HIV-1 and M-PMV RNA Nuclear Export Elements Program Viral Genomes for Distinct Cytoplasmic Trafficking Behaviors

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.逆转录病毒（Retroviruses）编码顺式作用RNA核输出元件，此类元件可克服含内含子的病毒mRNA的核滞留现象，其中包括被包装至正在组装的病毒颗粒中的全长未剪接基因组RNA（gRNAs）。HIV-1 Rev应答元件（RRE）借助病毒蛋白Rev招募细胞内核输出受体CRM1（亦称为exportin-1/XPO1）；而简单逆转录病毒则编码组成型转运元件（CTEs），可直接招募NXF1(Tap)/NXT1(p15) mRNA核输出机器的组分。目前尚不清楚gRNA的核输出如何与病毒颗粒组装上游的细胞质运输机器相联系。本研究通过时长超过24小时的多色活细胞成像技术，实现了对单个细胞内HIV-1 gRNA的核输出、翻译、细胞质运输以及病毒颗粒生成的直接可视化观测。我们的研究结果表明，HIV-1 RRE可调控独特的、整体性的、依赖Rev与CRM1的mRNA“爆发式”转运过程，使mRNA从细胞核中批量涌出，以非定位的方式充盈细胞质。与之形成对比的是，源自梅森-菲舍猴病毒（Mason-Pfizer monkey virus, M-PMV）的CTE可将gRNA与细胞质中的微管相连，促使gRNA显著聚集于构成微管组织中心（MTOC）的中心粒周核心的中心体。将每种核输出元件添加至选定的异源mRNA中，即可赋予其各自独特的输出行为；通过位点特异性RNA锚定策略将Rev/CRM1或NXF1/NXT1运输模块定向至mRNA，同样可实现这一效果。此外，每条转录本携带多个CTE可增强其向MTOC的靶向定位能力，这表明存在一种协同机制将NXF1/NXT1与微管相连。综合来看，这些研究结果揭示了逆转录病毒gRNA核质运输中引人关注且出乎意料的特征，并证实了mRNA核输出元件的作用不仅局限于核孔，还可影响gRNA在细胞质中的分布。