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Recombination in enteroviruses: the genetics, cell biology and biochemistry of a biphasic replicative mechanism of virus evolution

肠道病毒重组：病毒进化双相复制机制的遗传学、细胞生物学和生物化学

基本信息

批准号：
BB/M009343/1
负责人：
David Evans
金额：
$ 51.35万
依托单位：
University of St Andrews
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2015
资助国家：
英国
起止时间：
2015 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FM009343%2F1
关键词：
Recombination enteroviruses genetics cell biology

项目摘要

Many of the most important human and animal virus pathogens have positive strand RNA genomes. These include, for example, poliovirus, foot and mouth disease virus and deformed wing virus of honeybees. The majority of these RNA viruses evolve very rapidly, generating large populations of highly divergent progeny. This variation helps the virus evade the host immune system - including escaping immunity induced by vaccines - and may enable the virus to spread more efficiently to new hosts, including cross-species transfer. The variation in the virus population is due to two things; the virus has an error-prone polymerase enzyme which, through mis-incorporation, results in imprecise copying during virus replication. This mis-incorporation can result in up to 0.1% divergence in newly synthesized virus genomes. Secondly, if two related viruses co-infect the same cell, the virus can recombine. Recombination facilitates very much more extensive changes of the virus genome - up to 60% in many cases. Recombinant viruses are essentially hybrids, with part of the virus genome derived from one parent, and part from the other parent. Clearly, by combining such extensive regions of two different viruses there are opportunities for very significant changes in the phenotype i.e. the host range, tissues tropism or pathogenic potential, of the resulting virus.Our laboratory has demonstrated the evolution of a virulent recombinant form of deformed wing virus of honeybees which appears to be associated with global disease. Other studies have shown the evolution of neurovirulent poliovirus in a poorly vaccinated population following recombination with a related co-circulating virus. The error-prone polymerases of positive strand RNA viruses are well characterised. In contrast, the process of recombination is only poorly understood. We have developed an assay that, for the first time, allows the recombination process to be divided into two parts - an initial strand transfer event and a secondary resolution event that markedly increases the fitness of the recombinant virus. We propose to use this assay to provide the first detailed analysis of the mechanism of recombination.Our assay unequivocally demonstrates that the generation of a recombinant virus goes via an intermediate in which there are duplications of parts of the virus genome. "Evolution by duplication" is one of the fundamental processes in virus evolution, in which individual genes are duplicated and then can subsequently independently evolve through acquisition of point mutations. The assay we have developed allows this process to be studied experimentally. We will use enteroviruses as a model system; these viruses are generally well-characterised, there are good laboratory systems for their analysis and they are representative of the types of viruses of humans and animals in which recombination is both observed and problematic. We have over 25 years experience studying this group of viruses, in humans and animals.We will study how the sequence of the virus contributes to the frequency and site of recombination. We will analyse where this process occurs in cells and the contribution made to this event by defined cellular proteins. Finally we will investigate the biochemical mechanism involved in the primary recombination (strand transfer) and resolution events. These studies will contribute to an understanding of the fundamental mechanism of the recombination process which is a major driving force in virus evolution. Additionally, by defining the viral and cellular proteins that are involved it will enable the future control, exploitation or inhibition, of recombination - for example, in vaccines of the future.

许多最重要的人类和动物病毒病原体都具有正链 RNA 基因组。这些包括例如脊髓灰质炎病毒、口蹄疫病毒和蜜蜂变形翅病毒。这些 RNA 病毒中的大多数进化非常迅速，产生大量高度分化的后代。这种变异有助于病毒逃避宿主免疫系统，包括逃避疫苗诱导的免疫，并可能使病毒更有效地传播到新宿主，包括跨物种转移。病毒种群的变化有两个原因：该病毒具有容易出错的聚合酶，通过错误掺入，导致病毒复制过程中复制不精确。这种错误掺入可能会导致新合成的病毒基因组出现高达 0.1% 的差异。其次，如果两种相关的病毒共同感染同一个细胞，病毒就可以重组。重组促进了病毒基因组更广泛的变化——在许多情况下高达 60%。重组病毒本质上是杂种，病毒基因组的一部分来自一个亲本，一部分来自另一个亲本。显然，通过将两种不同病毒的如此广泛的区域相结合，有机会在表型上发生非常显着的变化，即所得病毒的宿主范围、组织向性或致病潜力。我们的实验室已经证明了蜜蜂变形翼病毒的剧毒重组形式的进化，这种病毒似乎与全球疾病有关。其他研究表明，神经毒性脊髓灰质炎病毒在疫苗接种不良的人群中与相关的共循环病毒重组后发生了进化。正链 RNA 病毒的易错聚合酶已得到很好的表征。相比之下，人们对重组过程知之甚少。我们开发了一种检测方法，首次将重组过程分为两部分——初始链转移事件和显着提高重组病毒适应性的二次分解事件。我们建议使用该测定来对重组机制进行首次详细分析。我们的测定明确地表明重组病毒的产生经历了一个中间体，其中病毒基因组的部分存在重复。 “复制进化”是病毒进化的基本过程之一，其中个体基因被复制，然后通过获得点突变而独立进化。我们开发的检测方法可以通过实验研究这一过程。我们将使用肠道病毒作为模型系统；这些病毒通常具有良好的特征，有良好的实验室系统进行分析，并且它们代表了人类和动物的病毒类型，在这些病毒中观察到重组并且存在重组问题。我们在人类和动物身上研究这组病毒拥有超过 25 年的经验。我们将研究病毒序列如何影响重组的频率和位点。我们将分析该过程在细胞中发生的位置以及特定的细胞蛋白对该事件的贡献。最后，我们将研究初级重组（链转移）和解析事件中涉及的生化机制。这些研究将有助于理解重组过程的基本机制，重组过程是病毒进化的主要驱动力。此外，通过定义所涉及的病毒和细胞蛋白，它将能够在未来控制、利用或抑制重组——例如，在未来的疫苗中。