Retrovirus Assembly and Maturation

我们继续分析HIV-1和小鼠白血病病毒(MLV)颗粒的组装和成熟。具体地说，我们已经表征了HIV-1 Gag蛋白的特性，它是HIV-1病毒颗粒的基本构件。我们发现GAG蛋白在溶液中处于单体-二聚体平衡状态。我们确定了负责二聚化的界面；它是以前描述的HIV-1衣壳蛋白(形成Gag的一部分)二聚体的位置。我们还发现，当加入核酸时，六聚肌醇(IP6)调节了由GAG组装的病毒样颗粒的性质，导致了从单体-二聚体到单体-三聚体平衡的转变。多条证据表明，当核酸和IP6同时存在时，核酸与蛋白质的C-末端核衣壳(NC)结构域结合，而IP6与N-末端基质(MA)结构域结合。通过突变GAG中的二聚体界面，我们产生了一种在溶液中保持单体的蛋白质。然后，我们使用了各种实验方法，包括凝胶过滤、静态和准弹性光散射、沉降速度分析、小角中子散射以及分子模拟，来表征该蛋白质的构象。所有的数据都表明，蛋白质在溶液中折叠，其末端在三维空间中彼此靠近。相反，这种蛋白质是已知的在组装的病毒颗粒中高度伸展的杆状；因此，当它组装时，它必须经历重大的构象变化。我们分析了HIV-1 Gag的C-末端结构域，包括NC结构域被亮氨酸拉链结构域取代的嵌合蛋白的组装特性。这些蛋白质在体内组装成类似病毒的颗粒；这些颗粒在形态上与由野生型GAG组装的颗粒几乎相同，但似乎不包含RNA。这些蛋白可以与野生型Gag共组装。当从细菌中纯化时，这些嵌合蛋白是低聚的，但除非添加辅助因子，否则不会组装；辅助因子可以是RNA或肌醇磷酸，可能具有中和MA和/或CA结构域的正电荷的功能。野生型Gag的正常组装可能也需要这些中和辅助因子，但这一要求在野生型Gag的实验中并不明显，因为在野生型情况下，NC结合也需要RNA。我们正在对MLV Gag蛋白进行类似的分析。值得注意的是，我们发现该蛋白的性质与HIV-1 Gag的性质有很大不同。因此，它不会在溶液中齐聚，也不会在溶液中折叠。相反，它看起来像是一根延伸的杆子，在溶液中的尺寸与组装的病毒颗粒中的尺寸大致相同。[对应于2007年4月艾滋病毒耐药计划实地考察报告中的Rein项目1]

We have continued our analysis of assembly and maturation of HIV-1 and murine leukemia virus (MLV) particles. Specifically, we have characterized the properties of the HIV-1 Gag protein, the fundamental building block of the HIV-1 virus particle. We found that the Gag protein is in monomer-dimer equilibrium in solution. We identified the interface responsible for the dimerization; it is a site previously described as the site at which the HIV-1 capsid protein (which forms a portion of Gag) dimerizes. We also found that inositol hexakisphosphate (IP6), which modulates the nature of the virus-like particles assembled from Gag when nucleic acid is added, causes a shift from monomer-dimer to monomer-trimer equilibrium. Several lines of evidence indicate that when both nucleic acid and IP6 are present, the nucleic acid binds to the C-terminal, nucleocapsid (NC) domain of the protein while the IP6 binds to the N-terminal matrix (MA) domain. By mutating the dimer interface in Gag, we generated a protein which remains monomeric in solution. We then used a wide variety of experimental approaches, including gel filtration, static and quasi-elastic light-scattering, sedimentation velocity analysis, and small-angle neutron scattering, as well as molecular modeling, to characterize the conformation of this protein. All of the data indicate that the protein is folded over in solution, with its ends near each other in 3-dimensional space. In contrast, the protein is known to be a highly extended rod in assembled virus particles; thus, it must undergo a major conformational change when it assembles. We have analyzed the assembly properties of chimeric proteins in which the C-terminal domains of HIV-1 Gag, including the NC domain, is replaced by a leucine-zipper domain. These proteins assemble into virus-like particles in vivo; these particles are morphologically almost identical to those assembled from wild-type Gag, but appear to contain no RNA. These proteins can coassemble with wild-type Gag. When purified from bacteria, these chimeric proteins are oligomeric, but do not assemble unless a cofactor is added; the cofactors can be either RNA or inositol phosphates, and probably function to neutralize positive charges in the MA and/or CA domains. Normal assembly by wild-type Gag presumably also requires these neutralizing cofactors, but this requirement was not apparent in experiments with wild-type Gag because RNA is also required for NC binding in the wild-type case. We are performing a similar analysis of the MLV Gag protein. Remarkably, we find that the properties of this protein are quite different from those of HIV-1 Gag. Thus, it does not oligomerize in solution, and is not folded over in solution. Rather, it appears to be an extended rod, with approximately the same dimensions in solution as in the assembled virus particle. [Corresponds to Rein Project 1 in the April 2007 site visit report of the HIV Drug Resistance Program]