DNA RECOMBINATION IS SUFFICIENT FOR RETROVIRAL TRANSDUCTION
DNA RECOMBINATION IS SUFFICIENT FOR RETROVIRAL TRANSDUCTION
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DOI：
10.1073/pnas.92.7.2460
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发表时间：
1995-03-28
影响因子：
11.1
通讯作者：
DUESBERG, PH
中科院分区：
文献类型：
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作者：
SCHWARTZ, JR;DUESBERG, S;DUESBERG, PH
致癌逆转录病毒携带从细胞原癌基因转导的编码序列。自然转导涉及两次非同源重组，因此极为罕见。由于转导从未在实验上重现过，因此其机制已根据两个假设进行了研究：(i) DNA 模型，假设两次 DNA 重组，以及 (ii) RNA 模型，假设病毒和原癌基因 RNA 逆转录过程中发生 5' DNA 重组和 3' RNA 重组。在这里，我们使用两种病毒DNA构建体来测试DNA模型的预测，即通过常规整合染色体原癌基因编码区的逆转录病毒DNA 3'来实现3'DNA重组。为了使 DNA 模型可行，此类重组病毒必须具有感染性，并且在所有逆转录病毒的 3' 长末端重复序列 (LTR) 之前不存在据称必需的多聚嘌呤区 (ppt)。我们的构建体由来自哈维肉瘤病毒的 ras 编码区组成，该区域在 5' 端天然连接到逆转录病毒 LTR，并在 3' 端直接（构建体 NdN）或通过细胞序列（构建体 SU）人工连接到逆转录病毒的 5' LTR。两种构建体都缺乏ppt，并且NdN的LTR甚至在5'端缺乏30个核苷酸。两种构建体都具有感染性，以每ml 10(5)焦点形成单位的滴度产生病毒。序列分析证明，两种病毒均与输入DNA共线，并且NdN病毒缺乏ppt和LTR的5'30核苷酸。结果表明 DNA 重组足以进行逆转录病毒转导，并且 ppt 和完整的 LTR 都不是逆转录病毒复制所必需的。 DNA 重组解释了其他人的以下观察结果，这些观察结果与 RNA 模型不一致：(i) 实验转导独立于病毒 RNA 的包装效率，(ii) 实验转导可能会相对于其他序列反转序列，正如转染期间 DNA 重组所预期的那样。
Oncogenic retroviruses carry coding sequences that are transduced from cellular protooncogenes. Natural transduction involves two nonhomologous recombinations and is thus extremely rare. Since transduction has never been reproduced experimentally, its mechanism has been studied in terms of two hypotheses: (i) the DNA model, which postulates two DNA recombinations, and (ii) the RNA model, which postulates a 5' DNA recombination and a 3' RNA recombination occurring during reverse transcription of viral and protooncogene RNA. Here we use two viral DNA constructs to test the prediction of the DNA model that the 3' DNA recombination is achieved by conventional integration of a retroviral DNA 3' of the chromosomal protooncogene coding region. For the DNA model to be viable, such recombinant viruses must be infectious without the purportedly essential polypurine tract (ppt) that precedes the 3' long terminal repeat (LTR) of all retroviruses. Our constructs consist of a ras coding region from Harvey sarcoma virus which is naturally Linked at the 5' end to a retroviral LTR and artificially linked at the 3' end either directly (construct NdN) or by a cellular sequence (construct SU) to the 5' LTR of a retrovirus. Both constructs lack the ppt, and the LTR of NdN even lacks 30 nucleotides at the 5' end, Both constructs pro, ed to be infectious, producing viruses at titers of 10(5) focus-forming units per ml. Sequence analysis proved that both viruses were colinear with input DNAs and that NdN virus lacked a ppt and the 5' 30 nucleotides of the LTR. The results indicate that DNA recombination is sufficient for retroviral transduction and that neither the ppt nor the complete LTR is essential for retrovirus replication. DNA recombination explains the following observations by others that cannot be reconciled with the RNA model: (i) experimental transduction is independent of the packaging efficiency of viral RNA, and (ii) experimental transduction may invert sequences with respect to others, as expected for DNA recombination during transfection.