Evolutionary potential of an RNA virus
Evolutionary potential of an RNA virus
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DOI：
10.1128/jvi.78.4.2114-2120.2004
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发表时间：
2004-02-01
影响因子：
5.4
通讯作者：
Bamford, DH
中科院分区：
文献类型：
--
作者：
Makeyev, EV;Bamford, DH
RNA病毒对变化的环境具有显著的适应性。这在医学上很重要，因为它使致病病毒能够逃避免疫反应和化疗，并且具有相当大的理论意义，因为它允许在方便的时间尺度内研究进化过程。一些早期的研究已经解决了适应RNA病毒种群的动态。然而，一直难以监测RNA基因组中响应于选择压力的分子变化的轨迹。为了解决这个问题，我们开发了一种新的体外进化系统的基础上的重组双链RNA噬菌体，phi 6，含有β-内酰胺酶（bla）基因标记。携带者状态的细菌细胞对氨苄青霉素有抗性，经过几次传代后，由于病毒携带的bla基因发生突变，它们对另一种高浓度的β-内酰胺抗生素头孢噻肟具有抗性。我们监测了头孢噻肟选择诱导的blacDNA的变化，并观察到整个基因中具有多个突变的序列变体的初始爆炸。在四次传代后，建立了含有三个特异性非同义突变的bla序列的稳定、同质群体。其中，之前曾报道过来自头孢他啶耐药细菌分离株的β-内酰胺酶的两种突变（E104 K和G238 S）。这些结果扩展了我们对病毒适应的分子机制的理解，也证明了使用RNA病毒作为异源蛋白定向进化的载体的可能性。
RNA viruses are remarkably adaptable to changing environments. This is medically important because it enables pathogenic viruses to escape the immune response and chemotherapy and is of considerable theoretical interest since it allows the investigation of evolutionary processes within convenient time scales. A number of earlier studies have addressed the dynamics of adapting RNA virus populations. However, it has been difficult to monitor the trajectory of molecular changes in RNA genomes in response to selective pressures. To address the problem, we developed a novel in vitro evolution system based on a recombinant double-stranded RNA bacteriophage, phi6, containing a beta-lactamase (bla) gene marker. Carrier-state bacterial cells are resistant to ampicillin, and after several passages, they become resistant to high concentrations of another beta-lactam antibiotic, cefotaxime, due to mutations in the virus-borne bla gene. We monitored the changes in bla cDNAs induced by cefotaxime selection and observed an initial explosion in sequence variants with multiple mutations throughout the gene. After four passages, a stable, homogeneous population of bla sequences containing three specific nonsynonymous mutations was established. Of these, two mutations (E104K and G238S) have been previously reported for beta-lactamases from cefotaxime-resistant bacterial isolates. These results extend our understanding of the molecular mechanisms of viral adaptation and also demonstrate the possibility of using an RNA virus as a vehicle for directed evolution of heterologous proteins.