Department of Microbiology and Immunology, School of Medicine and Dentistry, University of Rochester , Rochester, New York 14642, United States.
Biochemistry. 2014 Apr 29;53(16):2581-93. doi: 10.1021/bi4016692. Epub 2014 Apr 15.
Genomic regions rich in G residues are prone to adopt G-quadruplex structure. Multiple Sp1-binding motifs arranged in tandem have been suggested to form this structure in promoters of cancer-related genes. Here, we demonstrate that the G-rich proviral DNA sequence of the HIV-1 U3 region, which serves as a promoter of viral transcription, adopts a G-quadruplex structure. The sequence contains three binding elements for transcription factor Sp1, which is involved in the regulation of HIV-1 latency, reactivation, and high-level virus expression. We show that the three Sp1 binding motifs can adopt different forms of G-quadruplex structure and that the Sp1 protein can recognize and bind to its site folded into a G-quadruplex. In addition, a c-kit2 specific antibody, designated hf2, binds to two different G-quadruplexes formed in Sp1 sites. Since U3 is encoded at both viral genomic ends, the G-rich sequence is also present in the RNA genome. We demonstrate that the RNA sequence of U3 forms dimers with characteristics known for intermolecular G-quadruplexes. Together with previous reports showing G-quadruplex dimers in the gag and cPPT regions, these results suggest that integrity of the two viral genomes is maintained through numerous intermolecular G-quadruplexes formed in different RNA genome locations. Reconstituted reverse transcription shows that the potassium-dependent structure formed in U3 RNA facilitates RT template switching, suggesting that the G-quadruplex contributes to recombination in U3.
富含 G 残基的基因组区域易于形成 G-四链体结构。多个串联排列的 Sp1 结合基序被认为在癌症相关基因的启动子中形成这种结构。在这里，我们证明 HIV-1 U3 区域的富含 G 的前病毒 DNA 序列作为病毒转录的启动子，采用 G-四链体结构。该序列包含三个转录因子 Sp1 的结合元件，Sp1 参与 HIV-1 潜伏期、激活和高水平病毒表达的调节。我们表明，三个 Sp1 结合基序可以采用不同形式的 G-四链体结构，并且 Sp1 蛋白可以识别并结合其折叠成 G-四链体的位点。此外，一种称为 hf2 的 c-kit2 特异性抗体结合到 Sp1 位点形成的两个不同的 G-四链体。由于 U3 编码在病毒基因组的两个末端，富含 G 的序列也存在于 RNA 基因组中。我们证明 U3 的 RNA 序列形成具有已知的分子间 G-四链体特征的二聚体。结合先前报道显示 gag 和 cPPT 区域存在 G-四链体二聚体，这些结果表明通过在不同的 RNA 基因组位置形成大量的分子间 G-四链体来维持两个病毒基因组的完整性。重建的逆转录表明，U3 RNA 中形成的钾依赖性结构促进 RT 模板转换，表明 G-四链体有助于 U3 中的重组。
