Kotwal G J, Moss B
Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20892.
Virology. 1988 Dec;167(2):524-37.
The principal objectives of this study were to analyze the structure and coding potential of a long segment of DNA missing from a previously isolated (B. Moss, E. Winters, and J. A. Cooper (1981) J. Virol. 40, 387-395) attenuated variant of vaccinia virus strain WR and to examine the precise changes in the genome accompanying the deletion. The sequences of a 14.5-kbp region located at the left end of the standard vaccinia virus genome, extending from within the inverted terminal repetition (ITR) of the HindIII C fragment to the end of the HindIII N fragment, and of a 3-kbp segment from a corresponding region of the variant genome were determined. A comparison of these sequences revealed that the variant contained a deletion of 12 kbp and an insertion of 2.1 kbp. The origin of the inserted DNA was traced to the HindIII B region by using oligonucleotide probes indicating that a transposition of unique DNA located adjacent to the right ITR had occurred. Structural analysis indicated no extensive homologies, nucleotide substitutions, additions, or deletions at the boundaries of the transposed DNA. Examination of the right end of the variant genome indicated that a copy of the transposed DNA was still present and, therefore, the length of the ITR had been increased by 2.1 kbp. The variant genome could have formed by a mechanism that resulted in the replacement of a 22-kbp left-terminal fragment with a 12-kbp right-terminal fragment. The DNA missing from the variant and contained within the standard vaccinia virus WR genome contains 17 contiguous open reading frames (ORFs), all of which are directed leftward and apparently not required for replication in cultured cells. One deleted ORF has a 60% sequence similarity to another gene encoding a 42,000-Da protein present within the ITR suggesting that duplications have previously occurred during the evolution of vaccinia virus. Another deleted ORF has a 39% sequence similarity to a complement 4b binding protein. The transposed DNA contains two complete ORFs one of which has a 40% identity to a cowpox gene and a 30% identity to a family of plasma serine protease inhibitors.
本研究的主要目的是分析痘苗病毒株WR先前分离出的（B.莫斯、E.温特斯和J.A.库珀（1981年）《病毒学杂志》40卷，387 - 395页）减毒变异株中缺失的一段长DNA片段的结构和编码潜力，并检查伴随缺失在基因组中发生的精确变化。测定了位于标准痘苗病毒基因组左端的一个14.5千碱基对区域的序列，该区域从HindIII C片段的反向末端重复序列（ITR）内部延伸至HindIII N片段的末端，以及变异基因组相应区域的一个3千碱基对片段的序列。这些序列的比较显示，该变异株包含一个12千碱基对的缺失和一个2.1千碱基对的插入。通过使用寡核苷酸探针将插入DNA的起源追溯到HindIII B区域，表明发生了位于右ITR相邻处的独特DNA的转座。结构分析表明在转座DNA的边界处没有广泛的同源性、核苷酸取代、添加或缺失。对变异基因组右端的检查表明转座DNA的一个拷贝仍然存在，因此ITR的长度增加了2.1千碱基对。变异基因组可能是通过一种机制形成的，该机制导致用一个12千碱基对的右末端片段替换了一个22千碱基对的左末端片段。变异株中缺失且包含在标准痘苗病毒WR基因组内的DNA含有17个连续的开放阅读框（ORF），所有这些阅读框都向左定向，并且显然不是在培养细胞中复制所必需的。一个缺失的ORF与编码存在于ITR内的一种42,000道尔顿蛋白质的另一个基因有60%的序列相似性，这表明痘苗病毒进化过程中先前发生过重复。另一个缺失的ORF与一种补体4b结合蛋白有39%的序列相似性。转座DNA包含两个完整的ORF，其中一个与牛痘基因有40%的同一性，与一组血浆丝氨酸蛋白酶抑制剂有30%的同一性。
