Fidelity, robustness, and diversity in RNA virus evolution and pathogenesis

 描述（由申请人提供）：病毒学的一个基本问题是了解病毒种群的进化动力学以及在单个宿主中驱动它们的力量。这些宿主内动态决定了病毒适应新宿主、开发新传播途径或产生新抗原变体的速度。目前的模式是，对于RNA病毒，高突变率导致遗传多样性增加，从而允许更快速的适应和增强的毒力。然而，大多数新产生的突变对病毒复制是致命的或有害的。这些数据表明， 耐受性或稳健性可能比突变率对病毒多样性具有更大的影响。多样性的有益影响的机制基础也是不确定的，因为宿主内的少数变异体往往以极低的频率存在。因此，突变率、突变稳健性和多样性对急性RNA病毒感染毒力的重要性尚不清楚。这项研究的长期目标是阐明新病毒变体在宿主中产生和维持的基本机制。该项目的目的是定义形成体内出现的病毒突变谱的进化力量，并确定其与发病机制的相关性。核心假设是，对于许多急性病毒感染，观察到的多样性模式是复制速度和繁殖成功的选择的结果，而不是高突变率。这一假设是根据脊髓灰质炎病毒系统的初步数据提出的，这些数据表明：（i）高保真变体表现出生长缺陷，（ii）少数变体在感染宿主中很少见，（ii）突变稳健性决定了病毒的多样性、适应性和毒力。在这些强有力的初步数据的基础上，我们将从三个方面对这一假设进行检验。(Aim 1）定义复制速度和保真度之间的权衡。快速复制的变异体将通过实验进化得到，它们的复制动力学将与突变率、体外适应性和感染宿主的毒力相关。(Aim 2）定义突变鲁棒性对群体多样性和可进化性的影响。将通过基于密码子的合理设计获得突变耐受性不同的脊髓灰质炎病毒群体。将确定鲁棒性、多样性和自适应能力之间的关系。(Aim 3）确定宿主选择压力导致病毒多样性变化的程度。为了成功地在宿主体内定殖和传播，脊髓灰质炎病毒必须在先天免疫反应中存活并通过几个严格的瓶颈。深度测序和高级建模将用于确定这一过程是否伴随着群体中的遗传变化。该方法是创新的，因为新的概念，实验工具和分析方法被用来严格测试假设的病毒多样性的原因及其后果的宿主内进化。这项研究意义重大，因为它将确定复制速度、突变率、突变鲁棒性和多样性与RNA病毒发病机制的相关性。

 DESCRIPTION (provided by applicant): A fundamental problem in virology is to understand the evolutionary dynamics of virus populations and the forces that drive them within an individual host. These intrahost dynamics determine the rate at which a virus will adapt to new hosts, develop new routes of transmission, or generate novel antigenic variants. The current paradigm is that, for RNA viruses, high mutation rates lead to increased genetic diversity, which allows for more rapid adaptation and enhanced virulence. However, most newly generated mutations are either lethal or detrimental to viral replication. These data suggest that mutational tolerance, or robustness, may have a greater impact on viral diversity than mutation rate. The mechanistic basis for the beneficial effect of diversity is also uncertain, since intrahost minorit variants are often present at an extremely low frequency. Thus, the importance of mutation rate, mutational robustness, and diversity to the virulence of acute RNA virus infections is unclear. The long-term goal of this research is to elucidate the fundamental mechanisms through which novel viral variants are generated and maintained within a host. The objective of this project is to define the evolutionary forces that shape the spectrum of viral mutants that arise in vivo and to determine their relevance to pathogenesis. The central hypothesis is that for many acute viral infections, the observed patterns of diversity are a consequence of selection for replicative speed and reproductive success as opposed to high mutation rate. This hypothesis has been formulated on the basis of preliminary data in the poliovirus system, which show that (i) high fidelity variants exhibit a growth defect, (ii) minority variants are rare in infected hosts, and (ii) mutational robustness determines a virus' diversity, fitness, and virulence. Building on these strong preliminary data, the hypothesis will be tested with three aims. (Aim 1) Define the trade-off between replicative speed and fidelity. Fast replicating variants will be derived by experimental evolution, and their replication kinetics will be correlated with mutation rates, fitness in vitro, and virulence in infected hosts. (Aim 2) Define the impact of mutational robustness on population diversity and evolvability. Poliovirus populations that vary in mutational tolerance will be derived by codon-based rational design. The relationship between robustness, diversity and adaptive capacity will be determined. (Aim 3) Determine the extent to which host selective pressure causes shifts in viral diversity. To successfully colonize and spread within a host, poliovirus must survive the innate immune response and transit several stringent bottlenecks. Deep sequencing and advanced modeling will be used to determine whether this process is accompanied by genetic changes in the population. The approach is innovative since new concepts, experimental tools, and analytic methodologies are employed to rigorously test assumptions about the causes of viral diversity and its consequences for intrahost evolution. The proposed research is significant because it will define the relevance of replicative speed, mutation rate, mutational robustness, and diversity to the pathogenesis of RNA viruses.