Xu Z Z, Hyatt A, Boyle D B, Both G W
Division of Biomolecular Engineering, CSIRO, North Ryde, NSW, Australia.
Virology. 1997 Mar 31;230(1):62-71. doi: 10.1006/viro.1997.8452.
An ovine adenovirus which may be the prototype for a new group of adenoviruses has been engineered as a gene transfer vector. One recombinant containing a 0.95-kb insertion expressed a sheep parasite antigen from the ovine adenovirus major late promoter and tripartite leader sequences. It was shown that insertions of at least 4.3 kb were tolerated at either one of two sites in the genome without the introduction of a compensating deletion. The unique structure of this viral genome was further emphasized by the discovery that four open reading frames at the right hand end show significant identity to each other but not to other sequences in the databases. Two other unrelated open reading frames were also present. RT-PCR analysis identified two transcripts in this region which were derived from a promoter which was located very close to, or within the ITR sequence. Splicing removed all but the first and last of the ORFs from these RNAs, suggesting that some sequences might be nonessential for replication in vitro. A approximately 2-kb deletion, which removed or truncated the internal reading frames was introduced into the region without affecting virus viability. The carrying capacity of OAV recombinants should therefore be at least 6.3 kb. The relative packaging capacity of OAV (114%) therefore exceeds that of Ad5 (105%), although a comparison of virus particle sizes by electron microscopy showed that OAV was smaller than Ad5. These studies improve the potential utility of OAV as a gene transfer vector.
一种可能是新型腺病毒原型的绵羊腺病毒已被改造为基因转移载体。一种含有0.95 kb插入片段的重组体从绵羊腺病毒主要晚期启动子和三联前导序列表达了一种绵羊寄生虫抗原。结果表明，在基因组的两个位点中的任何一个位点，至少4.3 kb的插入片段都能被耐受，而无需引入补偿性缺失。该病毒基因组的独特结构通过以下发现得到进一步强调：右手端的四个开放阅读框彼此之间具有显著的同源性，但与数据库中的其他序列没有同源性。还存在另外两个不相关的开放阅读框。RT-PCR分析在该区域鉴定出两个转录本，它们来源于一个位于非常靠近ITR序列或在ITR序列内的启动子。剪接从这些RNA中去除了除第一个和最后一个开放阅读框之外的所有开放阅读框，这表明某些序列可能对于体外复制是非必需的。将一个大约2 kb的缺失片段引入该区域，该缺失片段去除或截断了内部阅读框，但不影响病毒的活力。因此，绵羊腺病毒重组体的携带能力至少应为6.3 kb。因此，绵羊腺病毒（114%）的相对包装能力超过了腺病毒5型（105%），尽管通过电子显微镜对病毒颗粒大小进行比较显示绵羊腺病毒比腺病毒5型小。这些研究提高了绵羊腺病毒作为基因转移载体的潜在实用性。
