Johnson P A, Wang M J, Friedmann T
Department of Pediatrics, University of California, San Diego, La Jolla 92093-0634.
J Virol. 1994 Oct;68(10):6347-62. doi: 10.1128/JVI.68.10.6347-6362.1994.
Derivatives of herpes simplex virus type 1 (HSV-1) have elicited considerable interest as gene transfer vectors because of their ability to infect a wide range of cell types efficiently, including fully differentiated neurons. However, it has been found that infection of many types of cell with vectors derived from replication-defective mutants of HSV-1 is associated with cytopathic effects (CPE). We have previously shown that viral gene expression played an important role in the induction of CPE caused by an HSV-1 mutant deleted for the essential immediate-early gene 3 (IE 3) (P.A. Johnson, A. Miyanohara, F. Levine, T. Cahill, and T. Friedmann, J. Virol. 66:2952-2965, 1992). We have investigated which viral genes might be responsible for CPE by comparing the ability of each of the individual genes expressed by an IE 3 deletion mutant during a nonproductive infection to inhibit biochemical transformation after cotransfection of BHK or CV-1 cells with a selectable marker gene. Transfection of IE genes 1,2, and 4 individually all caused a marked inhibition of colony formation, while transfection of IE 5 and the large subunit of ribonucleotide reductase had little effect. These results suggested that it would be necessary to mutate or reduce the expression of nearly all HSV-1 IE genes to reduce virus-induced CPE. Therefore, we have used VP16 mutants, which are unable to transinduce IE gene expression (C. I. Ace, T. A. McKee, J. M. Ryan, J. M. Cameron, and C. M. Preston, J. Virol. 63:2260-2269, 1989), to derive two replication-defective strains: 14H delta 3, which is deleted for both copies of IE 3, and in 1850 delta 42, which has a deletion in the essential early gene UL42. The IE 3-VP16 double mutant, 14H delta 3, is significantly less toxic than a single IE 3 deletion mutant over a range of multiplicities of infection, as measured in a cell-killing assay, and has an enhanced ability to persist in infected cells in a biologically retrievable form. In contrast, the UL42-VP16 double mutant, in 1850 delta 42, showed reduced toxicity only at low multiplicities of infection. To test the role of the virion host shutoff function as an additional candidate to influence virus-induced CPE, we have introduced a large insertion mutation into the virion host shutoff gene of an IE 3 deletion mutant and the double mutant 14H delta 3.(ABSTRACT TRUNCATED AT 400 WORDS)
1型单纯疱疹病毒（HSV - 1）的衍生物作为基因转移载体引起了广泛关注，因为它们能够有效感染多种细胞类型，包括完全分化的神经元。然而，已发现用源自HSV - 1复制缺陷型突变体的载体感染多种细胞会导致细胞病变效应（CPE）。我们之前已表明病毒基因表达在由缺失必需立即早期基因3（IE 3）的HSV - 1突变体引起的CPE诱导中起重要作用（P.A.约翰逊、A.宫原、F.莱文、T.卡希尔和T.弗里德曼，《病毒学杂志》66:2952 - 2965，1992）。我们通过比较在非生产性感染期间由IE 3缺失突变体表达的各个基因与选择标记基因共转染BHK或CV - 1细胞后抑制生化转化的能力，研究了哪些病毒基因可能导致CPE。单独转染IE基因1、2和4均显著抑制集落形成，而转染IE 5和核糖核苷酸还原酶大亚基的影响较小。这些结果表明，可能有必要使几乎所有HSV - 1 IE基因发生突变或降低其表达以减少病毒诱导的CPE。因此，我们使用了不能反式诱导IE基因表达的VP16突变体（C.I.艾斯、T.A.麦基、J.M.瑞安、J.M.卡梅伦和C.M.普雷斯顿，《病毒学杂志》63:2260 - 2269，1989）来获得两种复制缺陷型毒株：14H delta 3，其两个IE 3拷贝均缺失；以及1850 delta 42，其必需早期基因UL42存在缺失。在细胞杀伤试验中测定，IE 3 - VP16双突变体14H delta 3在一系列感染复数下的毒性明显低于单个IE 3缺失突变体，并且以生物学可回收形式在感染细胞中持续存在的能力增强。相比之下，UL42 - VP16双突变体1850 delta 42仅在低感染复数下显示出毒性降低。为了测试病毒体宿主关闭功能作为影响病毒诱导CPE的另一个候选因素的作用，我们在IE 3缺失突变体和双突变体14H delta 3的病毒体宿主关闭基因中引入了一个大的插入突变。（摘要截短于400字）
