Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.
Promiscuous RNA binding ensures effective encapsidation of APOBEC3 proteins by HIV-1.
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DOI：
10.1371/journal.ppat.1004609
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发表时间：
2015-01
期刊：
影响因子：
6.7
通讯作者：
Malim MH
中科院分区：
文献类型：
--
作者：
Apolonia L;Schulz R;Curk T;Rocha P;Swanson CM;Schaller T;Ule J;Malim MH
载脂蛋白 B mRNA 编辑酶催化多肽样 3 (APOBEC3) 蛋白是细胞编码的胞苷脱氨酶，其中一些蛋白，如 APOBEC3G (A3G) 和 APOBEC3F (A3F)，可作为有效的人类免疫缺陷病毒 1 型 (HIV-1) 限制因子。这些蛋白质需要包装成 HIV-1 颗粒才能发挥其抗病毒活性，但其发生的分子机制尚不完全清楚。 HIV-1 Gag 的核衣壳 (NC) 区域是 A3G 和 A3F 有效掺入所必需的，并且 A3G 和 NC 之间的相互作用先前已被证明是 RNA 依赖性的。在这里，我们首先使用无偏的个体核苷酸分辨率紫外交联和免疫沉淀 (iCLIP) 方法确定哪些 RNA 能够与 HV-1 感染的细胞以及无细胞病毒体中的 A3G 和 A3F 结合，从而详细解决这个问题。我们发现 A3G 和 A3F 结合许多不同类型的 RNA，包括 HIV-1 RNA、细胞 mRNA 和小非编码 RNA，例如 Y 或 7SL RNA。有趣的是，当包装的 HIV-1 基因组 RNA (gRNA) 和 7SL RNA 的水平降低时，A3G/F 掺入不受影响，这意味着这些 RNA 对于有效的 A3G/F 包装不是必需的。证实了早期的工作，由缺乏 NC 结构域的 Gag (Gag ΔNC) 形成的 HIV-1 颗粒未能包裹 A3G/F。在这里，我们通过证明向 Gag ΔNC 添加各种异源 RNA 结合蛋白和结构域有效恢复 A3G/F 包装来利用该系统，表明 A3G 和 A3F 具有接合多个 RNA 以确保病毒衣壳化的能力。我们提出，A3G 和 A3F 相当不加区别的 RNA 结合特性通过招募到各种逆转录病毒颗粒中来促进功能，这些逆转录病毒颗粒的包装 RNA 基因组包含不同的序列。 APOBEC3 蛋白是细胞编码的限制因子，可以抵抗感染，特别是 HIV-1 等逆转录病毒和逆转录转座子的感染。当包装到 HIV-1 颗粒中时，APOBEC3G 和 APOBEC3F 都会抑制逆转录并诱导病毒 DNA 发生破坏性超突变。尽管已经确定了 APOBEC3 病毒颗粒包装对 RNA 结合的依赖性，但其机制仍有待阐明。在这里，我们采用交联和下一代测序方法来确定哪些 RNA 与 HIV-1 感染细胞中的 A3G 和 A3F 结合。我们表明，这两种蛋白都能与多种不同的 RNA 结合，包括病毒 RNA 以及细胞编码和非编码 RNA，且选择性证据相对较少。然后，我们开发了一种互补测定法来解决可作为 A3G/F 病毒粒子包装底物的 RNA 的多样性。与 RNA 结合谱一致，许多 RNA 可以促进包装，前提是这些 RNA 本身被包装。这些观察结果表明 APOBEC3 包装缺乏选择性，并且仅由这些蛋白质的非特异性 RNA 结合能力驱动。我们推测该模型解释了容易受到单个 APOBEC3 蛋白抑制的广泛逆转录元件，并且此类底物无法通过序列变异逃脱 APOBEC3 介导的抑制。
The apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3 (APOBEC3) proteins are cell-encoded cytidine deaminases, some of which, such as APOBEC3G (A3G) and APOBEC3F (A3F), act as potent human immunodeficiency virus type-1 (HIV-1) restriction factors. These proteins require packaging into HIV-1 particles to exert their antiviral activities, but the molecular mechanism by which this occurs is incompletely understood. The nucleocapsid (NC) region of HIV-1 Gag is required for efficient incorporation of A3G and A3F, and the interaction between A3G and NC has previously been shown to be RNA-dependent. Here, we address this issue in detail by first determining which RNAs are able to bind to A3G and A3F in HV-1 infected cells, as well as in cell-free virions, using the unbiased individual-nucleotide resolution UV cross-linking and immunoprecipitation (iCLIP) method. We show that A3G and A3F bind many different types of RNA, including HIV-1 RNA, cellular mRNAs and small non-coding RNAs such as the Y or 7SL RNAs. Interestingly, A3G/F incorporation is unaffected when the levels of packaged HIV-1 genomic RNA (gRNA) and 7SL RNA are reduced, implying that these RNAs are not essential for efficient A3G/F packaging. Confirming earlier work, HIV-1 particles formed with Gag lacking the NC domain (Gag ΔNC) fail to encapsidate A3G/F. Here, we exploit this system by demonstrating that the addition of an assortment of heterologous RNA-binding proteins and domains to Gag ΔNC efficiently restored A3G/F packaging, indicating that A3G and A3F have the ability to engage multiple RNAs to ensure viral encapsidation. We propose that the rather indiscriminate RNA binding characteristics of A3G and A3F promote functionality by enabling recruitment into a wide range of retroviral particles whose packaged RNA genomes comprise divergent sequences. APOBEC3 proteins are cell-encoded restriction factors that counteract infections, particularly by retroviruses such as HIV-1, and retrotransposons. When packaged into HIV-1 particles, APOBEC3G and APOBEC3F both inhibit reverse transcription and induce destructive hypermutation in viral DNA. The mechanism of APOBEC3 virion packaging awaits elucidation, though a dependency on RNA binding has been established. Here, we employed a cross-linking and next generation sequencing approach to determine which RNAs are bound to A3G and A3F in HIV-1 infected cells. We show that both proteins bind to multiple different RNAs, including viral RNA as well as cellular coding and non-coding RNAs, with relatively little evidence of selectivity. We then developed a complementation assay to address the diversity of RNAs that can act as substrates for A3G/F virion packaging. Consistent with the RNA binding profiles, many RNAs can promote packaging provided that those RNAs are, themselves, packaged. These observations suggest that APOBEC3 packaging lacks selectivity and is driven simply by the non-specific RNA binding capabilities of these proteins. We speculate that this model accounts for the broad range of retro-elements that are susceptible to repression by individual APOBEC3 proteins, and also that such substrates cannot escape APOBEC3-mediated inhibition through sequence variation.
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