Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging
Genetic Evidence for a Connection between Rous Sarcoma Virus Gag Nuclear Trafficking and Genomic RNA Packaging
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DOI：
10.1128/jvi.00101-09
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发表时间：
2009-07-01
影响因子：
5.4
通讯作者：
Parent, Leslie J.
中科院分区：
文献类型：
--
作者：
Garbitt-Hirst, Rachel;Kenney, Scott P.;Parent, Leslie J.
逆转录病毒基因组RNA（gRNA）的包装需要RNA内的顺式作用元件和Gag多蛋白内的反式作用元件。在病毒gRNA的5'端的包装信号psi通过与核衣壳中的碱性残基和Cys-His盒RNA结合基序相互作用而结合到Gag。虽然Gag和gRNA之间的特异性相互作用已经在之前得到证实，但它们在何时何地发生还没有很好的理解。我们发现劳斯肉瘤病毒（RSV）Gag蛋白短暂定位于细胞核，尽管Gag核运输在病毒复制中的作用尚未完全阐明。与野生型颗粒相比，具有增强的Gag质膜靶向的RSV突变体（Myr 1 E）无法进行核运输，并且在病毒颗粒中掺入的gRNA水平也降低。基于这些结果，我们假设Gag核进入可能促进gRNA包装。为了通过使用功能获得性遗传学方法来测试这一想法，将来自核质蛋白的二分核定位信号（NLS）插入Myr 1 E Gag序列（产生突变Myr1E.NLS）以试图恢复核运输。在这里，我们报告说，插入NLS增强Myr1E.NLS Gag的核定位相比，Myr 1 E Gag。此外，NLS序列在含有Myr1E.NLS Gag的病毒中将gRNA包装恢复到接近野生型的水平，提供了将逆转录病毒Gag蛋白的核运输与gRNA掺入联系起来的遗传证据。
The packaging of retroviral genomic RNA (gRNA) requires cis-acting elements within the RNA and transacting elements within the Gag polyprotein. The packaging signal psi, at the 5' end of the viral gRNA, binds to Gag through interactions with basic residues and Cys-His box RNA-binding motifs in the nucleocapsid. Although specific interactions between Gag and gRNA have been demonstrated previously, where and when they occur is not well understood. We discovered that the Rous sarcoma virus (RSV) Gag protein transiently localizes to the nucleus, although the roles of Gag nuclear trafficking in virus replication have not been fully elucidated. A mutant of RSV (Myr1E) with enhanced plasma membrane targeting of Gag fails to undergo nuclear trafficking and also incorporates reduced levels of gRNA into virus particles compared to those in wild-type particles. Based on these results, we hypothesized that Gag nuclear entry might facilitate gRNA packaging. To test this idea by using a gain-of-function genetic approach, a bipartite nuclear localization signal (NLS) derived from the nucleoplasmin protein was inserted into the Myr1E Gag sequence (generating mutant Myr1E.NLS) in an attempt to restore nuclear trafficking. Here, we report that the inserted NLS enhanced the nuclear localization of Myr1E.NLS Gag compared to that of Myr1E Gag. Also, the NLS sequence restored gRNA packaging to nearly wild-type levels in viruses containing Myr1E.NLS Gag, providing genetic evidence linking nuclear trafficking of the retroviral Gag protein with gRNA incorporation.