MECHANISMS IN RETROVIRAL REPLICATION AND PATHOGENESIS

逆转录病毒组装科的研究目标是扩展我们对逆转录病毒复制和发病机制的基本机制的理解。这一认识可能提供对基因治疗有用的新技术或试剂，也可能导致对抗逆转录病毒引起的疾病（包括艾滋病）的新方法。逆转录病毒蛋白和核酸之间似乎有几种不同的相互作用模式，每种模式对病毒复制都有重要的功能影响。首先，Gag多蛋白在病毒组装过程中对病毒RNA进行精确的特异性识别。这种识别涉及到蛋白质中的锌指。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。我们的研究强烈表明，识别信号涉及基因组RNA分子二聚体形成的三维结构。我们的突变研究还表明，锌指除了在识别过程中发挥作用外，还有其他重要功能。目前正在调查这些额外的职能。其次，Gag多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，它们瞬间破坏碱基对的稳定，催化核酸分子的构象转变为最佳碱基对结构。当Gag多蛋白促进细胞tRNA分子向病毒RNA退火时，这种序列无关的活性在病毒组装之前或期间被使用；当病毒感染新的宿主细胞时，tRNA是逆转录的引物。在病毒成熟期间（即Gag被病毒蛋白酶切割后），当NC诱导颗粒内病毒RNA二聚体的构象重排时，该活性再次被使用。这种活性在逆转录过程中似乎也至关重要，在原病毒DNA合成过程中促进聚合和链转移步骤；最近的数据表明，在DNA整合到宿主染色体的过程中，它可能也很重要。我们正在研究这些蛋白质的核酸伴侣活性的分子机制。我们还发现HIV-1 Gag多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。这种组装需要核酸的存在，并且必须涉及GaguGag和gagunicacid的相互作用。核酸需求可以通过短至10u15个核苷酸的寡脱氧核苷酸来满足。病毒样颗粒的直径仅为25 ~ 30 nm，而在哺乳动物细胞中形成的真正病毒粒子的核心直径约为100 nm。值得注意的是，如果在网织红细胞裂解物存在的情况下进行组装反应，则形成100 nm的颗粒，而不是25 ~ 30 nm。因此，哺乳动物细胞中含有一种因子，可以改变Gag多蛋白分子在组装过程中相互作用的曲率半径。这个因子可能在细胞的正常组装过程中起作用；我们正在努力查明它的身份。值得注意的是，核酸是体外组装所必需的。这一发现表明，核酸可能对病毒颗粒在体内的组装也是必不可少的。然而，多年来人们已经知道，病毒的基因组RNA对于组装是完全没有必要的。我们最近发现RNA在逆转录病毒颗粒中起支架的作用。在没有基因组RNA的颗粒中，来自细胞的mrna执行这一功能。这些实验利用基于阿尔法病毒的载体高水平表达逆转录病毒基因产物。我们还用冷冻电子显微镜分析了未成熟和成熟小鼠白血病病毒颗粒的结构。我们发现这些粒子没有可检测到的二十面体对称，这与文献中的许多陈述相反。未成熟颗粒含有可见的亚基（可能是Gag多蛋白分子），它们以六角形的准晶填料排列。成熟粒子缺乏这种可见的结构规律性。二十面体对称的缺失意味着逆转录病毒粒子的组装原理与其他球形病毒不同。此外，我们还利用表面等离子体共振技术分析了HIV-1 NC蛋白与极短寡核苷酸的结合。尽管NC可能能够与任何单链DNA或RNA结合，但这些研究表明它具有深刻的序列偏好。我们正在对NC和Gag与短而明确的寡核苷酸的结合进行详细的研究；这些信息将有助于我们理解上述与核酸的各种相互作用（即，NC和Gag的伴侣活性，Gag对病毒样颗粒的组装，以及在体内病毒组装过程中Gag对基因组RNA的精细特异性封装）。最后，NC促进逆转录的事实意味着它是抗病毒治疗的潜在靶点。事实上，小鼠白血病病毒和HIV-1都可以被氧化NC锌指的化合物灭活。这些化合物可能是抗病毒治疗有用的先导化合物。

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 provide new techniques or reagents that could be useful in gene therapy, and may also 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 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. 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. We have also found that the HIV-1 Gag polyprotein is able to assemble into minute spherical virus-like particles in a completely defined system in vitro. The assembly requires the presence of nucleic acid, and must involve both GaguGag and Gagunucleic acid interactions. The nucleic acid requirement can be fulfilled by oligodeoxynucleotides as short as 10u15 nucleotides. The virus-like particles are only 25u30 nm in diameter, whereas the cores of authentic virions formed in mammalian cells are ~100 nm in diameter. Remarkably, if assembly reactions are performed in the presence of reticulocyte lysates, particles of 100 nm, rather than 25u30 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. This factor presumably acts during the normal assembly process in the cell; we are now trying to identify it. It is noteworthy that nucleic acid is required for assembly in vitro. This finding suggests that nucleic acid might be essential for assembly of viral particles in vivo as well. However, it has been known for many years that the genomic RNA of the virus is completely dispensable for assembly. We have recently found that RNA acts as scaffolding in retrovirus particles. In particles assembled without genomic RNA, mRNAs from the cell perform this function. These experiments have exploited alphavirus-based vectors for high-level expression of retroviral gene products. We have also analyzed the structure of immature and mature murine leukemia virus particles by cryoelectron microscopy. We found that these particles exhibit no detectable icosahedral symmetry, contrary to many statements in the literature. The immature particles contain visible subunits (presumably Gag polyprotein molecules), which are arranged with hexagonal paracrystalline packing. The mature particles lack this visible structural regularity. The absence of icosahedral symmetry implies that retrovirus particles are assembled according to different principles from those used by other spherical viruses. 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). Finally, the fact that NC facilitates reverse transcription means that it is a potential target for antiviral therapy. In fact, both murine leukemia virus and HIV-1 can be inactivated by compounds that oxidize the zinc fingers in NC. These compounds may be useful lead compounds for antiviral therapy.