APOBEC3 proteins APOBEC3F (A3F), APOBEC3G (A3G), and APOBEC3H (A3H) are host restriction factors that inhibit HIV-1 through DNA cytidine deaminase-dependent and -independent mechanisms and have either one (A3H) or two (A3F and A3G) zinc-binding domains. A3H antiviral activity encompasses multiple molecular functions, all of which depend on recognition of RNA or DNA. A3H crystal structures revealed an unusual interaction with RNA wherein an RNA duplex mediates dimerization of two A3H proteins. In this study, we sought to determine the importance of RNA-binding amino acids in the antiviral and biochemical properties of A3H. We show that the wild-type A3H-RNA interaction is essential for A3H antiviral activity and for two deaminase-independent processes: encapsidation into viral particles and inhibition of reverse transcription. Furthermore, an extensive mutagenesis campaign revealed distinct roles for two groups of amino acids at the RNA binding interface. C-terminal helix residues exclusively bind RNA, and loop 1 residues play a dual role in recognition of DNA substrates and in RNA binding. Weakening the interface between A3H and RNA allows DNA substrates to bind with greater affinity and enhances deamination rates, suggesting that RNA binding must be disrupted to accommodate DNA. Intriguingly, we demonstrate that A3H can deaminate overhanging DNA strands of RNA/DNA heteroduplexes, which are early intermediates during reverse transcription and may represent natural A3H substrates. Overall, we present a mechanistic model of A3H restriction and a step-by-step elucidation of the roles of RNA-binding residues in A3H activity, particle incorporation, inhibition of reverse transcriptase inhibition, and DNA cytidine deamination.IMPORTANCE APOBEC3 proteins are host factors that protect the integrity of the host genome by inhibiting retroelements as well as retroviruses, such as HIV-1. To do this, the APOBEC3H protein has evolved unique interactions with structured RNAs. Here, we studied the importance of these interactions in driving antiviral activity of APOBEC3H. Our results provide a clear picture of how RNA binding drives the ability of APOBEC3H to infiltrate new viruses and prevent synthesis of viral DNA. We also explore how RNA binding by APOBEC3H influences recognition and deamination of viral DNA and describe two possible routes by which APOBEC3H might hypermutate the HIV-1 genome. These results highlight how one protein can sense many nucleic acid species for a variety of antiviral activities.

中文翻译：

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核酸结合的灵活性是 APOBEC3H 抗病毒活性的核心。


APOBEC3 蛋白 APOBEC3F (A3F)、APOBEC3G (A3G) 和 APOBEC3H (A3H) 是宿主限制因子，通过 DNA 胞苷脱氨酶依赖性和非依赖性机制抑制 HIV-1，并具有一个 (A3H) 或两个（A3F 和 A3G）锌结合域。 A3H 抗病毒活性涵盖多种分子功能，所有这些功能都依赖于 RNA 或 DNA 的识别。 A3H 晶体结构揭示了与 RNA 的不寻常相互作用，其中 RNA 双链体介导两个 A3H 蛋白的二聚化。在这项研究中，我们试图确定 RNA 结合氨基酸在 A3H 的抗病毒和生化特性中的重要性。我们表明，野生型 A3H-RNA 相互作用对于 A3H 抗病毒活性和两个不依赖脱氨酶的过程至关重要：衣壳化为病毒颗粒和抑制逆转录。此外，广泛的诱变活动揭示了 RNA 结合界面上两组氨基酸的不同作用。 C 端螺旋残基专门结合 RNA，环 1 残基在识别 DNA 底物和 RNA 结合中发挥双重作用。削弱 A3H 和 RNA 之间的界面可以使 DNA 底物以更大的亲和力结合并提高脱氨率，这表明必须破坏 RNA 结合才能容纳 DNA。有趣的是，我们证明 A3H 可以使 RNA/DNA 异源双链体的悬垂 DNA 链脱氨基，这是逆转录过程中的早期中间体，可能代表天然的 A3H 底物。总的来说，我们提出了 A3H 限制的机制模型，并逐步阐明了 RNA 结合残基在 A3H 活性、颗粒掺入、逆转录酶抑制和 DNA 胞苷脱氨中的作用。重要性 APOBEC3 蛋白是通过抑制逆转录元件和逆转录病毒（例如 HIV-1）来保护宿主基因组完整性的宿主因子。为此，APOBEC3H 蛋白进化出了与结构化 RNA 的独特相互作用。在这里，我们研究了这些相互作用在驱动 APOBEC3H 抗病毒活性中的重要性。我们的结果清楚地表明 RNA 结合如何驱动 APOBEC3H 渗透新病毒并阻止病毒 DNA 合成的能力。我们还探讨了 APOBEC3H 与 RNA 的结合如何影响病毒 DNA 的识别和脱氨基作用，并描述了 APOBEC3H 可能使 HIV-1 基因组发生超突变的两种可能途径。这些结果强调了一种蛋白质如何感知多种核酸种类以发挥多种抗病毒活性。