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，而不是在受感染细胞的细胞质或细胞核中产生的。衣壳蛋白作为Gag多聚蛋白的一部分被结合到前病毒中。在前病毒蛋白发芽后，病毒蛋白水解酶将Gag分解成基质(MA)、衣壳(CA)和核衣壳(NC)结构域以及两个间隔肽SP1和SP2。从Gag壳中释放出来的CA组装成病毒衣壳，包含RNA和NC。有证据表明，正确形成的核心对于传染性是必不可少的。干扰成熟可以通过三种方式抑制病毒。一种是蛋白酶抑制剂(PI)，这是第一种成功用于对抗艾滋病毒的药物。最近，成熟抑制物(MI)被发现通过阻止蛋白酶进入SP1间隔肽中的裂解位点来发挥作用。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感染细胞中分离出的病毒粒子大多没有衣壳，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，但缺少与NC/RNA相关的最内层。这些发现发表在2011年。在随后的工作中，我们记录了强烈支持这一观点的数据，即野生型HIV病毒粒子的双锥形衣壳是在成熟的病毒粒子内重新组装的，而不是通过置换转变。在第二个MI，PF-46396存在的情况下产生的病毒粒子类似于BVM处理的病毒粒子，尽管这种MI在CA-SP1裂解抑制和阻断感染性方面都不那么严格。经MI处理的病毒粒子的外壳类似于未成熟的Gag外壳的CA层，但不太完整。我们的结论是，像PF-46396和Bvm这样的抑制剂结合到部分加工的GAG晶格上，在那里它们阻止了蛋白酶进入CA-Sp1裂解位点，并阻止了CA的释放。这些发现发表于2013年。接下来，我们研究了整合酶(IN)抑制剂(ALLINIS变构IN抑制剂)和整合酶基因中某些突变体的作用模式。这两种病毒粒子都被发现含有位于空壳外的“偏心凝聚物”，这些衣壳通常是畸形的。我们能够通过层析气泡图成像显示偏心凝析油具有高的NC含量，这是一种利用NC对辐射损伤的敏感性的新型标记技术。断层气泡图还定位了野生型核心内的NC，并将未成熟病毒粒子中的球状搞笑外壳衬里。基于这些观察，我们提出了IN在启动核心组装和将vRNP掺入成熟核心中的作用。这些发现发表于2015年。 在与E.Freed(NCI)的继续合作中，我们一直在研究保守的Pro-Pro-Ile-Pro(PPIP)基序(CA残基122-125)在连接HIV-1组装中CA的6和7螺旋(H6-H7环)中的作用。数据表明，突变P122A和I124A损害了释放、感染性和复制，T58S/T107I/P122A突变恢复到野生型(WT)样感染性。我们使用冷冻-ET和亚断层图像平均来评估这些突变如何影响未成熟(PR-)病毒粒子的组装。我们的结果表明，P122A和I124A突变损害了Gag晶格的配位，T58s/T107I/P122A产生了类WT晶格。因此，PPIP基序在病毒组装过程中协调未成熟的Gag晶格具有重要作用。目前正在对PR+病毒粒子进行检测，以确定PPIP基序在病毒成熟过程中的作用。 2)HIV Rev是一种小的调节蛋白，它介导基因组病毒mRNAs的核输出，这是HIV复制周期中的一个重要步骤。在这个过程中，REV与一种结构化的RNA分子--REV反应元件(RRE)结合在一起进行寡聚。这个复合体与宿主细胞的核输出机制接合。这种相互作用的详细结构信息对于设计抑制REV的抗病毒药物是必不可少的。多年来，由于雷夫的聚集倾向，结晶学研究一直受阻。然而，我们能够构建一种杂交单抗，其Fab与REV形成稳定的络合物，并以0.32 nm的分辨率溶解这些共晶体。这些结果发表在2011财年。随着对该抗体的进一步开发，我们对HIV Rev的研究仍在继续。特别是，我们构建了一个单链版本(ScFv)，并发现它也与REV共晶，这些晶体衍射到了显著更高的分辨率。这些晶体分成四个不同的空间群。尽管REV二聚体的交叉角从90度到140度变化很大，但所有的都被解决了，并揭示了基本上相同的单体结构。我们还对REV在体外组装成的螺旋管进行了冷冻-EM研究。它们表现出多态，管径在11 nm到13 nm之间变化。管子宽度的这些变化与晶体中所看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，它解释了REV亚基的排列如何与出口活性复合体中的RRE的A型结构相匹配。描述这项工作的论文是在上一个报告期后期发表的：DiMattia等人，Structure，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(首席执行官P.T.Wingfield)的合作已转向对与相互作用伙伴复杂的全长或部分REV结构的结晶学研究。我们的重点一直是表达感兴趣的蛋白质和建立结晶试验。几个含REV的配合物已经获得了很有前途的晶体。

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.