoporins (Nups) in nuclear pore complexes (NPC) to inhibit nucleocytoplasmic transport pathways. Although they share similar structures and carry out the same functions during infection, the 2Apro sequences are highly diverse among different RV clades, which suggests that the proteases might have different enzymatic activities and carryout their anti-host functions with unique mechanistic signatures. The avidity and specificity with which different RV 2As cleave Nups and their ability to disrupt nuclear transport pathways could alter antiviral signaling and affect virus replication levels, ultimately triggering different disease phenotypes and host immune responses. In this thesis, we sought to compare the proteolytic activities of different RV 2Apros in NPCs.;In vitro assays with recombinant proteases showed that different RV 2A pros have different Nup substrate specificities and target and cleave Nup62, Nup98, and Nup153 at different relative rates. Relative Nup degradation kinetics in infected cells between different RVs (A16 and B14) were similar to the relative Nup degradation kinetics we observed in the in vitro assays between corresponding 2Apros. Development of a cellular time-lapse fluorescent assay of nuclear import and export allowed for the comparison of nucleocytoplasmic transport disruption efficiencies of different RV 2A pros. RV 2Apros expressed alone in cells inhibited importin alpha/beta, transportin-1, transportin-3, and Crm1 nuclear transport pathways. However, the proteases inhibited these nuclear transport pathways at different rates. The RV-B proteases, especially B52 2Apro, disrupted nuclear import more efficiently than RV-A or RV-C proteases. Comparison of results from 2Apro nucleocytoplasmic transport inhibition experiments and results of clinical studies on RV illness severities revealed an inverse correlation between RV 2Apro nuclear import disruption efficiency and RV illness severity, which suggested that RVs with 2A pros that more efficiently disrupt nuclear import pathways are less virulent.;Collectively, the results presented in this thesis show that different RV 2Apros differentially disrupt nucleocytoplasmic trafficking pathways by differentially targeting and cleaving nucleoporins. Additionally, correlations between results of 2Apro nucleocytoplasmic disruption experiments and results of clinical studies suggest that 2A proteolytic activity in NPCs affects RV virulence.
机译：在小核糖核酸病毒感染的细胞中，几个活跃的核质运输途径被破坏。最近的研究表明，鼻病毒（RV）使用编码的2A蛋白酶2Apro裂解核孔复合体（NPC）中的核孔蛋白（Nups），以抑制核质转运途径。尽管它们在感染期间具有相似的结构并执行相同的功能，但是2Apro序列在不同的RV进化枝之间差异很大，这表明蛋白酶可能具有不同的酶活性，并具有独特的机械特征来发挥其抗宿主功能。不同RV 2A裂解Nups的亲和力及其破坏核转运途径的能力可能会改变抗病毒信号并影响病毒复制水平，最终引发不同的疾病表型和宿主免疫反应。在本论文中，我们试图比较不同RV 2Apros在NPC中的蛋白水解活性。重组蛋白酶的体外测定表明，不同的RV 2A pros具有不同的Nup底物特异性，并以不同的相对速率靶向和切割Nup62，Nup98和Nup153。 。在不同RV（A16和B14）之间的感染细胞中，相对Nup降解动力学与我们在相应2Apros的体外测定中观察到的相对Nup降解动力学相似。核进出口的细胞延时荧光测定法的发展允许比较不同RV 2A优点的核质运输破坏效率。单独在细胞中表达的RV 2Apros抑制了importin alpha / beta，transportin-1，transportin-3和Crm1核转运途径。但是，蛋白酶以不同的速率抑制这些核转运途径。 RV-B蛋白酶，特别是B52 2Apro，比RV-A或RV-C蛋白酶更有效地破坏了核输入。比较2Apro核质运输抑制实验的结果和RV疾病严重程度的临床研究结果，发现RV 2Apro核输入干扰效率与RV疾病严重程度之间呈负相关，这表明具有2A亲的RV更有效地干扰核输入途径的RV较少。总体而言，本研究结果表明，不同的RV 2Apros通过差异靶向和切割核孔蛋白来差异性地破坏核质运输途径。此外，2Apro核质破坏实验的结果与临床研究结果之间的相关性表明，NPC中的2A蛋白水解活性会影响RV毒力。

著录项

  • 作者

    Watters, Kelly.;

  • 作者单位

    The University of Wisconsin - Madison.;

  • 授予单位 The University of Wisconsin - Madison.;
  • 学科 Chemistry Biochemistry.
  • 学位 Ph.D.
  • 年度 2014
  • 页码 122 p.
  • 总页数 122
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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