Analysis of functions of Epstein-Barr virus-encoded small RNA EBER

EBV基因组的转基因通常是在靶向构建体转染到EBV感染细胞后通过同源重组完成的。然而，引入重组EBV基因组的标记基因可能会影响其病毒学特征。同源重组效率低是另一个问题。我们利用Cre/ loxp介导的位点特异性重组从重组病毒中去除标记基因，并将转基因引入EBV基因组。我们的目标是制造重组EBV，缺乏编码小的非转录rna EBERs （EBER-KO EBV）的病毒基因，EBER-KO EBV似乎与伯基特淋巴瘤的肿瘤发生有关。我们利用了一个突变的loxP位点，loxP2272，它在间隔区有两个碱基对突变（基因216:55,1998）。由于loxP2272不能与野生型loxP位点重组，因此可以通过Cre…More介导的基因替换，将野生型loxP和loxP2272两侧的DNA序列替换为具有相同loxP位点组合的DNA序列。携带新霉素耐药基因插入BamHI X区（XneoEBV）的重组Akata EBV受到EBER靶向。首先，在Akata细胞中通过同源重组，将XneoEBV的EBER基因替换为与野生型loxP和loxP2272相连的hygromycin-resistant （hygR）基因[EBER(-)hygREBV]。其次，在宿主细胞中通过cre介导的基因替换，将EBER(-)hygREBV的潮霉素盒替换为短的非编码DNA序列。随后，诱导病毒产生，并将产生的病毒用于感染ebv阴性Akata细胞。用G418筛选感染细胞，检测耐药细胞克隆是否存在缺乏hygR基因的重组EBV。在76个克隆中，有2个克隆发生了cre介导的基因替换，这两个克隆产生了大量缺乏hygR基因的EBER-KO病毒。从脐带血中纯化的B淋巴细胞分别感染等量的XneoEBV和EBER-KO EBV。尽管这些病毒表现出相当的传染性，但EBER-KO EBV的转化滴度比XneoEBV低约100倍，这表明EBERs对有效转化的贡献。少

Transgenesis of EBV genomes are usually accomplished via homologous recombination after transfection of targeting constructs into EBV-infected cells. However, the marker genes introduced into recombinant EBV genomes may affect their virological features. Low efficiency of homologous recombination is another problem. We utilized Cre/loxP-mediated site-specific recombination for removing marker genes from recombinant viruses, as well as for introducing transgenes into EBV genomes. We aimed to make recombinant EBV lacking viral genes encoding small, non-transcribed RNAs, EBERs (EBER-KO EBV), which appear to be responsible for Burkitt lymphoma tumorigenesis. We took advantage of a mutated loxP site, loxP2272, which has two base-pair mutations in its spacer region (Gene 216: 55, 1998). Since loxP2272 cannot recombine with wild-type loxP site, the region flanked with wild-type loxP and loxP2272 can be replaced with DNA sequences flanked with the same combination of loxP sites by means of Cre … More -mediated gene replacement. Recombinant Akata EBV carrying a neomycin-resistant gene inserted into the BamHI X region (XneoEBV) was subject to EBER targeting. First, EBER genes of XneoEBV were replaced with hygromycin-resistant (hygR) gene flanked with wild-type loxP and loxP2272 by means of homologous recombination in Akata cell [EBER(-)hygREBV]. Second, the hygromycin cassette of EBER(-)hygREBV was replaced with a short non-coding DNA sequence by means of Cre-mediated gene replacement in the host cells. Subsequently, virus production was induced, and the produced viruses were used for infecting EBV-negative Akata cells. Infected cells were selected by G418, and drug-resistant cell clones were examined for the presence of recombinant EBV lacking the hygR gene. Cre-mediated gene replacement occurred in 2 of out 76 clones, and those two cell clones produced a large quantity of EBER-KO virus lacking the hygR gene. B lymphocytes purified from cord blood were infected with equivalent amounts of XneoEBV and EBER-KO EBV, respectively. Although these viruses exhibited comparable infectivity, transforming titer of EBER-KO EBV was approximately 100 fold less than XneoEBV, demonstrating the contribution of EBERs for efficient transformation. Less