Thomas S K, Lilley C E, Latchman D S, Coffin R S
Department of Molecular Pathology, The Windeyer Institute of Medical Sciences, University College London, London W1P 6DB, England, UK.
J Virol. 1999 Sep;73(9):7399-409. doi: 10.1128/JVI.73.9.7399-7409.1999.
Herpes simplex virus (HSV) has often been suggested for development as a vector, particularly for the nervous system. Considerable evidence has shown that for use of HSV as a vector, immediate-early (IE) gene expression must be minimized or abolished, otherwise such vectors are likely to be highly cytotoxic. Mutations of vmw65 which abolish IE promoter transactivating activity may also be included to reduce IE gene expression generally. However, when vmw65 mutations are combined with an IE gene deletion, such viruses are hard to propagate, even on cells which otherwise complement the IE gene deletion effectively. We have found that vmw65 mutants can be effectively grown on cell lines expressing equine herpesvirus 1 gene 12, a non-HSV homologue of vmw65 with little sequence similarity to its HSV counterpart. This prevents repair by homologous recombination of vmw65 mutations in the virus, which would occur if mutations were complemented by vmw65 itself. The gene 12 protein is not packaged into HSV virions, which is important if viruses grown on such cells are to be used as vectors. These results not only further strengthen the evidence for direct functional homology between and similar modes of action of the two proteins but have allowed the generation of gene 12-containing cell lines in which ICP4 and ICP27 expression is induced by virus infection (probably by ICP0) and which give efficient growth of viruses deficient in ICP27, ICP4, and vmw65 (the viruses also have ICP34.5/ORFP deleted). Efficient growth of such viruses has not previously been possible. As these viruses are highly deficient in IE gene expression generally, such virus-cell line combinations may provide an alternative to HSV vectors with deletions of all four of the regulatory IE genes which, for optimal growth, require cell lines containing all four IE genes but which are hard to generate due to the intrinsic cytotoxicity of each of the proteins.
单纯疱疹病毒（HSV）常被提议开发为一种载体，尤其是用于神经系统。大量证据表明，对于将HSV用作载体而言，立即早期（IE）基因表达必须降至最低或消除，否则此类载体可能具有高度细胞毒性。也可纳入消除IE启动子反式激活活性的vmw65突变，以总体上降低IE基因表达。然而，当vmw65突变与IE基因缺失相结合时，此类病毒很难增殖，即使在其他方面能有效互补IE基因缺失的细胞上也是如此。我们发现，vmw65突变体能够在表达马疱疹病毒1型基因12的细胞系上有效生长，该基因是vmw65的非HSV同源物，与它的HSV对应物几乎没有序列相似性。这可防止病毒中vmw65突变通过同源重组进行修复，如果突变由vmw65自身互补，这种修复就会发生。基因12蛋白不会被包装进HSV病毒粒子中，这对于在这类细胞上生长的病毒用作载体来说很重要。这些结果不仅进一步加强了两种蛋白之间直接功能同源性及其相似作用模式的证据，还使得能够产生含基因12的细胞系，在该细胞系中，ICP4和ICP27的表达可由病毒感染诱导（可能是由ICP0诱导），并且能使缺乏ICP27、ICP4和vmw65（这些病毒也缺失ICP34.5/ORFP）的病毒高效生长。此前这类病毒不可能实现高效生长。由于这些病毒总体上在IE基因表达方面高度缺陷，此类病毒 - 细胞系组合可能为所有四个调控性IE基因均缺失的HSV载体提供一种替代方案，对于最佳生长而言，这些载体需要含有所有四个IE基因的细胞系，但由于每种蛋白固有的细胞毒性，这些细胞系很难产生。
