Replicative fidelity of lentiviral vectors produced by transient transfection
Replicative fidelity of lentiviral vectors produced by transient transfection
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DOI：
10.1016/j.virol.2005.12.037
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发表时间：
2006-05-10
期刊：
影响因子：
3.7
通讯作者：
Sutton, Richard E.
中科院分区：
文献类型：
--
作者：
Laakso, Meg M.;Sutton, Richard E.
先前的研究估计，每轮病毒复制的人类免疫缺陷病毒 I 型 (HIV) 碱基对替换率约为 10(-4) 至 10(-5)，并且假设 HIV 比其他逆转录病毒更容易出错。使用单循环逆转试验，我们意外地发现HIV、禽白血病病毒和莫洛尼鼠白血病病毒的逆转率相同，且在统计误差内。由于病毒复制需要病毒酶逆转录酶 (RT) 和细胞 RNA 聚合酶 II (RNAP)，因此我们假设相似的回复率实际上反映了 RNAP 的内在错误率，RNAP 是回复测定中所有三种逆转录病毒所共有的酶。为了解决这种可能性，构建了 U3 区域被报告基因回复盒替换的 HIV 载体，并通过瞬时转染产生载体上清液。所有单一整合回复体细胞系在两个长末端重复序列处均显示出相同的突变。这表明 RNAP 或另一种细胞酶负责这些逆转，或者 HIV RT 仅在第一链合成过程中出错。此外，当从整合载体中拯救出HIV颗粒而不是通过瞬时转染产生时，逆转率显着降低，这表明病毒产生细胞中的一种或多种因素在逆转录病毒复制的保真度中发挥着作用。这些结果对慢病毒载体上清液瞬时转染产生后转基因的保真度有影响。 (c) 2006 Elsevier Inc. 保留所有权利。
Previous investigations have estimated the human immunodeficiency virus type I (HIV) base pair substitution rate to be approximately 10(-4) to 10(-5) per round of viral replication, and HIV has been hypothesized to be more error-prone than other retroviruses. Using a single cycle reversion assay, we unexpectedly found that the reversion rates of HIV, avian leukosis virus and Moloney murine leukemia virus were the same, within statistical error. Because both the viral enzyme reverse transcriptase (RT) and cellular RNA polymerase II (RNAP) are required for viral replication, we hypothesized that the similar reversion rates actually reflect the intrinsic error rate of RNAP, which is the enzyme common to all three retroviruses in the reversion assay. To address this possibility, HIV vectors with the U3 region replaced by a reporter reversion cassette were constructed and vector supernatant produced by transient transfection. All single integrant revertant cell lines showed the identical mutations at both long terminal repeats. This indicates that either RNAP or another cellular enzyme is responsible for these reversions, or that HIV RT only makes errors during first strand synthesis. Additionally, when HIV particles were rescued from an integrated vector as opposed to being produced by transient transfection, the reversion rate was significantly lower, suggesting that one or more factors in the virus-producing cells plays a role in the fidelity of retroviral replication. These results have implications regarding the fidelity of the transgene after transient transfection production of lentiviral vector supernatants. (c) 2006 Elsevier Inc. All rights reserved.