Mechanisms in Retroviral Replication and Pathogenesis

逆转录病毒组装部的研究工作的目标是扩大我们对逆转录病毒复制和发病机制的基本机制的理解。这一认识可能导致抗击包括艾滋病在内的逆转录病毒引起的疾病的新方法。 逆转录病毒蛋白质和核酸之间似乎有几种不同的相互作用模式，每一种模式都对病毒复制具有重要的功能后果。首先，在病毒组装过程中，Gag多蛋白(病毒颗粒的结构蛋白)通过精妙的特异性识别选择病毒RNA进行包装。这种识别涉及蛋白质中的锌指。我们正在研究Gag蛋白在体内组装过程中识别和包装病毒基因组RNA的机制。我们的研究有力地表明，识别信号涉及由基因组RNA分子的二聚体形成的三维结构。我们正在研究二聚体连接的结构及其在病毒RNA包装中的可能作用。我们的突变研究还表明，除了在识别过程中的作用外，锌指还有其他重要的功能。这些额外的功能目前正在调查中。 其次，Gag多聚蛋白及其裂解产物--核衣壳蛋白(NC)具有核酸伴侣活性。也就是说，它们瞬时地破坏碱基对的稳定，催化构象转变为核酸分子中最优的碱基配对结构。这种序列无关的活性在病毒组装之前或期间使用，当Gag多蛋白促进细胞tRNA分子对病毒RNA的退火时；当病毒感染新的宿主细胞时，tRNA是逆转录的引物。当NC诱导病毒RNA二聚体在颗粒内的构象重排时，在病毒成熟期间(即，在GAG被病毒蛋白酶切割后)，该活性再次被使用。该活性在逆转录过程中似乎也是至关重要的，促进了前病毒DNA合成中的聚合和链转移步骤；最近的数据表明，它在DNA整合到宿主染色体中也可能是重要的。我们正在研究这些蛋白质核酸伴侣活性的分子机制。 第三，Gag多聚蛋白与病毒RNA或细胞mRNA分子相互作用，将它们用作组装病毒颗粒的“脚手架”。当产生病毒的细胞含有甲型病毒载体时，缺乏病毒RNA的颗粒中的细胞mRNA分子的存在尤其明显。这是因为甲型病毒载体将其RNA复制到非常高的水平，导致细胞中的mRNA群几乎是单分散的；在这种条件下，很容易在逆转录病毒颗粒中检测到取代逆转录病毒RNA的甲型病毒来源的mRNA。 我们还发现，HIV-1 Gag多聚蛋白能够在体外完全确定的系统中组装成微小的球形病毒样颗粒(在核酸存在的情况下)。短至10-15个核苷酸的寡聚脱氧核苷酸可以满足核酸需求。病毒样颗粒的直径仅为25-30 nm，而哺乳动物细胞中形成的真病毒粒子的核心直径约为100 nm。我们发现，如果在网织红细胞裂解物存在的情况下进行组装反应，形成的颗粒为100 nm，而不是25-30 nm。因此，哺乳动物细胞包含一个因子，它改变了GAG多蛋白分子在组装过程中相互作用的曲率半径。我们现在已经确定这个因素是五磷酸肌醇(IP5)。我们现在正在分析IP5对HIV-1 Gag分子之间相互作用的分子机制。我们还试图证明IP5或相关化合物有助于HIV-1病毒颗粒在体内的正确组装。 在所有逆转录病毒中，Gag多聚蛋白都以病毒产生细胞的质膜为靶点。虽然我们对这些蛋白质如何与膜结合有了一些了解，但质膜特异性的原因是完全未知的。我们还在探索磷脂酰肌醇参与将这些蛋白质靶向质膜的可能性。这些实验中的许多都使用了共聚焦显微镜。 此外，我们还利用表面等离子体共振技术分析了HIV-1 NC蛋白与非常短的寡核苷酸的结合。尽管NC可能能够与任何单链DNA或RNA结合，但这些研究表明它表现出深刻的序列偏好。我们从事了详细的研究NC和GAG与短的，定义明确的寡核苷酸的结合；这些信息应该有助于我们理解上面讨论的各种与核酸的相互作用(即NC和GAG的伴侣活性，GAG组装病毒样颗粒，以及在体内病毒组装过程中GAG对基因组RNA的精致特异性包裹)。 通过NC结合特定核酸和通过GAG组装病毒的简便体外检测的存在，使筛选这些功能的抑制剂的大型化合物文库成为可能。能够干扰这些功能的化合物已经被确定，现在正在测试它们防止HIV-1在培养细胞中复制的能力。

The goal of the research efforts in the Retrovirus Assembly Section is to extend our understanding of basic mechanisms in retroviral replication and pathogenesis. This understanding may lead to new methods of combatting retrovirus-induced disease, including AIDS. There appear to be several different modes of interaction between retroviral proteins and nucleic acids, each with important functional consequences for viral replication. First, an exquisitely specific recognition by the Gag polyprotein (the structural protein of the virus particle) selects the viral RNA for packaging during virus assembly. This recognition involves zinc fingers in the protein. We are studying the mechanism by which the Gag protein recognizes and packages the genomic RNA of the virus during assembly in vivo. Our research strongly suggests that the recognition signal involves the three-dimensional structure formed by a dimer of genomic RNA molecules. We are studying the structure of the dimer linkage and its possible role in packaging of viral RNA. Our mutational studies also show that the zinc fingers have other crucial functions, in addition to their role in the recognition process. These additional functions are now under investigation. Second, the Gag polyprotein and its cleavage product, the nucleocapsid (NC) protein, exhibit nucleic acid chaperone activity. That is, they transiently destabilize base pairs, catalyzing conformational transitions to the optimally base-paired structure in a nucleic acid molecule. This sequence-independent activity is used before or during virus assembly, when the Gag polyprotein promotes the annealing of a cellular tRNA molecule to the viral RNA; the tRNA is the primer for reverse transcription when the virus infects a new host cell. The activity is used again during virus maturation (i.e., after Gag is cleaved by the viral protease), when NC induces a conformational rearrangement in the viral RNA