Structural Biology of HIV Assembly and Maturation

1)逆转录病毒衣壳是不寻常的，因为它们是在成熟的病毒粒子内产生的，而不是在感染细胞的细胞质或细胞核中。衣壳蛋白作为Gag多聚蛋白的一部分并入前病毒粒子中。在前病毒体出芽后，病毒蛋白酶将Gag分解成其基质（MA）、衣壳（CA）和核衣壳（NC）结构域以及两个间隔肽SP1和SP2。从Gag壳释放的CA组装到病毒衣壳中，容纳RNA和NC。有证据表明，一个正确形成的核心是必不可少的传染性。有三种方式可以干扰成熟抑制病毒。一种是蛋白酶抑制剂（PI），这是第一种成功用于抗HIV的药物。最近，已经发现成熟抑制剂（MI）通过阻断蛋白酶接近其在SP1间隔肽中的切割位点而起作用。类成员MI是一种称为Beviramat（BVM）的化合物。我们使用冷冻电子断层扫描显示，从BVM治疗后的HIV感染细胞中分离的病毒粒子大多缺乏衣壳，但在病毒包膜下有一个不完整的蛋白质外壳，具有类似于未成熟HIV的Gag晶格的蜂窝结构，但缺乏与NC/RNA相关的最内层。这些研究结果于2011年发表。在随后的工作中，我们记录的数据强烈支持这样的观点，即野生型HIV病毒体的双锥形衣壳是在成熟的病毒体内从头组装的，而不是通过置换过渡。在存在第二种MI PF-46396的情况下产生的病毒体类似于SVM处理的病毒体，尽管这种MI在CA-SP1切割抑制和阻断感染性方面的严格性较低。MI处理的病毒体具有类似于未成熟Gag壳的CA层的壳，但不太完整。我们得出结论，PF-46396和BVM等抑制剂与部分加工的Gag晶格结合，在那里它们阻止蛋白酶进入CA-SP1切割位点并阻止CA的释放。这些研究结果于2013年发表。接下来，我们研究了整合酶（IN）抑制剂（ALLINI变构IN抑制剂）和它们表型复制的整合酶基因中的某些突变体的作用模式。这两种病毒体都被发现含有位于空的和通常畸形的衣壳外部的“偏心浓缩物”。 我们能够表明，偏心冷凝物具有高NC含量的断层气泡成像，一种新的标记技术，利用NC的敏感性辐射损伤。断层气泡图也定位NC内的野生型核心和衬里的球形加格壳在未成熟的病毒粒子。基于这些观察，我们提出了一个作用，在启动核心组件和纳入vRNP到成熟的核心。这些发现于2015年发表。 在我们与E. Freed（NCI）等人的研究中，我们一直在研究连接CA的螺旋6和7的环（H6-H7环）中的保守的Pro-Pro-Ile-Pro（PPIP）基序（CA残基122-125）在HIV-1组装中的作用。数据表明，突变P122 A和I124 A损害释放，感染性和复制，T58 S/T107 I/P122 A突变体恢复野生型（WT）样感染性。我们已经使用冷冻ET和亚断层平均来评估这些突变如何影响未成熟（PR-）病毒体的组装。我们的研究结果表明，P122 A和I124 A突变损害Gag晶格协调和T58 S/T107 I/P122 A产生WT样晶格。因此，PPIP基序在病毒组装过程中协调未成熟的Gag晶格中具有重要作用。PR+病毒体目前正在检查，以确定PPIP基序在病毒成熟中的作用。 2)HIV Rev是一种小的调节蛋白，介导基因组病毒mRNA的核输出，这是HIV复制周期中的重要步骤。在这个过程中，Rev寡聚化与结构化RNA分子，Rev反应元件（RRE）。这种复合物与宿主细胞的核输出机制相结合。这种相互作用的详细结构信息对于设计Rev抑制性抗病毒药物是必不可少的。许多年来，晶体学研究都被Rev的聚集倾向所阻碍。然而，我们能够构建杂交单克隆抗体，其Fab与Rev形成稳定的复合物，并在0.32 nm分辨率下解决这些共晶体。这些结果于2011财年公布。我们对HIV Rev的研究继续进一步开发这种抗体。特别是，我们构建了一个单链版本（scFv），并发现它也与Rev共结晶，这些晶体衍射到显着更高的分辨率。这些晶体分为四个不同的空间组。所有这些都得到了解决，并揭示了基本上相同的单体结构，尽管Rev二聚体的交叉角从90度到140度变化很大。我们还进行了冷冻电镜研究的螺旋管，Rev组装成体外。它们表现出多态性，管径在11 nm和13 nm之间变化。管宽度的这些变化与晶体中观察到的交叉角的变化相关。我们的数据还揭示了Rev之间的第三个界面，解释了Rev亚基的排列如何与输出活性复合物中RRE的A形结构相匹配。描述这项工作的论文发表在上一个报告期的后期：DiMattia等人， 结构，24：1068-80 2016。 3)在本报告所述期间，我们与NIAMS蛋白质表达实验室（P. T。温菲尔德，首席）已转向与相互作用伴侣复合的全长或部分Rev结构的晶体学研究。我们的重点一直是表达感兴趣的蛋白质和建立结晶试验。 有前途的晶体已经获得了几个Rev含配合物。

1) Retrovirus capsids are unusual in that they are produced inside the maturing virion, not in the cytoplasm or nucleus of the infected cell. Capsid protein is incorporated into the provirion as part of the Gag polyprotein. After the provirion has budded off, maturation ensues whereby the viral protease dissects Gag into its matrix (MA), capsid (CA), and nucleocapsid (NC) domains and two spacer peptides, SP1 and SP2. CA released from the Gag shell assembles into the viral capsid, housing the RNA and NC. Evidence suggests that a correctly formed core is essential for infectivity. There are three ways in which interference with maturation can inhibit the virus. One is Protease Inhibitors (PI), the first drugs to be used successfully against HIV. More recently, maturation inhibitors (MI) have been discovered that act by blocking the protease from access to its cleavage site in the SP1 spacer peptide. The class member MI is a compound called Beviramat (BVM). We used cryo-electron tomography to show that virions isolated from HIV-infected cells after BVM treatment mostly lack capsids but have an incomplete shell of protein underlying the viral envelope, with a honeycomb structure resembling the Gag lattice of immature HIV but lacking the innermost layer that is associated with NC/RNA. These findings were published in 2011. In subsequent work, we recorded data that strongly support the view that the biconical capsids of wild-type HIV virions are assembled de novo inside maturing virions and not by a displacive transition. Virions produced in the presence of a second MI, PF-46396, resemble BVM-treated virions although this MI is less stringent in both CA-SP1 cleavage inhibition and in blocking infectivity. MI-treated virions have a shell that resembles the CA layer of the immature Gag shell but is less complete. We concluded that inhibitors like PF-46396 and BVM bind to the partially processed Gag lattice where they deny the