Parcells M S, Anderson A S, Morgan T W
Department of Animal Science and Agricultural Biochemistry, College of Agricultural Sciences, University of Delaware, Newark 19717-1303, USA.
J Virol. 1995 Dec;69(12):7888-98. doi: 10.1128/JVI.69.12.7888-7898.1995.
We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.
我们之前报道了马立克氏病病毒（MDV）毒株的构建，这些毒株在病毒基因组独特短区域（US）中映射的各种基因发生了突变（J.L. 坎特洛、A.S. 安德森、A. 弗朗切斯科尼和R.W. 摩根，《病毒学杂志》65:1584 - 1588，1991；M.S. 帕西尔斯、A.S. 安德森和R.W. 摩根，《病毒基因》9:5 - 13，1994；M.S. 帕西尔斯、A.S. 安德森和R.W. 摩根，《病毒学杂志》68:8239 - 8253，1994）。这些毒株是通过使用在鸡胚成纤维细胞中生长良好的高传代水平1型MDV毒株构建的。尽管亲本病毒和突变病毒在细胞培养中能够生长，但体内研究受到这些毒株在鸡体内生长不良的限制。所研究突变体之一缺失了4.5 kbp的US区域DNA，并在缺失位点插入了大肠杆菌的lacZ基因。该缺失去除了与1型单纯疱疹病毒US1、US2和US10基因同源的MDV基因以及三个MDV特异性开放阅读框。我们现在报道在高致瘤性RB1B毒株的US区域构建了一个具有类似缺失的突变MDV。这个突变体，RB1B delta 4.5lac，在已建立的鸡胚成纤维细胞中的生长受损情况与之前描述的缺乏功能性US1基因的MDV相似。在鸡体内，RB1B delta 4.5lac表现出早期溶细胞感染、死亡率、肿瘤发生率和水平传播降低。从RB1B delta 4.5lac诱导的肿瘤中建立了几个淋巴母细胞系，从这些细胞系重新激活的病毒是LacZ阳性。这些结果表明，缺失的基因对于鸡T细胞的转化或潜伏的建立和维持并非必需。基于在细胞培养和体内观察到的RB1B delta 4.5lac的生长受损情况，我们得出结论，这些基因的缺失影响了MDV的裂解复制。这是在RB1B致瘤毒株中构建的首个MDV突变体，本文所述方法为直接研究MDV编码的致瘤性决定因素提供了途径。
