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

EBV基因组的转基因通常在将靶向构建体转染到EBV感染的细胞中后通过同源重组来完成。然而，标记基因导入重组EBV基因组可能会影响其病毒学特征。同源重组的低效率是另一个问题。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，以及将转基因引入EBV基因组。我们的目的是使重组EBV缺乏编码小的非转录RNA的病毒基因，EBERs（EBER-KO EBV），这似乎是负责伯基特淋巴瘤肿瘤发生。我们利用了突变的loxP位点loxP 2272，其在间隔区中具有两个碱基对突变（Gene 216：55，1998）。由于loxP 2272不能与野生型loxP位点重组，所以可以通过Cre将野生型loxP和loxP 2272侧翼的区域替换为侧翼具有相同loxP位点组合的DNA序列 关于我们 介导的基因置换。将携带插入BamHI X区的新霉素抗性基因的重组Akata EBV（XneoEBV）进行EBER靶向。首先，XneoEBV的EBER基因通过在Akata细胞中的同源重组被侧接野生型loxP和loxP 2272的潮霉素抗性（hygR）基因替换[EBER（-）hygREBV]。第二，通过Cre介导的宿主细胞中的基因置换，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。通过G418选择感染的细胞，并检查耐药细胞克隆是否存在缺乏hygR基因的重组EBV。Cre介导的基因置换发生在76个克隆中的2个中，并且这两个细胞克隆产生了大量缺乏hygR基因的EBER-KO病毒。分别用等量的XneoEBV和EBER-KO EBV感染从脐带血纯化的B淋巴细胞。尽管这些病毒表现出相当的感染性，但EBER-KO EBV的转化滴度比XneoEBV低约100倍，证明EBER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