Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.
Relationships between MA-RNA Binding in Cells and Suppression of HIV-1 Gag Mislocalization to Intracellular Membranes.
复制标题
细胞中 MA-RNA 结合与抑制 HIV-1 Gag 错误定位到细胞内膜之间的关系。
DOI：
10.1128/jvi.00756-19
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发表时间：
2019
影响因子：
5.4
通讯作者：
Ono,Akira
中科院分区：
文献类型：
--
作者：
Thornhill,Dishari;Olety,Balaji;Ono,Akira
HIV-1 Gag基质（MA）结构域介导Gag定位于质膜（PM），即感染性病毒体组装的位点。MA高碱性区（MA-HBR）与磷脂酰肌醇-（4，5）-二磷酸[PI（4，5）P2]（一种PM特异性酸性脂质）相互作用。MA-HBR还结合RNA。为了测试酸性脂质是否单独决定Gag的PM特异性定位或MA-RNA结合是否也起作用，我们比较了一组MA-HBR突变体，这些突变体在MA残基25和26或残基29和31处含有两种类型的取代：Lys→Arg（KR）（25/26 KR和29/31 KR）和Lys→Thr（KT）（25/26 KT和29/31 KT）。与HBR电荷在RNA结合中的重要性一致，两种KT突变体都不能有效地通过MA结合RNA，这与相应的KR突变体不同。25/26 KT Gag-yellow fluorescent protein（YFP）和29/31 KT Gag-YFP都与PM和细胞内膜非特异性结合，推测是通过肉豆蔻酰部分和剩余的MA碱性残基。相比之下，25/26 KR Gag-YFP特异性结合PM，表明总正电荷和/或MA结合的RNA在将Gag导航到PM中的作用。与29/31 KT Gag-YFP不同，29/31 KR Gag-YFP主要是胞质的，尽管具有较高的HBR电荷，但几乎没有显示出细胞内膜结合。因此，MA-RNA结合可能阻断细胞中混杂的Gag膜结合。值得注意的是，异源多聚化结构域的引入恢复了29/31 KR Gag-YFP的PI（4，5）P2依赖性PM特异性定位，表明PM结合的阻断比细胞内膜结合的阻断更容易逆转。总之，这些基于细胞的数据支持一个模型，其中MA-RNA结合确保PM特异性本地化的Gag通过抑制非特异性membrane binding.IMPORTANCEThe PM特异性本地化的HIV-1 Gag是一个重要的早期步骤，在感染性子代生产。Gag的MA高碱性区域（MA-HBR）和PM特异性脂质PI（4，5）P2之间的相互作用对于Gag定位到PM是关键的。此外，体外证据表明MA-RNA结合防止Gag与不含PI（4，5）P2的膜非特异性结合。然而，支持HIV-1 MA-RNA结合在PM特异性亚细胞定位中的作用的基于细胞的证据很少;因此，在细胞中，仅高碱性电荷或PI（4，5）P2结合能力就足以使MA特异性地将Gag引导至PM。本研究首次揭示了MA-HBR的RNA结合与细胞内混杂Gag定位的抑制之间的良好相关性，从而提供了基于细胞的证据，支持HIV-1 MA与RNA结合确保Gag特异性定位于PM的机制。
The HIV-1 Gag matrix (MA) domain mediates the localization of Gag to the plasma membrane (PM), the site for infectious virion assembly. The MA highly basic region (MA-HBR) interacts with phosphatidylinositol-(4,5)-bisphosphate [PI(4,5)P2], a PM-specific acidic lipid. The MA-HBR also binds RNAs. To test whether acidic lipids alone determine PM-specific localization of Gag or whether MA-RNA binding also plays a role, we compared a panel of MA-HBR mutants that contain two types of substitutions at MA residues 25 and 26 or residues 29 and 31: Lys→Arg (KR) (25/26KR and 29/31KR) and Lys→Thr (KT) (25/26KT and 29/31KT). Consistent with the importance of the HBR charge in RNA binding, both KT mutants failed to bind RNA via MA efficiently, unlike the corresponding KR mutants. Both 25/26KT Gag-yellow fluorescent protein (YFP) and 29/31KT Gag-YFP bound nonspecifically to the PM and intracellular membranes, presumably via the myristoyl moiety and remaining MA basic residues. In contrast, 25/26KR Gag-YFP bound specifically to the PM, suggesting a role for the total positive charge and/or MA-bound RNA in navigating Gag to the PM. Unlike 29/31KT Gag-YFP, 29/31KR Gag-YFP was predominantly cytosolic and showed little intracellular membrane binding despite having a higher HBR charge. Therefore, it is likely that MA-RNA binding blocks promiscuous Gag membrane binding in cells. Notably, the introduction of a heterologous multimerization domain restored PI(4,5)P2-dependent PM-specific localization for 29/31KR Gag-YFP, suggesting that the blocking of PM binding is more readily reversed than that of intracellular membrane binding. Altogether, these cell-based data support a model in which MA-RNA binding ensures PM-specific localization of Gag via suppression of nonspecific membrane binding.IMPORTANCEThe PM-specific localization of HIV-1 Gag is a crucial early step in infectious progeny production. The interaction between the MA highly basic region (MA-HBR) of Gag and the PM-specific lipid PI(4,5)P2 is critical for Gag localization to the PM. Additionally,in vitroevidence has indicated that MA-RNA binding prevents nonspecific binding of Gag to non-PI(4,5)P2-containing membranes. However, cell-based evidence supporting a role for HIV-1 MA-RNA binding in PM-specific subcellular localization has been scarce; thus, it remained possible that in cells, just the high basic charge or the PI(4,5)P2 binding ability is sufficient for MA to direct Gag specifically to the PM. The present study reveals for the first time an excellent correlation between RNA binding of the MA-HBR and inhibition of promiscuous Gag localization, both within the cells, and thereby provides cell-based evidence supporting a mechanism in which HIV-1 MA binding to RNA ensures the specific localization of Gag to the PM.