Department of Forensic Medicine and Molecular Pathology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto, Japan.
PLoS One. 2012;7(2):e31533. doi: 10.1371/journal.pone.0031533. Epub 2012 Feb 14.
Integration of the retroviral genome into host DNA is a critical step in the life cycle of a retrovirus. Although assays for in vitro integration have been developed, the actual DNA sequences targeted by murine leukemia retrovirus (MLV) during in vitro reproduction are unknown. While previous studies used artificial target sequences, we developed an assay using target DNA sequences from common MLV integration sites in Stat5a and c-myc in the genome of murine lymphomas and successfully integrated MLV into the target DNA in vitro. We calculated the free energy change during folding of the target sequence DNA and found a close correlation between the calculated free energy change and the number of integrations. Indeed, the integrations closely correlated with fluctuation of the structure of the target DNA segment. These data suggest that the fluctuation may generate a DNA structure favorable for in vitro integration into the target DNA. The approach described here can provide data on the biochemical properties of the integration reaction to which the target DNA structure may contribute.
逆转录病毒基因组整合到宿主 DNA 中是逆转录病毒生命周期中的一个关键步骤。虽然已经开发出用于体外整合的检测方法，但在体外繁殖过程中，鼠白血病病毒 (MLV) 实际靶向的 DNA 序列尚不清楚。虽然之前的研究使用了人工靶序列，但我们开发了一种使用来自鼠淋巴瘤基因组中 Stat5a 和 c-myc 中常见 MLV 整合位点的靶 DNA 序列的检测方法，并成功地将 MLV 体外整合到靶 DNA 中。我们计算了靶序列 DNA 折叠过程中的自由能变化，并发现计算出的自由能变化与整合的数量之间存在密切的相关性。事实上，整合与靶 DNA 片段结构的波动密切相关。这些数据表明，这种波动可能会产生一种有利于体外整合到靶 DNA 的 DNA 结构。这里描述的方法可以提供有关整合反应的生化特性的数据，靶 DNA 结构可能对此有贡献。
