Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.
Host cofactors and pharmacologic ligands share an essential interface in HIV-1 capsid that is lost upon disassembly.
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DOI：
10.1371/journal.ppat.1004459
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发表时间：
2014-10
期刊：
影响因子：
6.7
通讯作者：
James LC
中科院分区：
文献类型：
--
作者：
Price AJ;Jacques DA;McEwan WA;Fletcher AJ;Essig S;Chin JW;Halambage UD;Aiken C;James LC
HIV-1衣壳参与从反转录到整合位点选择的所有感染步骤，是多种宿主细胞和药物配体的靶标。然而，结构研究一直局限于衣壳单体(CA)，这些配体如何影响感染的机制基础还不清楚。在这里，我们证明了一个多亚单位界面，专门形成在CA六聚体内，介导与细胞辅因子NUP153和CPSF6内的线性表位的结合，并被抗逆转录病毒化合物PF74和BI-2竞争。每个配体都通过一个共享的苯丙氨酸-甘氨酸(FG)基序锚定到一个单体的N-末端结构域内的一个口袋上，除了BI-2之外，所有的配体也通过N-末端结构域：C-末端结构域(NTD：CTD)与第二个单体进行必要的相互作用。六聚体与CA单体的解离阻止了与CPSF6和PF74的高亲和力相互作用，并取消了与NUP153的结合。第二个界面是构象动态的，但NUP153或CPSF6多肽的结合仅由一种构象调节。NUP153和CPSF6有重叠的结合部位，但每个都产生独特的CA相互作用，当选择性突变时，扰乱辅因子依赖。这些结果表明，多个配体共享HIV-1衣壳中的重叠界面，该界面在病毒分解时丢失。人们对HIV-1感染的早期步骤知之甚少，部分原因是很难获得有关囊化病毒及其与宿主辅助因子相互作用的高分辨率信息。这反过来又使设计有效的抗衣壳(CA)药物变得困难。在我们目前的研究中，我们已经使用稳定的HIV-1CA的六聚体来获得含有两个细胞辅助因子的复杂晶体结构，这对HIV-1感染是重要的。这些结构和伴随的病毒学揭示了HIV-1衣壳中的一个基本界面，该界面在病毒剥离时丢失。该界面用于招募核靶向辅因子CPSF6和NUP153，NUP153是一种促进核进入的核孔成分。这些结构提供的高分辨率信息表明，界面是退化的，CA突变可以选择性地扰乱对每个辅因子的敏感性。这一界面也与两种抗病毒药物PF74和BI-2竞争，它们不同的作用机制尚不完全清楚。我们发现，PF74，但不是BI-2，跨多聚体衣壳内的单体结合，影响六聚体间的界面，这对维持完整的病毒粒子至关重要，并且添加饱和浓度的PF74会导致不可逆转的阻断病毒逆转录。
The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly. The early steps of HIV-1 infection are poorly understood, in part because of the difficulty in obtaining high-resolution information on encapsidated virus and its interaction with host cofactors. This, in turn, has made it difficult to design effective anti-capsid (CA) drugs. In our present study, we have used stabilized hexamers of HIV-1 CA to obtain complexed crystal structures with two cellular cofactors that are important for HIV-1 infection. These structures and accompanying virology reveal an essential interface in the capsid of HIV-1 that is lost upon viral uncoating. This interface is used to recruit both the nuclear targeting cofactor CPSF6 and NUP153, a nuclear pore component that facilitates nuclear entry. The high-resolution information provided by these structures reveals that the interface is degenerate and CA mutations can be made that selectively perturb sensitivity to each cofactor. This interface is also competed by two antiviral drugs, PF74 and BI-2, whose different mechanisms of action are not fully understood. We show that PF74, but not BI-2, binds across monomers within multimerized capsid affecting an inter-hexamer interface that is crucial for maintaining intact virions and that the addition of saturating concentrations of PF74 causes an irreversible block to viral reverse transcription.
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