Schmidt M, Rethwilm A
Institut für Virologie und Immunbiologie, Universität Würzburg, Germany.
Virology. 1995 Jun 20;210(1):167-78. doi: 10.1006/viro.1995.1328.
Replication-competent retroviral vectors (pFOV-1 to -3 and -7) were constructed on the basis of an infectious human foamy virus molecular clone which has deletions in the U3 region of the long terminal repeat and in the 3' region of the genome, previously identified to be nonessential for virus replication in vitro. The CAT and luciferase indicator genes were expressed as C-terminal fusion proteins to 215 amino acids of the viral Bet protein in the pFOV-1 vector. Introduction of the foot-and-mouth disease 2A protease sequence between the truncated bet coding sequence and the cloning site for the insertion of foreign genes in the pFOV-7 vector resulted in self-cleaving of the recombinant fusion protein. Alternatively, an internal ribosomal binding site was introduced, allowing expression of authentic foreign protein (pFOV-2 and -3 vectors). DNA fragments derived from the mouse hepatitis virus surface gene up to the length of 1.3 kb were inserted into pFOV-1. The vector constructs gave rise to viruses which were fully infectious in diploid human fibroblasts and recombinant viruses stably expressed high levels of foreign protein indicating that the pFOV vectors may be useful tools to study the effects of proteins of interest at least in tissue culture cells.
具有复制能力的逆转录病毒载体（pFOV - 1至 - 3和 - 7）是基于一种感染性人类泡沫病毒分子克隆构建的，该克隆在长末端重复序列的U3区域和基因组的3'区域存在缺失，此前已确定这些区域对病毒体外复制并非必需。在pFOV - 1载体中，氯霉素乙酰转移酶（CAT）和荧光素酶报告基因作为病毒Bet蛋白215个氨基酸的C末端融合蛋白表达。在pFOV - 7载体中，在截短的bet编码序列与用于插入外源基因的克隆位点之间引入口蹄疫2A蛋白酶序列，导致重组融合蛋白的自我切割。另外，引入了内部核糖体结合位点，允许表达天然外源蛋白（pFOV - 2和 - 3载体）。将源自小鼠肝炎病毒表面基因、长度达1.3 kb的DNA片段插入pFOV - 1。这些载体构建体产生的病毒在二倍体人成纤维细胞中具有完全感染性，并且重组病毒稳定表达高水平的外源蛋白，这表明pFOV载体可能是至少在组织培养细胞中研究感兴趣蛋白质作用的有用工具。
