Multiloci Manipulation of Baculovirus Genome Reveals the Pivotal Role of Homologous Regions in Viral DNA Replication, Progeny Production, and Enhancing Transcription
Multiloci Manipulation of Baculovirus Genome Reveals the Pivotal Role of Homologous Regions in Viral DNA Replication, Progeny Production, and Enhancing Transcription
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杆状病毒基因组的多位点操作揭示了同源区域在病毒 DNA 复制、后代产生和增强转录中的关键作用
DOI：
10.1021/acssynbio.1c00303
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发表时间：
2021-12-21
影响因子：
4.7
通讯作者：
Hu, Zhihong
中科院分区：
文献类型：
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作者：
Hu, Hengrui;Pan, Kai;Hu, Zhihong
病毒基因组的工程设计促进了对病毒的基础研究和应用研究。然而，具有大DNA基因组的病毒（如杆状病毒、疱疹病毒和痘病毒）的DNA的多位点操作在技术上是具有挑战性的，特别是对于高度同源或重复的序列。同源异型区（hrs）在许多大型DNA病毒中具有多个拷贝，在病毒的生命周期中起着关键作用。在这里，我们使用合成生物学研究杆状病毒hrs的基本功能，通过对苜蓿银纹夜蛾多核型多角体病毒（AcMNPV）DNA进行多位点操作，该DNA包含分散在基因组中的8个hrs。使用酵母中的转化相关重组，我们产生了重组AcMNPV基因组，其中我们删除了所有hr或保留了单个hr（hr 1，hr 2或hr 3）。将合成的病毒基因组转染到宿主细胞中后，拯救了感染性病毒，并表征了它们的复制特征。结果表明，删除所有hr严重损害病毒DNA复制和子代产生，而仅保留单个hr对于有效的病毒DNA复制和子代产生是必需的。具有hr 2或hr 3的合成病毒显示出与亲本病毒相似的生长曲线。转录组学分析表明，hr1，hr2和hr3可以增强基因转录的14.6 kb，13.8 kb和29.8 kb的周围区域内，分别。总体而言，这项研究揭示了合成生物学在大型DNA病毒的多位点工程和功能研究中的优势。此外，我们对hrs的研究结果将有助于杆状病毒表达载体的设计和改进。
The engineering of viral genomes facilitates both fundamental and applied research on viruses. However, the multiloci manipulation of DNAs of viruses with large DNA genomes, such as baculoviruses, herpesviruses, and poxviruses, is technically challenging, particularly for highly homologous or repetitive sequences. Homologous regions (hrs) have multiple copies in many large DNA viruses and play pivotal roles in the viral life cycle. Here, we used synthetic biology to investigate the fundamental function of baculoviral hrs by conducting multiloci manipulation of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) DNA that contains eight hrs scattered in the genome. Using transformation-associated recombination in yeast, we generated recombinant AcMNPV genomes in which we deleted all hrs or retained a single hr (hr1, hr2, or hr3). Infectious viruses were rescued after transfecting the synthetic viral genomes into host cells, and their replication features were characterized. The results demonstrated that deletion of all hrs severely compromised viral DNA replication and progeny production, whereas retaining only a single hr was essential for efficient viral DNA replication and progeny production. The synthetic virus with hr2 or hr3 showed a growth curve similar to that of the parental virus. Transcriptomic analysis revealed that hr1, hr2, and hr3 could enhance gene transcription within a surrounding region of 14.6 kb, 13.8 kb, and 29.8 kb, respectively. Overall, this study revealed the advantages of synthetic biology in multiloci engineering and functional studies of large DNA viruses. In addition, our findings on hrs will be helpful for the design and improvement of baculovirus-based expression vectors.