Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9
Nucleolar Trafficking of the Mouse Mammary Tumor Virus Gag Protein Induced by Interaction with Ribosomal Protein L9
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DOI：
10.1128/jvi.02463-12
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发表时间：
2013-01-01
影响因子：
5.4
通讯作者：
Parent, Leslie J.
中科院分区：
文献类型：
--
作者：
Beyer, Andrea R.;Bann, Darrin V.;Parent, Leslie J.
小鼠乳腺肿瘤病毒（MMTV）Gag蛋白指导未成熟病毒衣壳在细胞质中的组装，所述未成熟病毒衣壳随后从感染细胞的质膜出芽。MMTV Gag定位于小鼠乳腺上皮细胞中的离散细胞质病灶，与胞质衣壳的形成一致。出乎意料的是，我们还观察到Gag在来源于乳腺肿瘤的感染细胞的核仁中积累。为了检测可能影响其亚细胞定位的GAG相互作用蛋白，进行酵母双杂交筛选。核糖体蛋白L9（RPL9或L9）是核糖体大亚基的重要组成部分，是公认的肿瘤抑制因子，被鉴定为Gag结合伴侣。在表达MMTV（C3H）前病毒的细胞中L9的过表达导致Gag在核仁中特异性的、稳健的积累。Forster共振能量转移（FRET）和免疫共沉淀分析表明，Gag和L9在核仁内相互作用，CA结构域是相互作用的主要位点。此外，分离的NC结构域的Gag定位到核仁，这表明它包含一个核仁定位信号（NoLS）。为了确定L9是否在病毒装配中起作用，进行小干扰RNA（siRNA）介导的敲低。虽然Gag表达没有随着L9敲低而降低，但病毒产生显著受损。因此，我们的数据支持的假设，有效的MMTV颗粒组装是依赖于感染细胞的核仁中的Gag和L9的相互作用。
The mouse mammary tumor virus (MMTV) Gag protein directs the assembly in the cytoplasm of immature viral capsids, which subsequently bud from the plasma membranes of infected cells. MMTV Gag localizes to discrete cytoplasmic foci in mouse mammary epithelial cells, consistent with the formation of cytosolic capsids. Unexpectedly, we also observed an accumulation of Gag in the nucleoli of infected cells derived from mammary gland tumors. To detect Gag-interacting proteins that might influence its subcellular localization, a yeast two-hybrid screen was performed. Ribosomal protein L9 (RPL9 or L9), an essential component of the large ribosomal subunit and a putative tumor suppressor, was identified as a Gag binding partner. Overexpression of L9 in cells expressing the MMTV(C3H) provirus resulted in specific, robust accumulation of Gag in nucleoli. Forster resonance energy transfer (FRET) and coimmunoprecipitation analyses demonstrated that Gag and L9 interact within the nucleolus, and the CA domain was the major site of interaction. In addition, the isolated NC domain of Gag localized to the nucleolus, suggesting that it contains a nucleolar localization signal (NoLS). To determine whether L9 plays a role in virus assembly, small interfering RNA (siRNA)-mediated knockdown was performed. Although Gag expression was not reduced with L9 knockdown, virus production was significantly impaired. Thus, our data support the hypothesis that efficient MMTV particle assembly is dependent upon the interaction of Gag and L9 in the nucleoli of infected cells.