Khalique Hena, López Marco Jorge, Lim Filip
Departamento de Biología Molecular, Universidad Autónoma de Madrid, Cantoblanco, Madrid, Spain.
J Gene Med. 2016 Oct;18(10):302-311. doi: 10.1002/jgm.2929.
Although herpes simplex virus type 1 (HSV-1) has outstanding properties for gene delivery vectors and its genome is available in bacterial artificial chromosomes (BACs) for mutagenesis studies, one impediment is the presence of approximately 15.4 kb of DNA sequences that are duplicated in the HSV-1 genome, complicating vector construction and stability.
As a useful platform for building HSV-1 vectors, we have constructed a fully haploid HSV-1 genome BAC by deletion of one of these repeats, confirming that viral propagation in culture is not impaired. We used this ΔIR mutant to subsequently investigate whether the insertion of tetracycline-responsive tetO elements into the ICP34.5-ICP0 gene region can be used to control HSV-1 lytic replication.
The results of the present study show that ΔIR mutants deleted for ICP34.5 are viable for replication but not when the ICP0 promoter is also disrupted, thus indicating that regulation of infected cell protein 0 (ICP0) levels in the absence of ICP34.5 could be a viable means for controlling growth of HSV-1 vectors. Surprisingly, however, the tetO elements inserted into the ICP0 promoter did not confer ligand responsiveness to growth or ICP0 expression. Further analysis by transfection experiments revealed that ICP0 itself interferes with the tetracycline switch and reduces the the inducibility of this system.
Our new haploid HSV-1 BAC is a useful platform for building multiply deleted HSV-1 vectors. Deletion of the gene for ICP34.5 in this backbone renders viral growth dependent on ICP0, although ICP0 expression could not be regulated by tet-responsive transcriptional regulators.
尽管单纯疱疹病毒1型（HSV-1）作为基因传递载体具有出色的特性，并且其基因组可用于细菌人工染色体（BAC）进行诱变研究，但一个障碍是HSV-1基因组中存在约15.4 kb的重复DNA序列，这使载体构建和稳定性变得复杂。
作为构建HSV-1载体的有用平台，我们通过删除其中一个重复序列构建了一个完全单倍体的HSV-1基因组BAC，证实其在培养中的病毒传播未受损害。我们使用这个ΔIR突变体随后研究将四环素反应性tetO元件插入ICP34.5-ICP0基因区域是否可用于控制HSV-1的裂解复制。
本研究结果表明，缺失ICP34.5的ΔIR突变体可进行复制，但当ICP0启动子也被破坏时则不能，这表明在没有ICP34.5的情况下调节感染细胞蛋白0（ICP0）水平可能是控制HSV-1载体生长的可行方法。然而，令人惊讶的是，插入ICP0启动子的tetO元件并未赋予对生长或ICP0表达的配体反应性。通过转染实验进一步分析发现，ICP0本身会干扰四环素开关并降低该系统的诱导性。
我们新的单倍体HSV-1 BAC是构建多重缺失HSV-1载体的有用平台。在这个骨架中删除ICP34.5基因使病毒生长依赖于ICP0，尽管ICP0的表达不能由四环素反应性转录调节因子调控。
