Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity
Transcription factor binding sites in the pol gene intragenic regulatory region of HIV-1 are important for virus infectivity
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DOI：
10.1093/nar/gki720
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Van Lint, C
中科院分区：
文献类型：
--
作者：
Goffin, W;Demonté, D;Van Lint, C
我们之前在人类免疫缺陷病毒 1 型 (HIV-1) 的 pol 基因中发现了一个新的正转录调控元件 (nt 4481-4982)，其中包含核蛋白识别位点（位点 B、C、D 和 GC-box）[C. Van Lint、J. Ghysdael、P. Paras, Jr、A. Burny 和 E. Verdin (1994) J. Virol 68, 2632-2648]。在本研究中，我们进一步对每个结合位点进行了物理表征，并表明转录因子 Oct-1、Oct-2、PU.1、Sp1 和 Sp3 在体外与 pol 区域相互作用。使用 HIV 感染细胞系进行的染色质免疫沉淀测定表明，在染色质背景下，Sp1、Sp3、Oct-1 和 PU.1 在体内被招募到 HS7 区域。对于每个位点，我们已经确定了突变，这些突变废除了与其同源 DNA 序列结合的因子，而不改变整合酶的基本氨基酸序列。通过瞬时转染测定，我们证明了 pol 结合位点参与基因内区域的转录增强活性。我们分别对多聚化野生型和突变 pol 结合位点（即在不存在其他位点的情况下）进行的功能结果表明，PU.1、Sp1、Sp3 和 Oct-1 转录因子通过各自的 HS7 结合位点调节异源启动子的转录活性。最后，我们研究了 HS7 结合位点在 HIV-1 复制中的生理作用，并表明这些位点对于病毒感染性很重要。
We have previously identified in the pol gene of human immunodeficiency virus type 1 (HIV-1) a new positive transcriptional regulatory element (nt 4481-4982) containing recognition sites for nuclear proteins (sites B, C, D and a GC-box) [C. Van Lint, J. Ghysdael, P. Paras, Jr, A. Burny and E. Verdin (1994) J. Virol 68, 2632-2648]. In this study, we have further physically characterized each binding site and have shown that the transcription factors Oct-1, Oct-2, PU.1, Sp1 and Sp3 interact in vitro with the pol region. Chromatin immunoprecipitation assays using HIV-infected cell lines demonstrated in the context of chromatin that Sp1, Sp3, Oct-1 and PU.1 are recruited to the HS7 region in vivo. For each site, we have identified mutations abolishing factor binding to their cognate DNA sequences without altering the underlying amino acid sequence of the integrase. By transient transfection assays, we have demonstrated the involvement of the pol binding sites in the transcriptional enhancing activity of the intragenic region. Our functional results with multimerized wild-type and mutated pol binding sites separately (i.e. in the absence of the other sites) have demonstrated that the PU.1, Sp1, Sp3 and Oct-1 transcription factors regulate the transcriptional activity of a heterologous promoter through their respective HS7 binding sites. Finally, we have investigated the physiological role of the HS7 binding sites in HIV-1 replication and have shown that these sites are important for viral infectivity.