HIV 1 GAG AND ENV PROTEINS IN VIRUS ASSEMBLY AND INFECTION

HIV-1 Gag基质的功能表征 (MA)蛋白并定义参与这些功能的结构域，我们 已经引入了80多个单氨基酸和双氨基酸突变 在整个马萨诸塞州。生物和生化分析确定 突变类型：i)GAG膜结合受损或 肉豆蔻化作用，II)阻止Env掺入病毒粒子，III) 病毒组装受阻，iv)病毒进入受损，或v)重定向 从质膜组装到细胞质隔间。 在这些研究过程中，所有类别的病毒逆转株 获得了突变，并对其进行了鉴定。大多数人 在这些回复突变体的MA区中发现的第二位点变化 病毒位于距离原始突变很远的地方 主序列。当引入MA时， 原始突变，这些第二位点的变化补偿了 由原来的替换引起的缺陷，暗示直接或 远位氨基对之间的功能相互作用 酸。第二个站点的变化也是在上下文中介绍的 野生型MA，其中一些导致HIV-1缺陷 复制，暗示结构或功能上的相互依赖 在第二位点突变和原始突变之间。突变者 进一步的回复菌株的分析和表征 对MA结构与结构关系的认识 功能，并使我们能够描绘MA在HIV-1生命中的作用 周而复始。几种灵长类慢病毒的序列比较 分离株显示MA具有非常高的序列保守性 氨基酸20(Leu)。这个位置的突变，特别是赖氨酸 和Arg替换，延迟或阻止T细胞中的病毒复制 系、原代PBMC和人巨噬细胞。生化 分析表明，剩余的20个突变不会影响病毒 组装和释放，环境病毒掺入病毒粒子，外源RT 活性，或RNA包埋。然而，病毒早期的一个缺陷 在单周期(MAGI)感染性试验中观察生命周期， 这表明病毒的传染性降低了10倍。动能 利用不同的引物进行逆转录分析的研究 感染后早期时间点原代巨噬细胞和 T细胞株表明细胞中病毒DNA合成受到损害 感染了变异病毒。有趣的是，病毒DNA合成 在内源性RT检测中表达减弱。一种病毒变异体 保持20个位置的变化并获得第二个站点的变化 在第73位和第82位。有趣的是，变异病毒的活性在 内源性RT检测恢复到野生型水平。 慢病毒，包括HIV-1，具有跨膜包膜 (Env)具有异常长的细胞质尾巴的糖蛋白。一个 HIV-1胞浆结构域的显著结构特征是 在中央存在两个预测的a-螺旋(a-螺旋2)和 细胞质区域的C-末端(a-螺旋1)部分。至 描述这些领域，我们已经介绍了一些 截断、缺失、单氨基酸和双氨基酸 两个α-螺旋结构域中的替换突变。这个 对Env表达、Env掺入病毒粒子、病毒的影响 MAGI细胞中的传染性、T细胞中的病毒复制和合胞体 编队已经进行了评估。结果表明，gp41 胞质尾部α-螺旋结构域在环境中的重要作用 掺入和病毒感染性，并鉴定特定的功能 这些领域内的决定因素。我们还观察到有几个 Gp41a-螺旋2的突变显著损害了GAG的加工，并且 通过矩阵(MA)消除该有缺陷的GAG处理 阻止环境病毒与病毒粒子结合的突变。这 这一现象支持MA和gp41相互作用的假设 在Gag和Env的运输和病毒组装过程中。

To characterize functions of the HIV-1 Gag matrix (MA) protein and define domains involved in these functions, we have introduced over 80 single and double amino acid mutations throughout MA. Biological and biochemical analysis identified classes of mutations which: i) impaired Gag membrane binding or myristylation, ii) blocked Env incorporation into virions, iii) blocked virus assembly, iv) impaired virus entry, or v) redirected assembly from the plasma membrane to cytoplasmic compartments. During the course of these studies, viral revertants of all classes of mutations were obtained and characterized. The majority of second-site changes identified in the MA region of these revertant viruses were located distantly from the original mutations in the primary sequence. When introduced into MA in the presence of the original mutations, these second-site changes compensated for the defects caused by the original substitutions, suggesting a direct or functional interaction between the pairs of distantly-located amino acids. The second-site changes were also introduced in the context of wild-type MA and some of them caused defects in HIV-1 replication, suggesting structural or functional interdependency between the second-site and original mutations. The mutational analysis and characterization of revertants further our understanding of the relationship between MA structure and function, and enable us to delineate the role of MA in the HIV-1 life cycle. Sequence comparison of a number of primate lentivirus isolates reveals a very high degree of sequence conservation at MA amino acid 20 (Leu). Mutations at this position, in particular Lys and Arg substitutions, delayed or blocked virus replication in T-cell lines, primary PBMC, and human macrophages. Biochemical analyses revealed that the residue 20 mutations did not affect virus assembly and release, Env incorporation into virions, exogenous RT activity, or RNA encapsidation. However, a defect early in the virus life cycle was observed in single cycle (MAGI) infectivity assays, which demonstrated a 10-fold reduction in virus infectivity. Kinetic analyses of reverse transcription by PCR using a range of primers at early time points post-infection in both primary macrophages and T-cell lines indicated that viral DNA synthesis was impaired in cells infected with the mutant virus. Intriguingly, viral DNA synthesis was diminished in endogenous RT assays. A viral revertant maintained the position 20 change and acquired second-site changes at residues 73 and 82. Interestingly, activity of the revertant virus in the endogenous RT assay was restored to wild-type levels. Lentiviruses, including HIV-1, possess transmembrane envelope (Env) glycoproteins with unusually long cytoplasmic tails. A prominent structural feature of the HIV-1 cytoplasmic domain is the presence of two predicted a-helices in the central (a-helix 2) and C-terminal (a-helix 1) portions of the cytoplasmic domain. To characterize these domains, we have introduced a number of truncations, deletions, and single and double amino acid substitution mutations throughout both a-helical domains. The effects on Env expression, Env incorporation into virions, virus infectivity in MAGI cells, virus replication in T-cells, and syncytium formation has been assessed. The results indicate that the gp41 cytoplasmic tail a-helical domains play an important role in Env incorporation and virus infectivity, and identify specific functional determinants within these domains. We also observed that several mutations in gp41 a-helix 2 markedly impair Gag processing, and that this defective Gag processing is relieved by a matrix (MA) mutation which blocks Env incorporation into virions. This phenomenon supports the hypothesis that MA and gp41 interact during Gag and Env transport and virus assembly.