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The role of SUMO and PIAS proteins in retrovirus replication and restriction.

机译：SUMO和PIAS蛋白在逆转录病毒复制和限制中的作用。

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摘要

Since its discovery in 1997, the SUMO (small ubiquitin-like modifier) protein has emerged as a key regulator of protein function. SUMOylation regulates a substrate's function by altering its intracellular localization, protein-protein interactions, or stability and enzymatic activity. Proteins targeted for SUMOylation include transcription factors, proteins associated with DNA recombination, replication and repair, and proteins involved in signal transduction. In recent years numerous viral proteins have been found to interact with the SUMOylation machinery. This research investigates the role of SUMO and the SUMOylation machinery in retrovirus replication and restriction.;First, we demonstrate the antiviral effect of overexpression of SUMO1 on N-tropic MLV (murine leukemia virus) infection. We determined that the SUMO1 block of N-tropic MLV viral transduction is specific to 293T cells and is not observed in murine cell lines, Mus Dunni tail fibroblasts (MDTF) or Sc-1 cells. Further characterization of the SUMO1-enhanced restriction of N-tropic MLV reveals that the viral block, like that mediated by Fv1, is dependent on capsid residue 110 and mutation of this residue from arginine to glutamine renders the virus resistant to the SUMO1 antiviral effect. We also demonstrate that the viral block occurs early in the virus life cycle, before reverse transcription, and this block can be saturated. The SUMO1 antiviral effect on N-tropic MLV appears to involve a 293T specific factor that is not present in MDTF or Sc-1 cells and could possibly be enhancing the activity of a restriction factor, such as the TRIM5alpha (Tripartite motif 5alpha) protein.;In order to determine whether SUMO1 is enhancing the activity of the human TRIM5alpha (hTRIM5alpha) restriction factor, we generated hTRIM5alpha knockdowns and found that reduction in hTRIM5alpha expression resulted in a loss of the SUMO1 antiviral effect. Furthermore, introduction of an shRNA-resistant hTRIM5alpha construct to the SUMO1/hTRIM5alpha knockdown cells successfully restored the SUMO1 antiviral effect. We also determined that treatment of cells with the proteasome inhibitor, MG132, had no effect on the SUMO1/hTRIM5alpha block of N-tropic MLV.;Finally, we observed that overexpression of human PIAS1 and PIAS3 proteins, members of the PIAS family of SUMO E3 ligases, have no effect on B-, N-, or NB-tropic MLV viral transduction. Interestingly, human PIASy overexpression resulted in a reduction of B-, N-, and NB-tropic MLV viral transduction, as opposed to overexpression of SUMO-1, which only reduced N-tropic MLV viral transduction. Knockdown of human PIASy in 293T cells appears to have no effect on viral transduction and this could possibly be due to insufficient knockdown or functional redundancy of PIAS proteins. However, knockdown of human PIASy in 293T cells stably overexpressing SUMO1 did result in ablation of the SUMO1 block of N-tropic MLV. This suggests that hPIASy is also involved in the SUMO1-enhanced TRIM5alpha-mediated restriction of N-MLV. We also observed enhancement of B-tropic, N-tropic, and NB-tropic MLV infectivity in Balb/c cells in response to knockdown of mPIASy.;These findings have identified novel roles for SUMO1 and PIASy in viral replication and restriction. SUMO1 enhances the antiviral effects of hTRIM5alpha and specifically blocks N-tropic MLV viral transduction, in a mechanism that requires hPIASy. Human and murine PIASy negatively impact all three subclasses of MLV viral transduction. The mechanisms of SUMO1-enhanced hTRIM5alpha restriction and PIASy inhibition of viral transduction remain to be determined.
机译：自1997年被发现以来，SUMO（小泛素样修饰剂）蛋白已成为蛋白质功能的关键调节剂。 SUMOylation通过改变底物的细胞内定位，蛋白质-蛋白质相互作用或稳定性和酶促活性来调节其功能。靶向SUMOylation的蛋白质包括转录因子，与DNA重组，复制和修复相关的蛋白质以及涉及信号转导的蛋白质。近年来，已发现许多病毒蛋白与SUMOylation机制相互作用。这项研究调查了SUMO和SUMOylation机制在逆转录病毒复制和限制中的作用。首先，我们证明了SUMO1过表达对N-向性MLV（鼠白血病病毒）感染的抗病毒作用。我们确定N-向性MLV病毒转导的SUMO1块是293T细胞特有的，在鼠细胞系，Mus Dunni尾部成纤维细胞（MDTF）或Sc-1细胞中未观察到。对N-向性MLV的SUMO1增强限制的进一步表征表明，与Fv1介导的类似，病毒阻滞依赖于衣壳残基110，并且该残基从精氨酸突变为谷氨酰胺使该病毒具有抗SUMO1抗病毒作用的能力。我们还证明了病毒阻滞发生在病毒生命周期的早期，即逆转录之前，并且该阻滞可以饱和。 SUMO1对N嗜性MLV的抗病毒作用似乎涉及一个不存在于MDTF或Sc-1细胞中的293T特异性因子，并且可能增强了限制性因子的活性，例如TRIM5alpha（三方基序5alpha）蛋白。 ；为了确定SUMO1是否增强人类TRIM5alpha（hTRIM5alpha）限制因子的活性，我们产生了hTRIM5alpha敲低，并发现hTRIM5alpha表达的减少导致SUMO1抗病毒作用的丧失。此外，向SUMO1 / hTRIM5alpha敲低细胞中引入耐shRNA的hTRIM5alpha构建体成功恢复了SUMO1抗病毒作用。我们还确定了用蛋白酶体抑制剂MG132处理细胞对N-向性MLV的SUMO1 / hTRIM5alpha嵌段没有影响。最后，我们观察到人PIAS1和PIAS3蛋白的过度表达，这是SUMO的PIAS家族的成员E3连接酶对B向，N向或NB向MLV病毒转导没有影响。有趣的是，人PIASy过表达导致B-，N-和NB-向性MLV病毒转导减少，而SUMO-1的过表达则仅减少N-向性MLV病毒转导。在293T细胞中敲除人PIASy似乎对病毒转导没有影响，这可能是由于敲除不足或PIAS蛋白的功能冗余所致。但是，稳定表达过SUMO1的293T细胞中人PIASy的敲低确实导致N-向性MLV的SUMO1阻滞消融。这表明hPIASy也参与了SUMO1增强的TRIM5alpha介导的N-MLV的限制。我们还观察到Balb / c细胞对mPIASy的敲低对B-tropic，N-tropic和NB-tropic MLV感染力的增强。这些发现确定了SUMO1和PIASy在病毒复制和限制中的新作用。 SUMO1增强hTRIM5alpha的抗病毒作用，并以一种需要hPIASy的机制特异性阻断N-向性MLV病毒的转导。人和鼠类PIASy会对MLV病毒转导的所有三个亚类产生负面影响。 SUMO1增强hTRIM5alpha限制和PIASy抑制病毒转导的机制仍有待确定。

著录项

  • 作者

    Muntean, Lucia.;

  • 作者单位

    Columbia University.;

  • 授予单位 Columbia University.;
  • 学科 Biology Virology.
  • 学位 Ph.D.
  • 年度 2009
  • 页码 150 p.
  • 总页数 150
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

  • 入库时间 2022-08-17 11:38:25

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