Department of Immunotherapeutics, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Yushima, 1-5-45, Bunkyo-ku, Tokyo, 113-8519, Japan.
Department of Life Science, Faculty of Advanced Engineering, Chiba Institute of Technology, 2-17-1 Tsudanuma, Narashino-shi, Chiba, 275-0016, Japan.
Biochem Biophys Res Commun. 2019 Sep 3;516(4):1145-1151. doi: 10.1016/j.bbrc.2019.06.152. Epub 2019 Jul 6.
Reverse transcription of retroviral RNA is accomplished through a minus-strand strong stop cDNA (-sscDNA) synthesis and subsequent strand-transfer reactions. We have previously reported a critical role of guanosine (G) number at 5'-terminal of HIV-1 RNA for successful strand-transfer of -sscDNA. In this study, role(s) of the cap consisting of 7-methyl guanosine (G), a hallmark of transcripts generated by RNA polymerase II, at the 5'-end G nucleotide (5'-G) of HIV-1 RNA were examined. In parallel, contribution of highly conserved GGG tract located at the U3/R boundary in 3' terminal region of viral RNA (3'-GGG tract) was also addressed. The in vitro reverse transcription analysis using synthetic HIV-1 RNAs possessing the 5'-G with cap or triphosphate form demonstrated that the 5'-cap significantly increased strand-transfer efficiency of -sscDNA. Meanwhile, effect of the 5'-cap on the strand-transfer was retained in the reaction using mutant HIV-1 RNAs in which two Gs were deleted from the 3'-GGG tract. Lack of apparent contribution of the 3'-GGG tract during strand-transfer events in vitro was reproduced in the context of HIV-1 replication within cells. Instead, we noticed that the 3'-GGG tract might be required for efficient gene expression from proviral DNA. These results indicated that G of the cap on HIV-1 RNA might not be reverse-transcribed and a possible role of the 3'-GGG tract to accept the non-template nucleotide addition during -sscDNA synthesis might be less likely. The 5'-G modifications of HIV-1 RNAs by the cap- or phosphate-removal enzyme revealed that the cap or monophosphate form of the 5'-G was preferred for the 1st strand-transfer compared to the triphosphate or non-phosphate form. Taken together, a status of the 5'-G determined strand-transfer efficiency of -sscDNA without affecting the non-template nucleotide addition, probably by affecting association of the 5'-G with 3'-end region of viral RNA.
逆转录病毒 RNA 的逆转录通过负链强终止 cDNA（-sscDNA）合成和随后的链转移反应来完成。我们之前报道过 HIV-1 RNA 5'端的鸟苷（G）数量对于 -sscDNA 成功的链转移至关重要。在这项研究中，我们研究了 HIV-1 RNA 5'端 G 核苷酸（5'-G）处的帽结构（由 RNA 聚合酶 II 产生的转录本的标志）7-甲基鸟苷（G）的作用。同时，还研究了位于病毒 RNA 3'末端区域 U3/R 边界处高度保守的 GGG 序列（3'-GGG 序列）的作用。使用具有 5'-G 帽或三磷酸形式的合成 HIV-1 RNA 的体外逆转录分析表明，5'-帽显著提高了 -sscDNA 的链转移效率。同时，在从 3'-GGG 序列中删除两个 G 的突变 HIV-1 RNA 的反应中，5'-帽对链转移的影响得以保留。在细胞内 HIV-1 复制的情况下，体外链转移事件中 3'-GGG 序列缺乏明显的作用得到了重现。相反，我们注意到 3'-GGG 序列可能是从前病毒 DNA 有效表达基因所必需的。这些结果表明，HIV-1 RNA 上帽结构的 G 可能不会被逆转录，并且 3'-GGG 序列在 -sscDNA 合成过程中接受非模板核苷酸添加的可能作用较小。通过帽或磷酸酶去除酶对 HIV-1 RNA 的 5'-G 修饰表明，与三磷酸或非磷酸形式相比，帽或 5'-G 的单磷酸形式更有利于第 1 次链转移。总之，5'-G 的状态确定了 -sscDNA 的链转移效率，而不影响非模板核苷酸的添加，可能通过影响 5'-G 与病毒 RNA 3'末端区域的结合来实现。
