Behrens Ryan T, Aligeti Mounavya, Pocock Ginger M, Higgins Christina A, Sherer Nathan M
McArdle Laboratory for Cancer Research, Institute for Molecular Virology, and Carbone Cancer Center, University of Wisconsin-Madison, Madison, Wisconsin, USA.
Morgridge Institute for Research, University of Wisconsin-Madison, Madison, Wisconsin, USA.
J Virol. 2017 Jan 18;91(3). doi: 10.1128/JVI.02107-16. Print 2017 Feb 1.
HIV-1's Rev protein forms a homo-oligomeric adaptor complex linking viral RNAs to the cellular CRM1/Ran-GTP nuclear export machinery through the activity of Rev's prototypical leucine-rich nuclear export signal (NES). In this study, we used a functional fluorescently tagged Rev fusion protein as a platform to study the effects of modulating Rev NES identity, number, position, or strength on Rev subcellular trafficking, viral RNA nuclear export, and infectious virion production. We found that Rev activity was remarkably tolerant of diverse NES sequences, including supraphysiological NES (SNES) peptides that otherwise arrest CRM1 transport complexes at nuclear pores. Rev's ability to tolerate a SNES was both position and multimerization dependent, an observation consistent with a model wherein Rev self-association acts to transiently mask the NES peptide(s), thereby biasing Rev's trafficking into the nucleus. Combined imaging and functional assays also indicated that NES masking underpins Rev's well-known tendency to accumulate at the nucleolus, as well as Rev's capacity to activate optimal levels of late viral gene expression. We propose that Rev multimerization and NES masking regulates Rev's trafficking to and retention within the nucleus even prior to RNA binding.
HIV-1 infects more than 34 million people worldwide causing >1 million deaths per year. Infectious virion production is activated by the essential viral Rev protein that mediates nuclear export of intron-bearing late-stage viral mRNAs. Rev's shuttling into and out of the nucleus is regulated by the antagonistic activities of both a peptide-encoded N-terminal nuclear localization signal and C-terminal nuclear export signal (NES). How Rev and related viral proteins balance strong import and export activities in order to achieve optimal levels of viral gene expression is incompletely understood. We provide evidence that multimerization provides a mechanism by which Rev transiently masks its NES peptide, thereby biasing its trafficking to and retention within the nucleus. Targeted pharmacological disruption of Rev-Rev interactions should perturb multiple Rev activities, both Rev-RNA binding and Rev's trafficking to the nucleus in the first place.
HIV-1的Rev蛋白形成一种同源寡聚衔接子复合物，通过Rev典型的富含亮氨酸的核输出信号（NES）的活性，将病毒RNA与细胞的CRM1/Ran-GTP核输出机制联系起来。在本研究中，我们使用一种功能性荧光标记的Rev融合蛋白作为平台，来研究调节Rev NES的特性、数量、位置或强度对Rev亚细胞转运、病毒RNA核输出以及感染性病毒粒子产生的影响。我们发现，Rev活性对多种NES序列具有显著的耐受性，包括超生理NES（SNES）肽，否则这些肽会在核孔处阻止CRM1转运复合物。Rev耐受SNES的能力既取决于位置，也取决于多聚化，这一观察结果与一种模型一致，即Rev自缔合作用可暂时掩盖NES肽，从而使Rev偏向转运至细胞核。联合成像和功能分析还表明，NES掩盖是Rev在核仁积累这一众所周知的倾向以及Rev激活晚期病毒基因表达最佳水平能力的基础。我们提出，Rev多聚化和NES掩盖在RNA结合之前就调节Rev向细胞核的转运及其在细胞核内的滞留。
HIV-1在全球感染了超过3400万人，每年导致超过100万人死亡。感染性病毒粒子的产生由关键的病毒Rev蛋白激活，该蛋白介导含内含子的晚期病毒mRNA的核输出。Rev穿梭进出细胞核受肽编码的N端核定位信号和C端核输出信号（NES）的拮抗活性调节。Rev及相关病毒蛋白如何平衡强大的输入和输出活性以实现病毒基因表达的最佳水平尚不完全清楚。我们提供的证据表明，多聚化提供了一种机制，通过该机制Rev可暂时掩盖其NES肽，从而使其偏向转运至细胞核并滞留在细胞核内。对Rev-Rev相互作用进行靶向药理学破坏应会干扰多种Rev活性，包括Rev-RNA结合以及Rev最初向细胞核的转运。