dimer within the particle. The activity also appears to be crucial during reverse transcription, facilitating both polymerization and strand-transfer steps during proviral DNA synthesis; recent data suggest that it may be important during the integration of the DNA into the host chromosome as well. We are studying the molecular mechanism underlying the nucleic acid chaperone activity of these proteins. Third, the Gag polyprotein interacts with either the viral RNA or, alternatively, cellular mRNA molecules, using them as "scaffolding" in the assembly of virus particles. The presence of cellular mRNA molecules in particles lacking the viral RNA was particularly obvious when the virus-producing cells contained an alphaviral vector. This is because alphaviral vectors replicate their RNAs to extraordinary levels, resulting in a nearly monodisperse population of mRNAs in the cell; under these conditions the alphavirus-derived mRNA replacing retroviral RNA was easy to detect in retroviral particles. We have also found that the HIV-1 Gag polyprotein is able to assemble (in the presence of nucleic acid) into minute spherical virus-like particles in a completely defined system in vitro. The nucleic acid requirement can be fulfilled by oligodeoxynucleotides as short as 10-15 nucleotides. The virus-like particles are only 25-30 nm in diameter, whereas the cores of authentic virions formed in mammalian cells are ~100 nm in diameter. We found that if assembly reactions are performed in the presence of reticulocyte lysates, particles of 100 nm, rather than 25-30 nm, are formed. Therefore, mammalian cells contain a factor that alters the radius of curvature with which Gag polyprotein molecules interact with each other during the assembly process. We have now identified this factor as inositol pentakisphosphate (IP5). We are now analyzing the molecular mechanism of this effect of IP5 on the interactions between HIV-1 Gag molecules. We are also attempting to demonstrate that IP5 or related compounds contribute to the correct assembly of HIV-1 virus particles in vivo. In all retroviruses, the Gag polyprotein is targeted to the plasma membrane of the virus-producing cell. While we have some understanding of how these proteins bind to membranes, the reason for the specificity for the plasma membrane is completely unknown. We are also exploring the possibility that phosphatidylinositides are involved in targeting these proteins to the plasma membrane. Many of these experiments are using confocal microscopy. In addition, we have used surface plasmon resonance technology to analyze the binding of HIV-1 NC protein to very short oligonucleotides. Although NC is probably capable of binding to any single-stranded DNA or RNA, these studies showed that it exhibits profound sequence preferences. We are engaged in a detailed investigation of the binding of NC and Gag to short, well-defined oligonucleotides; this information should help us understand the various interactions with nucleic acids discussed above (i.e., chaperone activity of both NC and Gag, assembly of virus-like particles by Gag, and the exquisitely specific encapsidation of genomic RNA by Gag during virus assembly in vivo). The existence of convenient in vitro assays for specific nucleic acid binding by NC and for virus assembly by Gag has made it possible to screen large libraries of compounds for inhibitors of these functions. Compounds capable of interfering with these functions have been identified and are now being assayed for their ability to prevent HIV-1 replication in cultured cells.