Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA.
Fate of HIV-1 cDNA intermediates during reverse transcription is dictated by transcription initiation site of virus genomic RNA.
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DOI：
10.1038/srep17680
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发表时间：
2015-12-03
影响因子：
4.6
通讯作者：
Kannagi M
中科院分区：
文献类型：
--
作者：
Masuda T;Sato Y;Huang YL;Koi S;Takahata T;Hasegawa A;Kawai G;Kannagi M
逆转录病毒逆转录是通过从病毒基因组RNA复制的部分cDNA中间体的顺序链转移来完成的。在这里，我们揭示了HIV-1基因组RNA的5 '端鸟苷(G)在逆转录中的前所未有的作用。根据目前对HIV-1转录起始位点的共识，HIV-1转录本在5 '端具有单个G （g1形式）。然而，我们发现在5 '端（G2和g3形式）具有额外Gs的HIV-1转录本通过使用替代转录起始位点在感染细胞中大量表达。在病毒颗粒中也检测到G2和g3形式，尽管g1形式占主导地位。为了解决5 ' -G数的生物学影响，我们通过删除替代起始位点生成了只表达g1形式的HIV克隆DNA。由该克隆产生的病毒具有显著高的负强链cDNA （-sscDNA）链转移。使用合成HIV-1 RNA的体外实验显示，从g3形式的RNA中大量产生-sscDNA的流产形式，但从g1形式的RNA中急剧减少。此外，-sscDNA从g1形式的链转移被HIV-1核衣壳显著地刺激。综上所述，我们的研究结果表明，在逆转录过程中，与HIV-1转录起始位点对应的5 ' -G数对-sscDNA链转移的成功至关重要。
Retroviral reverse transcription is accomplished by sequential strand-transfers of partial cDNA intermediates copied from viral genomic RNA. Here, we revealed an unprecedented role of 5′-end guanosine (G) of HIV-1 genomic RNA for reverse transcription. Based on current consensus for HIV-1 transcription initiation site, HIV-1 transcripts possess a single G at 5′-ends (G1-form). However, we found that HIV-1 transcripts with additional Gs at 5′-ends (G2- and G3-forms) were abundantly expressed in infected cells by using alternative transcription initiation sites. The G2- and G3-forms were also detected in the virus particle, although the G1-form predominated. To address biological impact of the 5′-G number, we generated HIV clone DNA to express the G1-form exclusively by deleting the alternative initiation sites. Virus produced from the clone showed significantly higher strand-transfer of minus strong-stop cDNA (-sscDNA). The in vitro assay using synthetic HIV-1 RNAs revealed that the abortive forms of -sscDNA were abundantly generated from the G3-form RNA, but dramatically reduced from the G1-form. Moreover, the strand-transfer of -sscDNA from the G1-form was prominently stimulated by HIV-1 nucleocapsid. Taken together, our results demonstrated that the 5′-G number that corresponds to HIV-1 transcription initiation site was critical for successful strand-transfer of -sscDNA during reverse transcription.