Swaminathan S, Tomkinson B, Kieff E
Department of Medicine, Harvard Medical School, Boston, MA 02115.
Proc Natl Acad Sci U S A. 1991 Feb 15;88(4):1546-50. doi: 10.1073/pnas.88.4.1546.
Strains of Epstein-Barr virus (EBV) with deletions of the small RNA (EBER) genes were made by homologous recombination using the EBV P3HR-1 strain, which has undergone deletion of the essential transforming gene that encodes the EBV nuclear antigen, EBNA-2, and a DNA fragment that was wild type at the EBNA-2 locus but from which the EBER genes had been deleted. Even though the EBER and EBNA-2 genes are separated by 40 kilobases, selection for transforming P3HR-1 recombinants that required a restored EBNA-2 gene resulted in 20% cotransfer of the EBER deletion. EBER-deleted recombinants transformed primary B lymphocytes into lymphoblastoid cell lines (LCLs), which were indistinguishable form LCLs transformed by wild-type EBV in their proliferation, in latency-associated EBV gene expression, and in their permissiveness for EBV replication cycle gene expression. EBER-deleted virus from infected LCL clones could infect and growth-transform primary B lymphocytes. These procedures should be applicable to the construction of other EBV recombinants within 40 kilobases of the EBNA-2 gene. The EBER-deleted EBV recombinants should be useful in further evaluating the role of EBERs in EBV infection.
通过同源重组，利用EBV P3HR - 1毒株构建了缺失小RNA（EBER）基因的爱泼斯坦-巴尔病毒（EBV）毒株。P3HR - 1毒株已缺失了编码EBV核抗原EBNA - 2的必需转化基因，以及一个在EBNA - 2基因座处为野生型但EBER基因已被删除的DNA片段。尽管EBER基因和EBNA - 2基因相隔40千碱基，但选择需要恢复EBNA - 2基因的转化P3HR - 1重组体时，导致EBER缺失的共转移率为20%。缺失EBER的重组体将原代B淋巴细胞转化为淋巴母细胞系（LCLs），这些LCLs在增殖、潜伏相关EBV基因表达以及对EBV复制周期基因表达的允许性方面，与野生型EBV转化的LCLs没有区别。来自感染LCL克隆的缺失EBER的病毒能够感染并生长转化原代B淋巴细胞。这些方法应适用于构建EBNA - 2基因40千碱基范围内的其他EBV重组体。缺失EBER的EBV重组体应有助于进一步评估EBERs在EBV感染中的作用。
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