HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.
HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors.
复制标题
DOI：
10.1371/journal.ppat.1005565
复制
发表时间：
2016-04
期刊：
影响因子：
6.7
通讯作者：
Sherer NM
中科院分区：
文献类型：
--
作者：
Pocock GM;Becker JT;Swanson CM;Ahlquist P;Sherer NM
逆转录病毒编码顺式作用RNA核输出元件，其覆盖包含内含子的病毒mRNA的核保留，包括包装成组装病毒体的全长未剪接基因组RNA（gRNA）。HIV-1 Rev反应元件（RRE）使用病毒蛋白Rev募集细胞核输出受体CRM 1（也称为输出蛋白-1/XPO 1），而简单的逆转录病毒编码直接募集NXF 1（Tap）/NXT 1（p15）mRNA核输出机制组分的组成型转运元件（CTE）。gRNA核输出如何与病毒颗粒组装上游的细胞质中的运输机制相关联尚不清楚。在这里，我们使用了长期（>24小时）的活体细胞成像来直接观察单细胞中HIV-1 gRNA的核输出、翻译、细胞质运输和病毒颗粒的产生。我们表明，HIV-1 RRE调节独特的，envelope，Rev-和CRM 1依赖的“突发式”的mRNA从细胞核的过渡，以非本地化的方式淹没细胞质。相比之下，源自梅森-辉瑞猴病毒（M-PMV）的CTE将gRNA连接到细胞质中的微管，驱使它们显著地聚集到形成微管组织中心（MTOC）的中心粒周围核心的中心体。将每种输出元件添加到所选异源mRNA中足以赋予每种不同的输出行为，就像使用位点特异性RNA拴系策略将Rev/CRM 1或NXF 1/NXT 1转运模块定向到mRNA一样。此外，每个转录本的多个CTE增强了MTOC靶向，表明合作机制将NXF 1/NXT 1与微管联系起来。结合起来，这些结果揭示了逆转录病毒gRNA核质转运的惊人的、意想不到的特征，并证明了延伸超出核孔的mRNA输出元件影响gRNA在细胞质中的分布的作用。细胞和病毒的mRNA如何在细胞内运输还知之甚少。在这项研究中，我们使用荧光显微镜观察单个活细胞中HIV-1 mRNA的核输出、翻译、运输和病毒颗粒组装，重点是包装成病毒颗粒的全长病毒基因组RNA（gRNA）。我们证明了HIV-1 gRNA在病毒颗粒组装开始之前表现出从细胞核到细胞质的戏剧性“爆发”样转变。该过程由病毒Rev蛋白和细胞核输出因子CRM 1调节。相比之下，梅森-辉瑞猴病毒（M-PMV）是一种不利用CRM 1的致病性非人类逆转录病毒，它以受替代细胞核输出因子NXF 1调节的机制将gRNA连接到微管细胞骨架。结合，我们的比较研究揭示了两个意想不到的mRNA核质转运行为编程对比mRNA核输出因子。支持这些独特的贩运行为的分子细节的进一步阐明可能会告知针对病毒基因表达和/或感染性病毒体组装的抗病毒策略的发展。
Retroviruses encode cis-acting RNA nuclear export elements that override nuclear retention of intron-containing viral mRNAs including the full-length, unspliced genomic RNAs (gRNAs) packaged into assembling virions. The HIV-1 Rev-response element (RRE) recruits the cellular nuclear export receptor CRM1 (also known as exportin-1/XPO1) using the viral protein Rev, while simple retroviruses encode constitutive transport elements (CTEs) that directly recruit components of the NXF1(Tap)/NXT1(p15) mRNA nuclear export machinery. How gRNA nuclear export is linked to trafficking machineries in the cytoplasm upstream of virus particle assembly is unknown. Here we used long-term (>24 h), multicolor live cell imaging to directly visualize HIV-1 gRNA nuclear export, translation, cytoplasmic trafficking, and virus particle production in single cells. We show that the HIV-1 RRE regulates unique, en masse, Rev- and CRM1-dependent “burst-like” transitions of mRNAs from the nucleus to flood the cytoplasm in a non-localized fashion. By contrast, the CTE derived from Mason-Pfizer monkey virus (M-PMV) links gRNAs to microtubules in the cytoplasm, driving them to cluster markedly to the centrosome that forms the pericentriolar core of the microtubule-organizing center (MTOC). Adding each export element to selected heterologous mRNAs was sufficient to confer each distinct export behavior, as was directing Rev/CRM1 or NXF1/NXT1 transport modules to mRNAs using a site-specific RNA tethering strategy. Moreover, multiple CTEs per transcript enhanced MTOC targeting, suggesting that a cooperative mechanism links NXF1/NXT1 to microtubules. Combined, these results reveal striking, unexpected features of retroviral gRNA nucleocytoplasmic transport and demonstrate roles for mRNA export elements that extend beyond nuclear pores to impact gRNA distribution in the cytoplasm. How cellular and viral mRNAs are trafficked inside the cell is poorly understood. In this study, we use fluorescence microscopy to visualize HIV-1 mRNA nuclear export, translation, trafficking, and virus particle assembly in single living cells, focusing on the full-length viral genomic RNAs (gRNAs) that are packaged into virus particles. We demonstrate that HIV-1 gRNAs exhibit dramatic “burst”-like transitions from the nucleus to fill the cytoplasm prior to the onset of virus particle assembly. This process is regulated by the viral Rev protein and the cellular nuclear export factor CRM1. By contrast, Mason-Pfizer monkey virus (M-PMV), a pathogenic non-human retrovirus that does not exploit CRM1, links gRNAs to the microtubule cytoskeleton in a mechanism regulated by an alternative cellular nuclear export factor, NXF1. Combined, our comparative studies reveal two unanticipated mRNA nucleocytoplasmic transport behaviors programmed by contrasting mRNA nuclear export factors. Further elucidation of the molecular details underpinning these unique trafficking behaviors may inform the development of antiviral strategies targeting viral gene expression and/or infectious virion assembly.