Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector
Human beta interferon scaffold attachment region inhibits de novo methylation and confers long-term, copy number-dependent expression to a retroviral vector
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DOI：
10.1128/jvi.74.6.2671-2678.2000
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发表时间：
2000-03-01
影响因子：
5.4
通讯作者：
Plavec, I
中科院分区：
文献类型：
--
作者：
Dang, Q;Auten, J;Plavec, I
基于莫洛尼鼠白血病病毒的逆转录病毒载体表达在CEMSS T细胞的长期体外培养期间逐渐丧失。然而，当将人β干扰素支架附着区（IFN-SAR）插入载体中紧邻3'长末端重复序列（LTR）的上游时，表达在研究期间（4个月）得以维持。逆转录病毒载体感染的CEMSS细胞的克隆分析显示，含SAR的逆转录病毒载体表达水平与前病毒拷贝数正相关（P < 0.0001），而对照载体感染的克隆中前病毒拷贝数与表达水平之间没有相关性。33%感染对照载体的CEMSS细胞克隆显示5' LTR区域部分或完全甲基化的证据。与此形成鲜明对比的是，我们在用含SAR的载体感染的克隆中没有检测到甲基化。为了证明甲基化对逆转录病毒载体表达的直接抑制作用，我们用体外甲基化前病毒DNA转染了293细胞。在瞬时转染的细胞中，甲基化LTR的表达减少，但不完全抑制，无论是否存在IFN-SAR序列。然而，在稳定转染的细胞中，甲基化完全消除了对照载体的表达，但不包括含有SAR的载体。此外，含SAR的载体的表达随时间稳定，表明SAR序列减轻载体的甲基化介导的转录抑制的能力。这项研究扩展了我们对逆转录病毒载体甲基化失活机制的理解，并为SAR元件的功能作用提供了见解。
Moloney murine leukemia virus-based retroviral vector expression is gradually lost during prolonged in vitro culture of CEMSS T cells. However, when the human beta interferon scaffold attachment region (IFN-SAR) was inserted into the vector immediately upstream of the 3' long terminal repeat (LTR), expression was maintained for the length of the study (4 months). Clonal analysis of the retrovirus vector-infected CEMSS cells showed that SAR-containing retroviral vector expression levels were positively correlated with the proviral copy numbers (P < 0.0001), while there was no correlation between the proviral copy numbers and expression levels in control vector-infected clones. Thirty-three percent of the CEMSS cell clones infected with the control vector showed evidence of partial or complete methylation in the 5' LTR region. In sharp contrast, we detected no methylation in the clones infected with the SAR-containing vector. To demonstrate a direct inhibitory effect of methylation on retroviral vector expression, we have transfected 293 cells with in vitro-methylated proviral DNA. In transiently transfected cells, expression of methylated LTR was reduced but not completely inhibited, irrespective of the presence of the IFN-SAR sequence, In stably transfected cells, however, methylation completely abolished expression of the control vector but not of the SAR-containing vector. Furthermore, the expression of the SAR-containing vector was stable over time, indicating the ability of the SAR sequence to alleviate methylation-mediated transcriptional repression of a vector. This study extends our understanding of the mechanisms of retroviral vector inactivation by methylation and provides insight into a functional role for the SAR elements.