Possible mechanism of adenovirus generation from a cloned viral genome tagged with nucleotides at its ends
Possible mechanism of adenovirus generation from a cloned viral genome tagged with nucleotides at its ends
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DOI：
10.1111/j.1348-0421.2006.tb03829.x
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发表时间：
2006-01-01
影响因子：
2.6
通讯作者：
Saito, Izmu
中科院分区：
文献类型：
--
作者：
Fukuda, Hiromitsu;Terashima, Miho;Saito, Izmu
已知整个克隆的人5型腺病毒（Ad 5）基因组在转染到293细胞中后，当通过用适当的限制酶消化暴露基因组的两端时，能够产生感染性病毒。然而，当基因组的一端或两端用核苷酸标记并且不完整时，病毒基因组的标记端在病毒克隆的产生期间是否保持标记或校正为完整尚不清楚，并且如果发生这样的寡核苷酸去除，病毒如何去除这些标记序列并从而恢复其正确的结构？在这里，我们在我们的半定量研究中表明，病毒克隆的生成效率根据两端核苷酸标签的长度而降低，并且在病毒生成过程中，两个寡核苷酸标签都被精确地去除，并恢复了适当的末端序列。有趣的是，病毒基因组的一端被标记，而另一端连接约12-kb的序列，确实以降低但显著的效率产生完整的病毒克隆。从这些结果中，我们在这里提出了一个可能的机制，即终端蛋白脱氧胞苷复合物进入酶切结束，并达到脱氧鸟嘌呤在体内DNA合成的起始位置。位于一端的复制起点深深嵌入双链DNA中，可以被两个循环的单向全长DNA合成激活，该合成由另一个暴露的复制起点启动，距离约30个酶。我们还描述了新的盒式蛋白酶，其不仅可以使用PacI，而且可以使用BstBI来构建腺病毒载体，而不会降低构建效率。
The entire cloned human adenovirus type 5 (Ad5) genome is known to be able to generate infectious virus after transfection into 293 cells when the both ends of the genome are exposed by digestion with appropriate restriction enzymes. However, when one or both ends of the genome are tagged with nucleotides and are not intact, whether the tagged end of the viral genome was remained tagged or corrected to be intact during the generation of viral clones has been unclear and, if such oligonucleotide removal occurs, how does the virus remove these tagged sequences and thereby restore its proper structure? Here, we show in our semi-quantitative study that the generation efficiency of virus clones decreases depending on the length of nucleotide tags at the both ends and that both the oligonucleotide tags were precisely removed during virus generation with restoration of the proper terminal sequences. Interestingly the viral genome of which one end was tagged, while the other was attached about 12-kb sequences, did generate intact viral clones at a reduced but significant efficiency. From these results, we here propose a possible mechanism whereby the terminal-protein-deoxycytidine complex enters from the enzyme-cleaved end and reaches deoxyguanine at the initiating position of DNA synthesis in vivo. A replication origin at one end, embedded deeply in double-stranded DNA, can be activated by two cycles of one-directional full-length DNA synthesis initiated by the other exposed replication origin about 30 kilobases away. We also describe new cassette cosmids which can use not only PacI but also BstBI for construction of an adenovirus vector, without reducing construction efficiency.