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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.
摘要：
人类免疫缺陷病毒I型（HIV-1）中的Gag-Pol多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)。公关的成熟形式，RT和IN是同二聚体，异二聚体和四聚体，分别。二聚体或四聚体形成的确切机制尚不清楚。这里,为了深入了解前体中PR和RT的二聚化，我们准备了一个模型前体，PR-RT,在PR活性位点掺入失活突变，D25A,并且在p6*区域中包括两个残基，融合到SUMO标签上，在PR区的N端。我们还制备了两个PR-RT突变体，在PR区含有二聚体解离突变，PR(T26A)-RT,或在RT区域，PR-RT（W401A）。尺寸排阻色谱显示PR-RT和PR（T26A）-RT中的单体和二聚体级分，但PR-RT中只有单体(W401A)。在蛋白酶抑制剂存在下PR-RT的SEC实验，darunavir,显著增强了二聚化。此外,SEC结果表明，估计的PR-RT二聚体解离常数高于成熟的RT异二聚体，p66/p51，但略低于早产RT同源二聚体，p66/p66.进行逆转录酶测定和RT成熟测定作为评估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二聚体界面在PR-RT的二聚化中起着至关重要的作用，而PR二聚体界面的作用较小。
