Granger S W, Fan H
Department of Molecular Biology and Biochemistry and Cancer Research Institute, University of California, Irvine, California 92697-3900, USA.
J Virol. 1998 Nov;72(11):8961-70. doi: 10.1128/JVI.72.11.8961-8970.1998.
The enhancer sequences in the Moloney murine leukemia virus (M-MuLV) long terminal repeat (LTR) are of considerable interest since they are crucial for virus replication and the ability of the virus to induce T lymphomas. While extensive studies have identified numerous nuclear factors that can potentially bind to M-MuLV enhancer DNA in vitro, it has not been made clear which of these factors are bound in vivo. To address this problem, we carried out in vivo footprinting of the M-MuLV enhancer in infected cells by in vivo treatment with dimethyl sulfate (DMS) followed by visualization through ligation-mediated PCR (LMPCR) and gel electrophoresis. In vivo DMS-LMPCR footprinting of the upstream LTR revealed evidence for factor binding at several previously characterized motifs. In particular, protection of guanines in the central LVb/Ets and Core sites within the 75-bp repeats was detected in infected NIH 3T3 fibroblasts, Ti-6 lymphoid cells, and thymic tumor cells. In contrast, factor binding at the NF-1 sites was found in infected fibroblasts but not in T-lymphoid cells. These results are consistent with the results of previous experiments indicating the importance of the LVb/Ets and Core sequences for many retroviruses and the biological importance especially of the NF-1 sites in fibroblasts and T-lymphoid cells. No evidence for factor binding to the glucocorticoid responsive element and LVa sites was found. Additional sites of protein binding included a region in the GC-rich sequences downstream of the 75-bp repeats (only in fibroblasts), a hypersensitive guanine on the minus strand in the LVc site (only in T-lymphoid cells), and a region upstream of the 75-bp repeats. These experiments provide concrete evidence for the differential in vivo binding of nuclear factors to the M-MuLV enhancers in different cell types.
莫洛尼鼠白血病病毒（M-MuLV）长末端重复序列（LTR）中的增强子序列备受关注，因为它们对病毒复制以及病毒诱导T淋巴瘤的能力至关重要。尽管大量研究已在体外鉴定出众多可能与M-MuLV增强子DNA结合的核因子，但尚未明确其中哪些因子在体内会与之结合。为解决这一问题，我们通过用硫酸二甲酯（DMS）对感染细胞进行体内处理，随后通过连接介导的PCR（LMPCR）和凝胶电泳进行可视化，对感染细胞中的M-MuLV增强子进行了体内足迹分析。对上游LTR进行的体内DMS-LMPCR足迹分析揭示了在几个先前已鉴定的基序处存在因子结合的证据。特别是，在感染的NIH 3T3成纤维细胞、Ti-6淋巴细胞和胸腺肿瘤细胞中检测到75 bp重复序列内中央LVb/Ets和核心位点的鸟嘌呤受到保护。相比之下，在感染的成纤维细胞中发现了NF-1位点处的因子结合，而在T淋巴细胞中未发现。这些结果与先前实验的结果一致，表明LVb/Ets和核心序列对许多逆转录病毒的重要性，以及NF-1位点在成纤维细胞和T淋巴细胞中的生物学重要性。未发现因子与糖皮质激素反应元件和LVa位点结合的证据。蛋白质结合的其他位点包括75 bp重复序列下游富含GC的序列中的一个区域（仅在成纤维细胞中）、LVc位点负链上的一个超敏鸟嘌呤（仅在T淋巴细胞中）以及75 bp重复序列上游的一个区域。这些实验为不同细胞类型中核因子与M-MuLV增强子的体内差异结合提供了确凿证据。
