Nuclear entry and CRM1-dependent nuclear export of the Rous sarcoma virus Gag polyprotein

The retroviral Gag polyprotein directs budding from the plasma membrane of infected cells. Until now, it was believed that Gag proteins of type C retroviruses, including the prototypic oncoretrovirus Rous sarcoma virus, were synthesized on cytosolic ribosomes and targeted directly to the plasma membrane. Here we reveal a previously unknown step in the subcellular trafficking of the Gag protein, that of transient nuclear localization. We have identified a targeting signal within the N-terminal matrix domain that facilitates active nuclear import of the Gag polyprotein. We also found that Gag is transported out of the nucleus through the CRM1 nuclear export pathway, based on observations that treatment of virus-expressing cells with leptomycin B resulted in the redistribution of Gag proteins from the cytoplasm to the nucleus. Internal deletion of the C-terminal portion of the Gag p10 region resulted in the nuclear sequestration of Gag and markedly diminished budding, suggesting that the nuclear export signal might reside within p10. Finally, we observed that a previously described matrix mutant, Myr1E, was insensitive to the effects of leptomycin B, apparently bypassing the nuclear compartment during virus assembly. Myr1E has a defect in genomic RNA packaging, implying that nuclear localization of Gag might be involved in viral RNA interactions. Taken together, these findings provide evidence that nuclear entry and egress of the Gag polyprotein are intrinsic components of the Rous sarcoma virus assembly pathway.

逆转录病毒Gag多聚蛋白（retroviral Gag polyprotein）介导感染细胞质膜处的病毒出芽过程。此前学界普遍认为，包括原型致癌逆转录病毒劳斯肉瘤病毒（Rous sarcoma virus）在内的C型逆转录病毒的Gag蛋白，均在胞质核糖体上合成并直接靶向质膜。本研究揭示了Gag蛋白亚细胞运输中此前未被发现的环节：暂时性核定位。我们在其N端基质结构域中鉴定出一段靶向信号，可介导Gag多聚蛋白的主动核输入。此外，基于“用亮霉素B（leptomycin B）处理表达病毒的细胞后，Gag蛋白从细胞质重新分布至细胞核”的实验结果，我们发现Gag可通过CRM1核输出通路完成核外运出。对Gag p10区域的C端片段进行内部缺失后，会导致Gag发生核滞留并显著削弱出芽效率，这提示核输出信号可能位于p10区域内。最后，我们观察到此前已报道的基质突变体Myr1E对亮霉素B的作用不敏感，其在病毒组装过程中可绕过核区室。Myr1E存在基因组RNA包装缺陷，这暗示Gag的核定位可能参与病毒RNA的相互作用。综上，这些研究结果证实，Gag多聚蛋白的核入与核出过程是劳斯肉瘤病毒组装通路的固有组成部分。