Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi-Do, South Korea.
Department of Pharmacy, College of Pharmacy and Institute of Pharmaceutical Sciences, CHA University, Gyeonggi-Do, South Korea
J Virol. 2021 Jan 13;95(3). doi: 10.1128/JVI.01643-20.
PP7 is a leviphage, with a single-stranded RNA genome, that infects PAO1. A reverse genetic system for PP7 was previously created by using reverse-transcribed cDNA (PP7) from a virion-derived RNA genome. Here, we have found that the PP7 cDNA contained 20 nucleotide differences from the PP7 genome sequence deposited in the database. We created another reverse genetic system exploiting chemically synthesized cDNA (PP7) based on the database sequence. Unlike PP7, which yielded infectious PP7 virions, PP7-derived particles were incapable of plaque formation on PAO1 cells, which was restored in the PAO1 cells expressing the maturation protein (MP) from PP7 Using this reverse genetic system, we revealed two amino acid residues involved in the known roles of MP (i.e., adsorption and genome replication), fortuitously providing a lesson that the viral RNA genome sequencing needs functional verification, possibly by a reverse genetic system. The biological significance of RNA phages has been largely ignored, ironically, because few studies have focused on RNA phages. As an initial attempt to properly represent RNA phages in the phageome, we previously created, by using reverse-transcribed cDNA, a reverse genetic system for the small RNA phage PP7, which infects the opportunistic human pathogen We report another system by using chemically synthesized cDNA based on the database genome that has 20 nucleotide differences from the previous cDNA. Investigation of those cDNA-derived phage virions revealed that two amino acids of the maturation protein are crucial for the normal phage lifecycle at different steps. Our study provides insight into the molecular basis for the RNA phage lifecycle and a lesson that the RNA genome sequencing needs to be carefully validated by cDNA-based phage assembly systems.
PP7 是一种单链 RNA 基因组的利福平噬菌体，感染 PAO1。先前已创建了用于 PP7 的反向遗传学系统，该系统使用源自病毒衍生 RNA 基因组的逆转录 cDNA (PP7)。在这里，我们发现 PP7 cDNA 与数据库中提交的 PP7 基因组序列有 20 个核苷酸差异。我们创建了另一个反向遗传学系统，该系统利用基于数据库序列的化学合成 cDNA (PP7)。与产生有感染力的 PP7 病毒粒子的 PP7 不同，源自 PP7 的粒子不能在 PAO1 细胞上形成噬菌斑，而在表达来自 PP7 的成熟蛋白 (MP) 的 PAO1 细胞中得以恢复。使用这种反向遗传系统，我们揭示了两个氨基酸残基参与了已知的 MP 作用（即吸附和基因组复制），这偶然提供了一个教训，即病毒 RNA 基因组测序需要功能验证，可能通过反向遗传系统。讽刺的是，由于很少有研究集中在 RNA 噬菌体上，因此 RNA 噬菌体的生物学意义在很大程度上被忽视了。作为在噬菌体组中正确表示 RNA 噬菌体的初步尝试，我们先前使用逆转录 cDNA 创建了一种用于感染机会性病原体的小型 RNA 噬菌体 PP7 的反向遗传学系统。我们报告了另一种基于数据库基因组的化学合成 cDNA 系统，该系统与以前的 cDNA 有 20 个核苷酸差异。对那些 cDNA 衍生的噬菌体病毒粒子的研究表明，成熟蛋白的两个氨基酸在不同步骤的正常噬菌体生命周期中至关重要。我们的研究提供了对 RNA 噬菌体生命周期的分子基础的深入了解，并提供了一个教训，即 RNA 基因组测序需要通过基于 cDNA 的噬菌体组装系统进行仔细验证。
