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

许多最重要的人类和动物病毒病原体具有正链RNA基因组。例如，这些病毒包括脊髓灰质炎病毒、口蹄疫病毒和蜜蜂的畸形翼病毒。这些RNA病毒中的大多数进化得非常快，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。病毒种群的变异有两个原因；病毒有一个容易出错的聚合酶，通过错误的结合，在病毒复制过程中导致不精确的复制。这种错误结合可导致新合成的病毒基因组出现高达0.1%的分化。其次，如果两个相关的病毒同时感染同一个细胞，病毒可以重组。重组促进了病毒基因组更广泛的变化——在许多情况下可达60%。重组病毒本质上是杂交病毒，病毒基因组的一部分来自一个亲本，另一部分来自另一个亲本。显然，通过结合两种不同病毒的如此广泛的区域，有可能在表型上发生非常重大的变化，即由此产生的病毒的宿主范围、组织亲和性或致病潜力。我们的实验室已经证明了一种强毒重组形式的蜜蜂变形翅膀病毒的进化，这似乎与全球疾病有关。其他研究表明，在接种疫苗不足的人群中，神经毒性脊髓灰质炎病毒在与相关的共循环病毒重组后会发生进化。正链RNA病毒的易出错聚合酶被很好地表征。相比之下，人们对重组的过程却知之甚少。我们已经开发了一种检测方法，首次将重组过程分为两个部分——初始链转移事件和二次分解事件，该事件显著增加了重组病毒的适应性。我们建议使用这个实验来提供重组机制的第一个详细分析。我们的实验明确地表明，重组病毒的产生是通过一个中间体进行的，在这个中间体中存在病毒基因组部分的重复。“复制进化”是病毒进化的基本过程之一，在这个过程中，单个基因被复制，然后通过获得点突变独立进化。我们开发的分析方法允许对这一过程进行实验研究。我们将使用肠病毒作为模型系统；这些病毒通常具有良好的特征，有良好的实验室系统对其进行分析，并且它们代表了既观察到重组又有问题的人类和动物病毒类型。我们在人类和动物身上研究这类病毒已有超过25年的经验。我们将研究病毒的序列如何影响重组的频率和位点。我们将分析这一过程在细胞中发生的位置，以及特定细胞蛋白对这一事件的贡献。最后，我们将探讨参与初级重组（链转移）和分解事件的生化机制。这些研究将有助于了解重组过程的基本机制，这是病毒进化的主要驱动力。此外，通过确定所涉及的病毒和细胞蛋白，它将使未来能够控制、利用或抑制重组，例如在未来的疫苗中。

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.