protease access to the CA-SP1 cleavage site and prevent the release of CA. These findings were published in 2013. Next, we Investigated the mode of action of integrase (IN) inhibitors (ALLINIs allosteric IN inhibitors) and certain mutants in the integrase gene that they phenocopy. Both kinds of virions were found to contain "eccentric condensates" located outside empty and often malformed capsids. We were able to show that eccentric condensates have a high NC content by tomo-bubblegram imaging, a novel labeling technique that exploits NC's susceptibility to radiation damage. Tomo-bubblegrams also localized NC inside wild-type cores and lining the spherical Gag shell in immature virions. Based on these observations, we have proposed a role for IN in initiating core assembly and the incorporation of vRNP into the mature core. These findings were published in 2015. In a continuation of our collaboration with E. Freed (NCI), we have been investigating the role of a conserved Pro-Pro-Ile-Pro (PPIP) motif (CA residues 122-125) in the loop connecting helices 6 and 7 (H6-H7 loop) of CA in HIV-1 assembly. Data suggest that the mutations P122A and I124A impair release, infectivity, and replication, and the T58S/T107I/P122A mutant reverts to wild type (WT)-like infectivity. We have used cryo-ET and subtomogram averaging to assess how these mutations affect assembly of the immature (PR-) virion. Our results suggest that the P122A and I124A mutations impair Gag lattice coordination and that T58S/T107I/P122A produces a WT-like lattice. The PPIP motif, then, has an important role in coordinating the immature Gag lattice during virus assembly. PR+ virions are now being examined to ascertain the role of the PPIP motif in virus maturation. 2) HIV Rev is a small regulatory protein that mediates the nuclear export of genomic viral mRNAs, an essential step in the HIV replication cycle. In this process, Rev oligomerizes in association with a structured RNA molecule, the Rev response element (RRE). This complex engages with the nuclear export machinery of the host cell. Detailed structural information on this interaction is essential for the purpose of designing Rev-inhibiting antiviral drugs. For many years, crystallographic studies were thwarted by Rev's tendency to aggregate. However, we were able to construct a hybrid monoclonal antibody whose Fab forms a stable complex with Rev, and solve these co-crystals at 0.32 nm resolution. These results were published in FY 11. Our research on HIV Rev continued with further exploitation of this antibody. In particular, we constructed a single-chain version (scFv) and found that it also co-crystallized with Rev and these crystals diffracted to significantly higher resolution. The crystals came in four different space groups. All were solved and revealed essentially the same structure of the monomer, although the crossing angle of the Rev dimer varies widely from 90 to 140 degrees. We also performed cryo-EM studies of helical tubes that Rev assembles into in vitro. They exhibited polymorphism, with the tube diameter varying between 11 nm and 13 nm. These variations in tube width correlated with the variations in crossing-angle seen in the crystals. Our data also revealed a third interface between Revs that explains how the arrangement of Rev subunits can be matched to the A-shaped architecture of the RRE in export-active complexes. The paper describing this work was published late in the last reporting period: DiMattia et al., Structure, 24:1068-80 2016. 3) Over the current reporting period, our collaboration with the NIAMS Protein Expression Laboratory (P. T. Wingfield, Chief) has shifted to crystallographic studies of full-length or partial Rev constructs complexed with interaction partners. Our emphasis has been on expressing proteins of interest and setting up crystallization trials. Promising crystals have already been obtained for several Rev-containing complexes.