Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA.
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland, USA
J Virol. 2018 Sep 26;92(20). doi: 10.1128/JVI.01089-18. Print 2018 Oct 15.
High-throughput DNA sequencing enables the study of experimental evolution in near real time. Until now, mutants with deletions of nonessential host range genes were used in experimental evolution of vaccinia virus (VACV). Here, we guided the selection of adaptive mutations that enhanced the fitness of a hybrid virus in which an essential gene had been replaced with an ortholog from another poxvirus genus. Poxviruses encode a complete system for transcription, including RNA polymerase and stage-specific transcription factors. The abilities of orthologous intermediate transcription factors from other poxviruses to substitute for those of VACV, as determined by transfection assays, corresponded with the degree of amino acid identity. VACV in which the A8 or A23 intermediate transcription factor subunit gene was replaced by the myxoma (MYX) virus ortholog exhibited decreased replication. During three parallel serial passages of the hybrid virus with the MYXA8 gene, plaque sizes and virus yields increased. DNA sequencing of virus populations at passage 10 revealed high frequencies of five different single nucleotide mutations in the two largest RNA polymerase subunits, RPO147 and RPO132, and two different Kozak consensus sequence mutations predicted to increase translation of the MYXA8 mRNA. Surprisingly, there were no mutations within either intermediate transcription factor subunit. Based on homology with RNA polymerase, the VACV mutations were predicted to be buried within the internal structure of the enzyme. By directly introducing single nucleotide substitutions into the genome of the original hybrid virus, we demonstrated that both RNA polymerase and translation-enhancing mutations increased virus replication independently. Previous studies demonstrated the experimental evolution of vaccinia virus (VACV) following deletion of a host range gene important for evasion of host immune defenses. We have extended experimental evolution to essential genes that cannot be deleted but could be replaced by a divergent orthologous gene from another poxvirus. Replacement of a VACV transcription factor gene with one from a distantly related poxvirus led to decreased fitness as evidenced by diminished replication. Serially passaging the hybrid virus at a low multiplicity of infection provided conditions for selection of adaptive mutations that improved replication. Notably, these included five independent mutations of the largest and second largest RNA polymerase subunits. This approach should be generally applicable for investigating adaptation to swapping of orthologous genes encoding additional essential proteins of poxviruses as well as other viruses.
高通量 DNA 测序使近实时研究实验进化成为可能。到目前为止，实验进化中使用的痘苗病毒（VACV）缺失非必需宿主范围基因的突变体。在这里，我们指导选择适应性突变，这些突变增强了一种嵌合病毒的适应性，该病毒的一个必需基因已被另一种痘病毒属的同源物替换。痘病毒编码完整的转录系统，包括 RNA 聚合酶和阶段特异性转录因子。通过转染测定确定，来自其他痘病毒的同源中间转录因子替代 VACV 中间转录因子的能力与氨基酸同一性程度相对应。用来自粘液瘤病毒（MYX）的 A8 或 A23 中间转录因子亚基基因替换 VACV 的 A8 或 A23 中间转录因子亚基基因的 VACV 显示出复制能力下降。在嵌合病毒的三个平行系列传代中，随着 MYXA8 基因的传代，噬菌斑大小和病毒产量增加。在第 10 代病毒群体的 DNA 测序中，在两个最大的 RNA 聚合酶亚基 RPO147 和 RPO132 中发现了五个不同的单核苷酸突变和两个不同的 Kozak 一致序列突变的高频，这些突变预计会增加 MYXA8mRNA 的翻译。令人惊讶的是，在两个中间转录因子亚基中都没有突变。基于与 RNA 聚合酶的同源性，预测 VACV 突变被包裹在酶的内部结构中。通过直接将单核苷酸替换引入原始嵌合病毒的基因组中，我们证明了 RNA 聚合酶和翻译增强突变都可以独立地增加病毒复制。先前的研究表明，在缺失对宿主免疫防御逃避至关重要的宿主范围基因后，痘苗病毒（VACV）的实验进化。我们已经将实验进化扩展到不能缺失但可以被来自另一种痘病毒的差异同源基因替换的必需基因。用来自远缘痘病毒的 VACV 转录因子基因替换导致适应性降低，这表现为复制能力下降。在低感染复数下连续传代杂交病毒为选择改善复制的适应性突变提供了条件。值得注意的是，这些突变包括两个最大和第二大 RNA 聚合酶亚基的五个独立突变。这种方法应该普遍适用于研究痘病毒以及其他病毒的同源基因交换对适应的影响，因为这些基因编码其他必需的蛋白质。
