Infectious plant virus clones are challenging to construct and manipulate due to the presence of cryptic promoter sequences that induce toxicity in bacteria. Common methods to overcome toxicity include intron insertion to interrupt toxic open reading frames and the use of Rhizobium or yeast species that do not recognize the same cryptic promoters. Unfortunately, intron insertion must be attempted on a trial and error basis within full-length clones and may change the infection characteristics of the virus. We have developed a facile method that can detect multiple cryptic bacterial promoters within large virus genomes. These promoters can then be silenced to obtain infectious clones that can be manipulated in E. coli. Our strategy relies on the generation of a viral library which is cloned upstream of either an eGFP open reading frame for low-throughput analysis or chloramphenicol for next generation sequencing. Pokeweed mosaic virus (PkMV), a 9.5 Kb ssRNA potyvirus, was used as a proof of concept. We found 16 putative promoter regions within 150-250 bp library fragments throughout the PkMV genome. 5’RACE allowed identification of the promoter sequence within each fragment, and subsequent silencing produced infectious clones. Our results indicate that cryptic promoters are ubiquitous within large viral genomes and that promoter screening is a desirable first step when constructing a viral clone. Our method can be applied to large plant and animal viruses as well as any DNA sequence for which low level of background transcriptional activity is required.

由于存在诱导细菌毒性的神秘启动子序列，感染性植物病毒克隆的构建和操作具有挑战性。克服毒性的常用方法包括插入内含子以中断有毒的开放阅读框，以及使用不识别相同神秘启动子的根瘤菌或酵母菌。不幸的是，内含子插入必须在全长克隆中反复试验，并且可能会改变病毒的感染特征。我们开发了一种简便的方法，可以检测大型病毒基因组中的多个隐秘细菌启动子。然后可以沉默这些启动子以获得可以在大肠杆菌中操作的感染性克隆. 我们的策略依赖于病毒文库的生成，该文库被克隆到用于低通量分析的 eGFP 开放阅读框或用于下一代测序的氯霉素的上游。商陆花叶病毒 (PkMV)，一种 9.5 Kb ssRNA 病毒，被用作概念证明。我们在整个 PkMV 基因组的 150-250 bp 文库片段中发现了 16 个推定的启动子区域。5'RACE 允许识别每个片段内的启动子序列，随后的沉默产生感染性克隆。我们的结果表明隐性启动子在大型病毒基因组中无处不在，并且启动子筛选是构建病毒克隆时理想的第一步。我们的方法可应用于大型植物和动物病毒以及需要低水平背景转录活性的任何 DNA 序列。