Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication.
Balanced splicing at the Tat-specific HIV-1 3'ss A3 is critical for HIV-1 replication.
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DOI：
10.1186/s12977-015-0154-8
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发表时间：
2015-03-28
期刊：
影响因子：
3.3
通讯作者：
Schaal H
中科院分区：
文献类型：
--
作者：
Erkelenz S;Hillebrand F;Widera M;Theiss S;Fayyaz A;Degrandi D;Pfeffer K;Schaal H
病毒调节蛋白 Tat 对于在病毒基因表达的早期阶段从 5'-LTR 启动子建立高效转录至关重要。 Tat 编码 mRNA 的形成需要在病毒 3'ss A3 处进行剪接，此前已证明该剪接受到下游剪接调节元件 (SRE) ESS2p 和 ESE2/ESS2 的负向和正向调节。然而，使用新颖的RESCUE型计算HEXplorer算法，我们最近能够识别位于ESS2p和ESE2/ESS2之间的另一个剪接增强子（ESE5807-5838，以下称为ESEtat）。在这里，我们表明 ESEtat 对病毒 tat-mRNA 剪接有很大影响，并且它对于调节 3'ss A3 使用至关重要。在具有复制能力的病毒中，ESEtat 序列的突变失活或锁核酸 (LNA) 指导的掩蔽与 (i) 激活病毒 3'ss A3 和 (ii) 积累编码 Tat 的 mRNA 种类失败相关。因此，由于 Tat 蛋白量不足，有效的病毒复制受到严重损害。 RNA 体外结合测定显示 SRSF2 和 SRSF6 作为候选剪接因子，通过 ESEtat 和 ESE2 作用，激活 3'ss A3。这一观点得到了共表达实验的支持，在共表达实验中，野生型而非 ESETat 阴性原病毒对较高水平的 SRSF2 和 SRSF6 蛋白以及较高水平的 tat-mRNA 剪接作出反应。值得注意的是，我们还发现 SRSF6 过表达在原病毒转染的细胞内建立了抗病毒状态，有效地阻止了病毒颗粒的产生。对于抗 HIV-1 活性，剪接因子中富含精氨酸-丝氨酸 (RS) 的结构域是可有可无的。根据我们的结果，我们提出 3'ss A3 处的剪接依赖于增强 SR 蛋白 SRSF2 和 SRSF6 与 ESEtat 和 ESE2 序列的结合。 LNA 定向掩蔽导致的突变失活或特异干扰 ESEtat 活性似乎可以解释病毒基因表达的早期缺陷，可能是通过切断 HIV 有效转录其基因组所需的 Tat 供应线来实现的。
The viral regulatory protein Tat is essential for establishing a productive transcription from the 5′-LTR promoter during the early phase of viral gene expression. Formation of the Tat-encoding mRNAs requires splicing at the viral 3′ss A3, which has previously been shown to be both negatively and positively regulated by the downstream splicing regulatory elements (SREs) ESS2p and ESE2/ESS2. However, using the novel RESCUE-type computational HEXplorer algorithm, we were recently able to identify another splicing enhancer (ESE5807-5838, henceforth referred to as ESEtat) located between ESS2p and ESE2/ESS2. Here we show that ESEtat has a great impact on viral tat-mRNA splicing and that it is fundamental for regulated 3′ss A3 usage. Mutational inactivation or locked nucleic acid (LNA)-directed masking of the ESEtat sequence in the context of a replication-competent virus was associated with a failure (i) to activate viral 3′ss A3 and (ii) to accumulate Tat-encoding mRNA species. Consequently, due to insufficient amounts of Tat protein efficient viral replication was drastically impaired. RNA in vitro binding assays revealed SRSF2 and SRSF6 as candidate splicing factors acting through ESEtat and ESE2 for 3′ss A3 activation. This notion was supported by coexpression experiments, in which wild-type, but not ESEtat-negative provirus responded to higher levels of SRSF2 and SRSF6 proteins with higher levels of tat-mRNA splicing. Remarkably, we could also find that SRSF6 overexpression established an antiviral state within provirus-transfected cells, efficiently blocking virus particle production. For the anti-HIV-1 activity the arginine-serine (RS)-rich domain of the splicing factor was dispensable. Based on our results, we propose that splicing at 3′ss A3 is dependent on binding of the enhancing SR proteins SRSF2 and SRSF6 to the ESEtat and ESE2 sequence. Mutational inactivation or interference specifically with ESEtat activity by LNA-directed masking seem to account for an early stage defect in viral gene expression, probably by cutting off the supply line of Tat that HIV needs to efficiently transcribe its genome.
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作者：
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影响因子：
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