Retrovirus Assembly and Maturation

HIV-1 Gag与IP5/6结合位点的定位分析IP5/6对HIV-1 Gag构象的影响 重组HIV-1 Gag蛋白在体外可以组装成病毒样颗粒(VLP)。然而，在标准条件下，这些VLP的曲率半径比真正的HIV-1颗粒小得多。在组装反应中加入肌醇五烷基磷酸(IP5)可形成大小正确的VLP。与突变蛋白和嵌合蛋白的结合研究表明，基质(MA)和核衣壳(NC)结构域都与IP5相互作用，尽管它们之间的区域具有灵活性。我们在这方面的研究旨在确定结合位点，并测试除非有核酸存在，否则GAG在IP5中在溶液中折叠的可能性。 肌醇磷酸在活细胞中HIV-1颗粒组装中的作用 我们正在研究IP5相关化合物是否在体内和体外调节HIV-1颗粒组装。这些实验是在培养的人类细胞和酵母中进行的，使用了各种扰乱IP5衍生物新陈代谢的试剂和技术。 HIV-1 Gag小分子寡聚体的分析 GAG寡聚可能是病毒组装的关键第一步。我们发现HIV-1Gag在0.5M的氯化钠溶液中以单体-二聚体的形式存在。它似乎也在短的寡核苷酸上进行寡聚，可能模仿了组装的早期步骤。我们正在测试Gag-Gag相互作用的可能位点上的突变对二聚化的影响。 寻找HIV-1 Gag分子在组装中的接触部位 要参与VLP组装，每个Gag分子必须与其他几个分子相互作用。文献中的证据表明，所有与Gag-Gag相互作用的相关位点都位于衣壳(CA)的C-末端结构域(CTD)内和/或在CA和NC之间的p2区域。我们正在通过分析该区域突变体的特性来测试这一想法，包括它们通过野生型Gag抑制病毒粒子组装的能力。我们还试图确定由于组装成VLP而防止氢-重离子交换的GAG区域。 MLV GAG活体组装气缸的结构分析 逆转录病毒颗粒是多形性的，缺乏全面的二十面体对称性。我们确定了Moloney小鼠白血病病毒(MLV)Gag蛋白在体内组装成圆柱形颗粒的条件。这些圆柱体的冷冻电子显微镜图像显示出周期性；我们正在利用这种规律性来推导出MLV Gag蛋白的中等分辨率结构。 MLV与HIV-1成熟的研究 逆转录病毒CA蛋白N端保守的脯氨酸似乎在感染过程中起重要作用。MLV突变蛋白的N-端不能从p12上切割，或者N-端是甘氨酸而不是脯氨酸，是野生型功能的有效反式显性抑制物。为了深入了解Pro的作用，我们比较了HIV-1 CA N端的一系列突变体对野生型蛋白功能的抑制能力，并分析了这些突变体和混合颗粒中的功能缺陷。

Localization of the Binding Site on HIV-1 Gag for IP5/6; Analysis of the Effect of IP5/6 on the Conformation of HIV-1 Gag Recombinant HIV-1 Gag protein can assemble into virus-like particles (VLPs) in a defined system in vitro. However, under standard conditions, these VLPs have a far smaller radius of curvature than authentic HIV-1 particles. Addition of inositol pentakisphosphate (IP5) to the assembly reaction leads to the formation of VLPs of the correct size. Binding studies with mutant and chimeric proteins suggest that both matrix (MA) and nucleocapsid (NC) domains interact with IP5, despite the flexibility of regions between them. Our research in this area is aimed at identifying the binding sites and testing the possibility that Gag is folded in solution in IP5 unless nucleic acid is present. Role of Inositol Phosphates in HIV-1 Particle Assembly in Living Cells We are investigating whether IP5-related compounds modulate HIV-1 particle assembly in vivo, as well as in vitro. These experiments are being carried out both in cultured human cells and in yeast, using a variety of reagents and techniques that disrupt the metabolism of IP5 derivatives. Analysis of Small Oligomers of HIV-1 Gag Gag oligomerization may be a crucial first step in virus assembly. We found that HIV-1 Gag is in monomer-dimer equilbrium in 0.5 M NaCl. It also appears to oligomerize on short oligonucleotides, perhaps mimicking an early step in assembly. We are testing mutations at possible sites of Gag-Gag interaction for effects on dimerization. Search for Sites of Contact Between HIV-1 Gag Molecules in Assembly To participate in VLP assembly, each Gag molecule must interact with several others. Evidence in the literature suggests that all the relevant sites of Gag-Gag interaction are within the C-terminal domain (CTD) of capsid (CA) and/or in the p2 region between CA and NC. We are testing this idea by analyzing properties of mutants in this region, including their ability to inhibit virion assembly by wild-type Gag. We are also attempting to identify regions of Gag that are protected from hydrogen-deuterium exchange as a result of assembly into VLPs. Structural Analysis of Cylinders Assembled in Vivo from MLV Gag Retrovirus particles are pleomorphic and lack overall icosahedral symmetry. We identified conditions under which the Moloney murine leukemia virus (MLV) Gag protein assembles into cylindrical particles in vivo. Cryo-electron microscopic images of these cylinders exhibit periodicity; we are using this regularity to derive a moderate-resolution structure of MLV Gag protein. Studies on MLV and HIV-1 Maturation The conserved proline at the N-terminus of retroviral CA proteins seems important in the infectious process. MLV mutant proteins in which the N-terminus cannot be cleaved from p12, or in which the N-terminus is glycine rather than proline, are potent trans-dominant inhibitors of wild-type function. To gain insight into the role of proline, we are comparing a series of mutants at the N-terminus of HIV-1 CA with respect to their ability to inhibit the function of wild-type protein, and we are analyzing the functional defects in these mutants and in the mixed particles.