Interplay between protease and reverse transcriptase dimerization in a model HIV-1 polyprotein.

IF 4.5 3区 生物学 Q1 BIOCHEMISTRY & MOLECULAR BIOLOGY
Protein Science Pub Date : 2024-07-01 DOI:10.1002/pro.5080
Brisa Caroline Alves Chagas, Xiaohong Zhou, Michel Guerrero, Tatiana V Ilina, Rieko Ishima
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引用次数: 0

Abstract

The Gag-Pol polyprotein in human immunodeficiency virus type I (HIV-1) encodes enzymes that are essential for virus replication: protease (PR), reverse transcriptase (RT), and integrase (IN). The mature forms of PR, RT and IN are homodimer, heterodimer and tetramer, respectively. The precise mechanism underlying the formation of dimer or tetramer is not yet understood. Here, to gain insight into the dimerization of PR and RT in the precursor, we prepared a model precursor, PR-RT, incorporating an inactivating mutation at the PR active site, D25A, and including two residues in the p6* region, fused to a SUMO-tag, at the N-terminus of the PR region. We also prepared two mutants of PR-RT containing a dimer dissociation mutation either in the PR region, PR(T26A)-RT, or in the RT region, PR-RT(W401A). Size exclusion chromatography showed both monomer and dimer fractions in PR-RT and PR(T26A)-RT, but only monomer in PR-RT(W401A). SEC experiments of PR-RT in the presence of protease inhibitor, darunavir, significantly enhanced the dimerization. Additionally, SEC results suggest an estimated PR-RT dimer dissociation constant that is higher than that of the mature RT heterodimer, p66/p51, but slightly lower than the premature RT homodimer, p66/p66. Reverse transcriptase assays and RT maturation assays were performed as tools to assess the effects of the PR dimer-interface on these functions. Our results consistently indicate that the RT dimer-interface plays a crucial role in the dimerization in PR-RT, whereas the PR dimer-interface has a lesser role.

模型 HIV-1 多聚蛋白中蛋白酶和逆转录酶二聚化之间的相互作用。
人类免疫缺陷病毒 I 型(HIV-1)中的 Gag-Pol 多聚蛋白编码病毒复制所必需的酶:蛋白酶(PR)、逆转录酶(RT)和整合酶(IN)。PR、RT 和 IN 的成熟形式分别是同源二聚体、异源二聚体和四聚体。二聚体或四聚体形成的确切机制尚不清楚。在此,为了深入了解 PR 和 RT 在前体中的二聚化,我们制备了一个模型前体 PR-RT,在 PR 活性位点 D25A 处加入了一个失活突变,并在 PR 区域的 N 端加入了 p6* 区域的两个残基,融合了 SUMO 标记。我们还制备了 PR-RT 的两个突变体,它们在 PR 区(PR(T26A)-RT)或 RT 区(PR-RT(W401A))都含有二聚体解离突变。尺寸排阻色谱法在 PR-RT 和 PR(T26A)-RT 中显示出单体和二聚体部分,但在 PR-RT(W401A)中仅显示出单体。在蛋白酶抑制剂达芦那韦存在下对 PR-RT 进行的 SEC 实验显著增强了二聚化。此外,SEC结果表明,PR-RT二聚体的估计解离常数高于成熟RT异源二聚体p66/p51的解离常数,但略低于未成熟RT同源二聚体p66/p66的解离常数。反转录酶测定和 RT 成熟测定是评估 PR 二聚体界面对这些功能的影响的工具。我们的结果一致表明,RT 二聚体表面在 PR-RT 的二聚化过程中起着关键作用,而 PR 二聚体表面的作用较小。
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来源期刊
Protein Science
Protein Science 生物-生化与分子生物学
CiteScore
12.40
自引率
1.20%
发文量
246
审稿时长
1 months
期刊介绍: Protein Science, the flagship journal of The Protein Society, is a publication that focuses on advancing fundamental knowledge in the field of protein molecules. The journal welcomes original reports and review articles that contribute to our understanding of protein function, structure, folding, design, and evolution. Additionally, Protein Science encourages papers that explore the applications of protein science in various areas such as therapeutics, protein-based biomaterials, bionanotechnology, synthetic biology, and bioelectronics. The journal accepts manuscript submissions in any suitable format for review, with the requirement of converting the manuscript to journal-style format only upon acceptance for publication. Protein Science is indexed and abstracted in numerous databases, including the Agricultural & Environmental Science Database (ProQuest), Biological Science Database (ProQuest), CAS: Chemical Abstracts Service (ACS), Embase (Elsevier), Health & Medical Collection (ProQuest), Health Research Premium Collection (ProQuest), Materials Science & Engineering Database (ProQuest), MEDLINE/PubMed (NLM), Natural Science Collection (ProQuest), and SciTech Premium Collection (ProQuest).
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