Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.
Critical Contribution of Tyr15 in the HIV-1 Integrase (IN) in Facilitating IN Assembly and Nonenzymatic Function through the IN Precursor Form with Reverse Transcriptase.
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HIV-1 整合酶 (IN) 中的 Tyr15 通过逆转录酶的 IN 前体形式促进 IN 组装和非酶功能。
作者：
Takahata T;Takeda E;Tobiume M;Tokunaga K;Yokoyama M;Huang YL;Hasegawa A;Shioda T;Sato H;Kannagi M;Masuda T.
已经报道了HIV-1整合酶（IN）在酶促整合步骤之前的步骤中的非酶促作用。为了获得结构和功能的洞察IN的非酶的作用，我们进行了遗传分析的HIV-1 IN，集中在一个高度保守的Tyr 15的N-末端结构域（NTD），这已被证明是调节两个NTD二聚体构象之间的平衡状态。用丙氨酸、组氨酸或色氨酸取代Tyr 15可预防HIV-1感染，并导致逆转录严重受损，而进入细胞后逆转录酶（RT）或衣壳解体动力学无明显缺陷。重组IN蛋白的交联分析表明，致命的Tyr 15突变严重损害IN结构的组装。值得注意的是，用苯丙氨酸取代Tyr 15对所有IN功能都是耐受的，这表明芳香族侧链的苯环是IN组装和功能的关键部分。基于先前提出的IN组装四聚体模型的其他诱变分析表明Tyr 15在促进IN亚基之间的疏水相互作用中起关键作用，连同亚基界面内的其他近端残基。用RT和IN缺失的突变HIV-1（ΔRT ΔIN）和IN和RT反式供应的拯救实验表明，非酶促IN功能可能通过IN前体与RT缀合（RT-IN）来发挥。重要的是，Tyr 15的致死突变显著降低了RT-IN的功能和组装。两者合计，Tyr 15似乎在促进通过RT-IN前体形式在病毒RNA上的IN和RT的正确组装中发挥关键作用。重要性IN酶链转移功能（TFTI）抑制剂已应用于联合抗逆转录病毒疗法来治疗HIV-1感染的患者。最近，已经发现了与HIV-1 IN残基相互作用的变构IN抑制剂（ALLINI），其位置不同于由ALLINI靶向的催化位点。重要的是，ALLINI影响HIV-1 IN的非酶促作用，为开发具有与ALLINI不同机制的下一代IN抑制剂提供了理论基础。在这里，我们证明了Tyr 15在HIV-1 IN NTD中通过促进NTD与IN的其他结构域的疏水相互作用在IN组装过程中起着关键作用。重要的是，我们发现IN通过其与RT的融合形式进行功能组装对于IN发挥其非酶功能至关重要。我们的研究结果为HIV-1 IN的非酶功能及其预防提供了一种新的机制。
Nonenzymatic roles for HIV-1 integrase (IN) at steps prior to the enzymatic integration step have been reported. To obtain structural and functional insights into the nonenzymatic roles of IN, we performed genetic analyses of HIV-1 IN, focusing on a highly conserved Tyr15 in the N-terminal domain (NTD), which has previously been shown to regulate an equilibrium state between two NTD dimer conformations. Replacement of Tyr15 with alanine, histidine, or tryptophan prevented HIV-1 infection and caused severe impairment of reverse transcription without apparent defects in reverse transcriptase (RT) or in capsid disassembly kinetics after entry into cells. Cross-link analyses of recombinant IN proteins demonstrated that lethal mutations of Tyr15 severely impaired IN structure for assembly. Notably, replacement of Tyr15 with phenylalanine was tolerated for all IN functions, demonstrating that a benzene ring of the aromatic side chain is a key moiety for IN assembly and functions. Additional mutagenic analyses based on previously proposed tetramer models for IN assembly suggested a key role of Tyr15 in facilitating the hydrophobic interaction among IN subunits, together with other proximal residues within the subunit interface. A rescue experiment with a mutated HIV-1 with RT and IN deleted (ΔRT ΔIN) and IN and RT supplied intransrevealed that the nonenzymatic IN function might be exerted through the IN precursor conjugated with RT (RT-IN). Importantly, the lethal mutations of Tyr15 significantly reduced the RT-IN function and assembly. Taken together, Tyr15 seems to play a key role in facilitating the proper assembly of IN and RT on viral RNA through the RT-IN precursor form.IMPORTANCEInhibitors of the IN enzymatic strand transfer function (INSTI) have been applied in combination antiretroviral therapies to treat HIV-1-infected patients. Recently, allosteric IN inhibitors (ALLINIs) that interact with HIV-1 IN residues, the locations of which are distinct from the catalytic sites targeted by INSTI, have been discovered. Importantly, ALLINIs affect the nonenzymatic role(s) of HIV-1 IN, providing a rationale for the development of next-generation IN inhibitors with a mechanism that is distinct from that of INSTI. Here, we demonstrate that Tyr15 in the HIV-1 IN NTD plays a critical role during IN assembly by facilitating the hydrophobic interaction of the NTD with the other domains of IN. Importantly, we found that the functional assembly of IN through its fusion form with RT is critical for IN to exert its nonenzymatic function. Our results provide a novel mechanistic insight into the nonenzymatic function of HIV-1 IN and its prevention.