Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.
Formation of RNA Granule-Derived Capsid Assembly Intermediates Appears To Be Conserved between Human Immunodeficiency Virus Type 1 and the Nonprimate Lentivirus Feline Immunodeficiency Virus.
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RNA 颗粒衍生的衣壳组装中间体的形成似乎在人类免疫缺陷病毒 1 型和非灵长类慢病毒猫免疫缺陷病毒之间是保守的。
DOI：
10.1128/jvi.01761-17
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发表时间：
2018
影响因子：
5.4
通讯作者：
Lingappa,JaisriR
中科院分区：
文献类型：
--
作者：
Reed,JonathanC;Westergreen,Nick;Barajas,BrookC;Ressler,DylanTB;Phuong,DarylJ;Swain,JohnV;Lingappa,VishwanathR;Lingappa,JaisriR
在细胞中未成熟衣壳组装过程中，人类免疫缺陷病毒1型（HIV-1） Gag会选择宿主RNA颗粒，形成一条含有宿主成分的细胞内组装中间体途径，包括两种细胞组装促进剂ABCE1和DDX6。在其他灵长类慢病毒中也观察到类似的组装途径。在这里，我们询问猫免疫缺陷病毒（FIV），一种非灵长类慢病毒，是否也形成RNA颗粒衍生的衣壳组装中间体。首先，我们发现在分析过程中，释放的FIV未成熟衣壳和含有大量FIV gag的细胞内复合物是不稳定的，这与HIV-1不同。我们确定了收获条件，包括在交叉连接中，克服了这个问题，揭示了一系列FIV Gag-containing复合物，其大小与HIV-1组装中间体相对应。先前，我们发现组装缺陷的HIV-1 Gag突变体在特定的组装中间体上被阻止；在这里，我们确定了四个装配缺陷的FIV Gag突变体，包括三个以前没有研究过的突变体，并证明它们似乎与同源的HIV-1突变体在相同的中间被捕获。来自共免疫沉淀的进一步证据表明，这些含有FIV Gag的复合物对应于组装中间体，这表明内源性ABCE1和RNA颗粒蛋白DDX6与FIV Gag相关，正如之前在HIV-1 Gag中所显示的那样，但在稳定状态下与核糖体蛋白无关。此外，我们发现FIV Gag与另一种RNA颗粒蛋白DCP2结合。最后，我们通过近距离连接实验验证了FIV Gag-ABCE1和FIV Gag-DCP2的相互作用，证明了它们在原位共定位。总之，这些数据支持一种模型，即灵长类动物和非灵长类动物慢病毒形成细胞内衣壳组装中间体，源自非翻译宿主RNA颗粒。与HIV-1 Gag一样，FIV Gag也组装成未成熟的衣壳；然而，尚不清楚FIV Gag是否通过来自宿主RNA颗粒的未成熟衣壳组装中间体的途径进行，如HIV-1 Gag所示。在这里，我们发现FIV Gag形成的复合物在大小上类似于HIV-1衣壳组装中间体，并且它们与ABCE1和DDX6 （HIV-1组装中间体中发现的两种HIV-1未成熟衣壳组装的宿主促进剂）的关联。我们的研究还表明，已知的和新的装配缺陷的FIV Gag突变体不能以类似于HIV-1 Gag突变体的模式进展到假定的中间体。最后，我们利用成像技术证明FIV Gag与ABCE1和RNA颗粒蛋白DCP2共定位。因此，我们得出结论，来自宿主RNA颗粒的组装中间体的形成在灵长类动物和非灵长类动物慢病毒之间可能是保守的，并且可以为未来的抗病毒策略提供靶点。
During immature capsid assembly in cells, human immunodeficiency virus type 1 (HIV-1) Gag co-opts a host RNA granule, forming a pathway of intracellular assembly intermediates containing host components, including two cellular facilitators of assembly, ABCE1 and DDX6. A similar assembly pathway has been observed for other primate lentiviruses. Here we asked whether feline immunodeficiency virus (FIV), a nonprimate lentivirus, also forms RNA granule-derived capsid assembly intermediates. First, we showed that the released FIV immature capsid and a large FIV Gag-containing intracellular complex are unstable during analysis, unlike for HIV-1. We identified harvest conditions, includingin situcross-linking, that overcame this problem, revealing a series of FIV Gag-containing complexes corresponding in size to HIV-1 assembly intermediates. Previously, we showed that assembly-defective HIV-1 Gag mutants are arrested at specific assembly intermediates; here we identified four assembly-defective FIV Gag mutants, including three not previously studied, and demonstrated that they appear to be arrested at the same intermediate as the cognate HIV-1 mutants. Further evidence that these FIV Gag-containing complexes correspond to assembly intermediates came from coimmunoprecipitations demonstrating that endogenous ABCE1 and the RNA granule protein DDX6 are associated with FIV Gag, as shown previously for HIV-1 Gag, but are not associated with a ribosomal protein, at steady state. Additionally, we showed that FIV Gag associates with another RNA granule protein, DCP2. Finally, we validated the FIV Gag-ABCE1 and FIV Gag-DCP2 interactions with proximity ligation assays demonstrating colocalizationin situ. Together, these data support a model in which primate and nonprimate lentiviruses form intracellular capsid assembly intermediates derived from nontranslating host RNA granules.IMPORTANCELike HIV-1 Gag, FIV Gag assembles into immature capsids; however, it is not known whether FIV Gag progresses through a pathway of immature capsid assembly intermediates derived from host RNA granules, as shown for HIV-1 Gag. Here we showed that FIV Gag forms complexes that resemble HIV-1 capsid assembly intermediates in size and in their association with ABCE1 and DDX6, two host facilitators of HIV-1 immature capsid assembly that are found in HIV-1 assembly intermediates. Our studies also showed that known and novel assembly-defective FIV Gag mutants fail to progress past putative intermediates in a pattern resembling that observed for HIV-1 Gag mutants. Finally, we used imaging to demonstrate colocalization of FIV Gag with ABCE1 and with the RNA granule protein DCP2. Thus, we conclude that formation of assembly intermediates derived from host RNA granules is likely conserved between primate and nonprimate lentiviruses and could provide targets for future antiviral strategies.