The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA
The C-terminal p6 domain of the HIV-1 Pr55Gag precursor is required for specific binding to the genomic RNA
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DOI：
10.1080/15476286.2018.1481696
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发表时间：
2018-01-01
期刊：
影响因子：
4.1
通讯作者：
Bernacchi, Serena
中科院分区：
文献类型：
--
作者：
Dubois, Noe;Khoo, Keith K.;Bernacchi, Serena
Pr55（Gag）前体从大量过量的细胞和部分或完全剪接的病毒RNA中专门选择HIV-1基因组RNA（gRNA），并驱动病毒在质膜上组装。在这些过程中，Pr55(Gag) 的 NC 结构域与 gRNA 相互作用，而其 C 端 p6 结构域结合细胞和病毒因子并协调病毒颗粒的释放。 Gagp6 是 Pr55(Gag) 的截短形式，缺乏 p6 结构域，通常用作体外分析中野生型 Pr55(Gag) 的默认替代物。随着全长重组 Pr55(Gag) 生产的最新进展，我们通过系统比较 Pr55(Gag) 和 Gagp6 与一组病毒和细胞 RNA 的结合来测试 p6 结构域是否也有助于 Pr55(Gag) 的 RNA 结合特异性。出乎意料的是，我们的荧光数据表明 p6 结构域对于 Pr55(Gag) 与 HIV-1 gRNA 的特异性结合绝对是必需的。它的缺失不仅导致对 gRNA 的亲和力降低，而且还导致对剪接的病毒和细胞 RNA 的亲和力增加。相比之下，Gagp6 对所有测试的 RNA 表现出相似的亲和力。从 Pr55(Gag) 和 Gagp6 中去除 C 端 His 标签均匀地将 RNA-蛋白质复合物的 Kd 值增加了 2.5 倍，但不影响这些蛋白质的结合特异性。总而言之，我们的结果证明了 p6 结构域在 Pr55(Gag)-RNA 相互作用的特异性中的新作用，并强烈表明 p6 结构域有助于区分 HIV-1 gRNA 与细胞和剪接病毒 mRNA，这对于其选择性衣壳化是必要的。
The Pr55(Gag) precursor specifically selects the HIV-1 genomic RNA (gRNA) from a large excess of cellular and partially or fully spliced viral RNAs and drives the virus assembly at the plasma membrane. During these processes, the NC domain of Pr55(Gag) interacts with the gRNA, while its C-terminal p6 domain binds cellular and viral factors and orchestrates viral particle release. Gagp6 is a truncated form of Pr55(Gag) lacking the p6 domain usually used as a default surrogate for wild type Pr55(Gag) for in vitro analysis. With recent advance in production of full-length recombinant Pr55(Gag), here, we tested whether the p6 domain also contributes to the RNA binding specificity of Pr55(Gag) by systematically comparing binding of Pr55(Gag) and Gagp6 to a panel of viral and cellular RNAs. Unexpectedly, our fluorescence data reveal that the p6 domain is absolutely required for specific binding of Pr55(Gag) to the HIV-1 gRNA. Its deletion resulted not only in a decreased affinity for gRNA, but also in an increased affinity for spliced viral and cellular RNAs. In contrast Gagp6 displayed a similar affinity for all tested RNAs. Removal of the C-terminal His-tag from Pr55(Gag) and Gagp6 uniformly increased the Kd values of the RNA-protein complexes by 2.5 fold but did not affect the binding specificities of these proteins. Altogether, our results demonstrate a novel role of the p6 domain in the specificity of Pr55(Gag)-RNA interactions, and strongly suggest that the p6 domain contributes to the discrimination of HIV-1 gRNA from cellular and spliced viral mRNAs, which is necessary for its selective encapsidation.