Detection, characterization and regulation of antisense transcripts in HIV-1.
Detection, characterization and regulation of antisense transcripts in HIV-1.
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DOI：
10.1186/1742-4690-4-71
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发表时间：
2007-10-02
期刊：
影响因子：
3.3
通讯作者：
Barbeau B
中科院分区：
文献类型：
--
作者：
Landry S;Halin M;Lefort S;Audet B;Vaquero C;Mesnard JM;Barbeau B
我们和其他人最近已经证明，人类逆转录病毒HTLV-Ⅰ产生了一个剪接的反义转录本，这导致了HBZ蛋白的合成。本研究的目的是证明HIV-1反义转录的存在，并提供一个更好的表征转录及其调控。在潜伏感染的J1.1细胞系和pNL4.3转染的293 T细胞中通过标准RT-PCR分析进行的初始实验证实了HIV-1中反义转录的存在。一个更适合的RT-PCR方案与有限的RT-PCR文物也导致了成功的检测反义转录在几个感染的细胞系。RACE分析表明，存在几个转录起始位点，定位在3 'LTR的5'边界附近（在反义链中）。有趣的是，在反义链上鉴定出一种新的polyA信号，并含有polyA信号共有序列。在293 T和Jurkat细胞中用表达NL4.3前病毒DNA的反义寡核苷酸酶的转染实验显示荧光素酶报告基因表达，其进一步由各种T细胞活化剂诱导。此外，病毒达特蛋白被发现是一个正的调节剂的反义转录的瞬时和稳定转染的这种前病毒DNA构建体。在用pNL4.3衍生的构建体稳定转染的293 T细胞中的RT-PCR分析进一步证实了这些结果。用表达反义寡核苷酸酶的NL4.3 DNA克隆产生的假型病毒体感染293 T、Jurkat、SupT 1、U937和CEMT 4细胞，导致产生AZT敏感的荧光素酶信号，然而，该信号不如来自NL 4.3Luc-infected细胞的信号明显。这些结果首次证明了在感染的情况下，反义转录存在于HIV-1中。因此，应重新检查该转录本中预测的反义ORF的可能翻译。
We and others have recently demonstrated that the human retrovirus HTLV-I was producing a spliced antisense transcript, which led to the synthesis of the HBZ protein. The objective of the present study was to demonstrate the existence of antisense transcription in HIV-1 and to provide a better characterization of the transcript and its regulation. Initial experiments conducted by standard RT-PCR analysis in latently infected J1.1 cell line and pNL4.3-transfected 293T cells confirmed the existence of antisense transcription in HIV-1. A more adapted RT-PCR protocol with limited RT-PCR artefacts also led to a successful detection of antisense transcripts in several infected cell lines. RACE analyses demonstrated the existence of several transcription initiation sites mapping near the 5' border of the 3'LTR (in the antisense strand). Interestingly, a new polyA signal was identified on the antisense strand and harboured the polyA signal consensus sequence. Transfection experiments in 293T and Jurkat cells with an antisense luciferase-expressing NL4.3 proviral DNA showed luciferase reporter gene expression, which was further induced by various T-cell activators. In addition, the viral Tat protein was found to be a positive modulator of antisense transcription by transient and stable transfections of this proviral DNA construct. RT-PCR analyses in 293T cells stably transfected with a pNL4.3-derived construct further confirmed these results. Infection of 293T, Jurkat, SupT1, U937 and CEMT4 cells with pseudotyped virions produced from the antisense luciferase-expressing NL4.3 DNA clone led to the production of an AZT-sensitive luciferase signal, which was however less pronounced than the signal from NL4.3Luc-infected cells. These results demonstrate for the first time that antisense transcription exists in HIV-1 in the context of infection. Possible translation of the predicted antisense ORF in this transcript should thus be re-examined.
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影响因子：
5.4
作者：
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通讯作者：
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影响因子：
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作者：
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