rentianyixu 发表于 2017-4-2 16:11:54

J Virol:突变的蛋白让病毒传播更加猖獗

某些遗传突变会诱发病毒疯长,而其它遗传突变则会让病毒“衰减”,从而失去正常的功能和作用,然而某些遗传突变则会在特定的状况下暴露出自己的真实意图。近日,一项刊登在国际杂志Journal of Virology上的研究报告中,来自弗吉尼亚理工大学的研究人员通过研究就发现有一种突变蛋白会在高温下失去功能,这或许就能够帮助研究人员理解轮状病毒的发病机制,这种病毒是诱发婴幼儿腹泻性痢疾的主要原因。文章中,研究者揭示了轮状病毒蛋白此前未知的一种功能性位点,该病毒会感染5岁以下的儿童,如果患儿没有及时治疗就会引发致死性的脱水;轮状病毒具有三层结构,其核心由遗传物质和酶类组成,这种特殊的聚合酶能够帮助病毒遗传物质进行复制,而该过程的详细机制目前仍然是一个谜题,当然这无形中也限制了科学家们对轮状病毒生物特性的研究进展。为了阐明上述过程发生的详细机制,研究人员对一种特殊类型的轮状病毒进行了研究,这种特殊病毒的聚合酶具有温度敏感性的突变特性,当暴露于高温下时病毒就无法进行繁殖,通过阐明该突变在高温下影响聚合酶的行为,研究人员或许就能够阐明突变和蛋白质行为之间的因果关系。研究者McDonald说道,当细胞在低温下孵育时,突变的聚合酶就会正常发挥功能,其酶活性同正常蛋白一样,但在高温下,这两种蛋白之间就会出现较大的偏差,这或许就给我们提供了更多的证据来表明,病毒聚合酶的温度敏感性特性或许是由特殊突变所诱发的。据研究者介绍,突变周围区域并没有聚合酶的已知功能,然而当将突变蛋白暴露于高温下时,其或许就不能同其它蛋白之间进行合适的相互作用了,而且也不能够对病毒的核酸进行有效的复制。此前研究中研究人员解释了病毒中温度敏感性突变的存在,这项研究中研究人员通过研究首次揭示了这种突变诱发病毒出现温度敏感性的分子机制。研究者McDonald认为,不论何时病毒中存在诱发蛋白功能缺陷的突变,蛋白结构的动力学特性都会发生改变,为了理解突变改变聚合酶结构的机制,研究者利用计算机模拟的技术将突变蛋白同正常蛋白进行了对比来阐明在高温下病毒蛋白的移动特性(动力学特性);研究者发现,聚合酶上远端环状结构的运动也会发生很多改变,这或许就表明,聚合酶最初的突变区域或许也非常重要,但其它位点对于聚合酶的功能发挥也很重要。研究者McDonald说道,目前我们对轮状病毒的复制进行了深入的探究,我们发此案了一些在功能上非常重要的区域,当然这部分研究是目前我们系列研究的一部分,研究者希望通过进行多项研究来阐明轮状病毒聚合酶的结构和功能特性,同时他们也希望更加清楚地阐明病毒在细胞内复制的分子机制。最后研究者指出,在研究病毒运转机制的过程中,病毒的聚合酶和温度敏感性病毒作为科学工具已经被科学家们使用了很多年了,本文研究或许能够帮助研究者们未来更好地指导人们进行病毒感染的预防以及开发更多治疗轮状病毒感染的新型疗法。

rentianyixu 发表于 2017-4-2 16:14:38

A Temperature-Sensitive Lesion in the N-Terminal Domain of the Rotavirus Polymerase Affects Its Intracellular Localization and Enzymatic Activity
Allison O. McKella,b, Leslie E. W. LaContea and Sarah M. McDonalda,b

ABSTRACT

Temperature-sensitive (ts) mutants of simian rotavirus (RV) strain SA11 have been previously created to investigate the functions of viral proteins during replication. One mutant, SA11-tsC, has a mutation that maps to the gene encoding the VP1 polymerase and shows diminished growth and RNA synthesis at 39°C compared to that at 31°C. In the present study, we sequenced all 11 genes of SA11-tsC, confirming the presence of an L138P mutation in the VP1 N-terminal domain and identifying 52 additional mutations in four other viral proteins (VP4, VP7, NSP1, and NSP2). To investigate whether the L138P mutation induces a ts phenotype in VP1 outside the SA11-tsC genetic context, we employed ectopic expression systems. Specifically, we tested whether the L138P mutation affects the ability of VP1 to localize to viroplasms, which are the sites of RV RNA synthesis, by expressing the mutant form as a green fluorescent protein (GFP) fusion protein (VP1L138P-GFP) (i) in wild-type SA11-infected cells or (ii) in uninfected cells along with viroplasm-forming proteins NSP2 and NSP5. We found that VP1L138P-GFP localized to viroplasms and interacted with NSP2 and/or NSP5 at 31°C but not at 39°C. Next, we tested the enzymatic activity of a recombinant mutant polymerase (rVP1L138P) in vitro and found that it synthesized less RNA at 39°C than at 31°C, as well as less RNA than the control at all temperatures. Together, these results provide a mechanistic basis for the ts phenotype of SA11-tsC and raise important questions about the role of leucine 138 in supporting key protein interactions and the catalytic function of the VP1 polymerase.

IMPORTANCE RVs cause diarrhea in the young of many animal species, including humans. Despite their medical and economic importance, gaps in knowledge exist about how these viruses replicate inside host cells. Previously, a mutant simian RV (SA11-tsC) that replicates worse at higher temperatures was identified. This virus has an amino acid mutation in VP1, which is the enzyme responsible for copying the viral RNA genome. The mutation is located in a poorly understood region of the polymerase called the N-terminal domain. In this study, we determined that the mutation reduces the ability of VP1 to properly localize within infected cells at high temperatures, as well as reduced the ability of the enzyme to copy viral RNA in a test tube. The results of this study explain the temperature sensitivity of SA11-tsC and shed new light on functional protein-protein interaction sites of VP1.
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