Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription
Role of post-transcriptional modifications of primer tRNALys,3 in the fidelity and efficacy of plus strand DNA transfer during HIV-1 reverse transcription
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DOI：
10.1074/jbc.274.7.4412
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发表时间：
1999-02-12
影响因子：
4.8
通讯作者：
Darlix, JL
中科院分区：
文献类型：
--
作者：
Auxilien, S;Keith, G;Darlix, JL
在HIV逆转录过程中，（+）链DNA合成由RNase h -抗性序列（多嘌呤通道）引物，并持续到与病毒引物结合位点（PBS）杂交的tRNA的3'端对应的18 nt双链RNA区域（Lys,3）。在（+）链DNA转移之前，逆转录酶（RT）需要解开tRNA- pbs双链RNA，以便逆转录引物tRNA的3'端（Lys,3）。由于（+）链DNA转移的详细机制尚不完全清楚，我们开发了一个体外系统来仔细研究这一机制，该系统由HIV 5' RNA，天然修饰tRNA(Lys,3)，合成未修饰tRNALys，3或与PBS互补的寡核苷酸（RNA或DNA）以及病毒蛋白RT和核壳蛋白（NCp7）组成。RT停留在双链tRNA- pbs RNA复合物上，能够逆转录天然tRNA修饰的核苷（Lys,3）。天然tRNA修饰的核苷m(1) a -58 （Lys,3）作为停止信号仅部分有效，因为RT可以转录到反密码子环中超修饰的腺苷(ms（2)t(6) a -37）。m(1)A-58几乎总是转录成A，而其他修饰的核苷则正确转录，除了m(7)G-46，有时转录成t。相反，合成tRNA(Lys,3)， RNA PBS引物和DNA PBS引物完全逆转录。在存在受体模板的情况下，（+）链DNA转移只有在含有天然tRNA(Lys,)3或RNA PBS引物的模板下才有效。转移产物的序列分析显示，合成tRNA在转移位点上经常出现错误（Lys,3），而天然tRNA则没有（Lys,3）。因此，存在于所有逆转录病毒tRNA引物中的修饰核苷m(1)A-58似乎对（+）链DNA转移的有效性和保真度都很重要。我们发现其他因素，如受体模板的（-）PBS的性质和RT的RNase PP活性也会影响（+）链DNA转移的效果。
During HIV reverse transcription, (+) strand DNA synthesis is primed by an RNase H-resistant sequence, the polypurine tract, and continues as far as a 18-nt double-stranded RNA region corresponding to the 3' end of tRNA(Lys,3) hybridized to the viral primer binding site (PBS). Before (+) strand DNA transfer, reverse transcriptase (RT) needs to unwind the double-stranded tRNA-PBS RNA in order to reverse-transcribe the 3' end of primer tRNA(Lys,3). Since the detailed mechanism of (+) strand DNA transfer remains incompletely understood, we developed an in vitro system to closely examine this mechanism, composed of HIV 5' RNA, natural modified tRNA(Lys,3), synthetic unmodified tRNALys,3 Or oligonucleotides (RNA or DNA) complementary to the PBS, as well as the viral proteins RT and nucleocapsid protein (NCp7), Prior to (+) strand DNA transfer, RT stalls at the double-stranded tRNA-PBS RNA complex and is able to reverse-transcribe modified nucleosides of natural tRNA(Lys,3). Modified nucleoside m(1)A-58 of natural tRNA(Lys,3) is only partially effective as a stop signal, as RT can transcribe as far as the hyper-modified adenosine (ms(2)t(6)A-37) in the anticodon loop. m(1)A-58 is almost always transcribed into A, whereas other modified nucleosides are transcribed correctly, except for m(7)G-46, which is sometimes transcribed into T. In contrast, synthetic tRNA(Lys,3), an RNA PBS primer, and a DNA PBS primer are completely reverse-transcribed, In the presence of an acceptor template, (+) strand DNA transfer is efficient only with templates containing natural tRNA(Lys,)3 or the RNA PBS primer, Sequence analysis of transfer products revealed frequent errors at the transfer site with synthetic tRNA(Lys,3), not observed with natural tRNA(Lys,3). Thus, modified nucleoside m(1)A-58, present in all retroviral tRNA primers, appears to be important for both efficacy and fidelity of (+) strand DNA transfer. We show that other factors such as the nature of the (-) PBS of the acceptor template and the RNase PP activity of RT also influence the efficacy of (+) strand DNA transfer.