HIV 1 GAG AND ENV PROTEINS IN VIRUS ASSEMBLY AND INFECTION

病毒Gag蛋白控制HIV-1复制周期的许多方面。Gag前体蛋白驱动病毒颗粒在感染细胞中的组装，并且通过与跨膜包膜（Env）糖蛋白gp 41的推定相互作用，指导Env掺入病毒颗粒中。感染后，Gag蛋白在脱壳中发挥核心作用，并协助逆转录过程。为了表征HIV-1 Gag基质（MA）蛋白的功能，我们在整个MA中引入了80多个单和双氨基酸突变。生物学和生化分析确定了在病毒生命周期的各个阶段显示缺陷的突变类别。我们还在整个Gag前体蛋白中引入了许多提前终止密码子以产生一系列截短的Gag蛋白。分析这些截短的Gag结合膜、直接组装和释放病毒样颗粒以及与全长Gag相互作用的能力提供了关于Gag中负责膜结合和病毒组装的结构域的信息。我们观察到的N-末端附近的MA的残基在Gag的膜结合的关键作用，并观察到截短的Gag蛋白含有MA和C-末端结构域的衣壳（CA）有效地结合膜全长Gag. MA氨基酸20突变引起的缺陷，在病毒的生命早期。在内源性RT测定中，病毒DNA合成也减少，并且含有MA残基20取代的Gag蛋白相对于野生型表现出增加的膜结合能力。残基20突变体的病毒回复突变体保持了位置20的变化，并在MA残基73和82处获得了第二个位点的变化。在内源性RT试验中，回复突变病毒的活性恢复至接近野生型水平。有趣的是，当病毒体与鼠白血病病毒Env糖蛋白假型化时，MA中的回复突变体变化阻断了HIV-1蛋白酶切割鼠白血病病毒跨膜Env蛋白的胞质结构域的能力，从而消除了病毒的感染性。我们已经观察到一个强大的细胞类型依赖性的要求在病毒复制的长胞质尾，一些细胞系是允许的和其他非允许的大gp 41胞质尾缺失。这种要求在病毒进入的水平上不强加，因为缺乏gp 41胞质尾的突变体有效地感染允许和非允许细胞类型。使用水泡性口炎病毒G蛋白假型，我们开发了一个高瞬时表达系统，使我们能够生物化学评估Env掺入非允许细胞类型。该分析表明，在非允许细胞中的阻断是在Env掺入的水平上施加的。这些结果表明，长的gp 41胞质尾区在Env掺入中以细胞类型依赖性方式起关键作用，这意味着宿主因子在Env掺入过程中的作用。HIV-1 gp 41胞质结构域的一个突出结构特征是在胞质结构域的中心（螺旋2）和C-末端（螺旋1）部分存在两个预测的α-螺旋。为了表征这些结构域，我们在两个α-螺旋结构域中引入了一些截短、缺失以及单和双氨基酸取代突变。评估了对Env表达、Env掺入病毒体、单循环试验中的病毒感染性、T细胞中的病毒复制和合胞体形成的影响。结果表明，gp 41胞质尾螺旋结构域在Env掺入和病毒感染性中起重要作用。分离并表征了几种α-螺旋2突变体的病毒回复突变体。有趣的是，其中一个回复突变体的变化映射到MA; MA的第二个位点的变化补偿了gp 41突变造成的Env掺入缺陷。这些结果支持在Env掺入过程中存在MA/gp 41相互作用。- Gag，基质，包膜糖蛋白，HIV-1，逆转录病毒，病毒组装，膜结合，gp 41，Gag靶向。

The viral Gag proteins control many aspects of the HIV-1 replication cycle. The Gag precursor protein drives the assembly of virus particles in the infected cell, and, through putative interactions with the transmembrane envelope (Env) glycoprotein gp41, directs Env incorporation into virus particles. Following infection, the Gag proteins play a central role in uncoating and assist in the reverse transcription process. To characterize functions of the HIV-1 Gag matrix (MA) protein, we have introduced over 80 single and double amino acid mutations throughout MA. Biological and biochemical analysis identified classes of mutations that displayed defects at various stages of the virus life cycle. We have also introduced a number of premature termination codons throughout the Gag precursor protein to generate a series of truncated Gag proteins. Analysis of the ability of these truncated Gags to bind membrane, direct assembly and release of virus-like particles, and to interact with full-length Gag has provided information concerning the domains in Gag responsible for membrane binding and virus assembly. We observed a critical role for residues near the N-terminus of MA in binding of Gag to membrane, and observed that truncated Gag proteins containing MA and the C-terminal domain of capsid (CA) bound membrane as efficiently as full-length Gag.Mutations at MA amino acid 20 caused a defect early in the virus life. Viral DNA synthesis was also diminished in endogenous RT assays, and Gag proteins containing MA residue 20 substitutions exhibited increased membrane binding capacity relative to wild-type. A viral revertant of a residue 20 mutant maintained the position 20 change and acquired second-site changes at MA residues 73 and 82. Activity of the revertant virus in the endogenous RT assay was restored to nearly wild-type levels. Intriguingly, when virions were pseudotyped with the murine leukemia virus Env glycoproteins, the revertant changes in MA blocked the ability of HIV-1 protease to cleave the cytoplasmic domain of the murine leukemia virus transmembrane Env protein, thereby abolishing virus infectivity.Lentiviruses, including HIV-1, possess transmembrane Env glycoproteins with unusually long cytoplasmic tails. We have observed a strong cell-type-dependent requirement for the long cytoplasmic tail in virus replication; some cell lines are permissive and others non-permissive for large gp41 cytoplasmic tail deletions. This requirement is not imposed at the level of virus entry, as mutants lacking the gp41 cytoplasmic tail efficiently infect both permissive and non-permissive cell types. Using vesicular stomatitis virus G protein pseudotypes, we developed a high transient expressions system that enabled us to biochemically assess Env incorporation in non- permissive cell types. This analysis demonstrated that the block in non-permissive cells was imposed at the level of Env incorporation. These results indicate that the long gp41 cytoplasmic tail plays a critical role in Env incorporation in a cell-type dependent manner, implying a role for host factors in the Env incorporation process. A prominent structural feature of the HIV-1 gp41 cytoplasmic domain is the presence of two predicted alpha-helices in the central (helix 2) and C-terminal (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 alpha-helical domains. The effects on Env expression, Env incorporation into virions, virus infectivity in single-cycle assays, virus replication in T-cells, and syncytium formation were assessed. The results indicate that the gp41 cytoplasmic tail helical domains play an important role in Env incorporation and virus infectivity. Viral revertants of several alpha-helix 2 mutants were isolated and characterized. Interestingly, one of the revertant changes mapped to MA; the second-site change in MA compensated for the Env incorporation defect imposed by the gp41 mutation. These results support the existence of a MA/gp41 interaction during Env incorporation. - Gag, matrix, envelope glycoproteins, HIV-1, retrovirus, virus assembly, membrane binding, gp41, Gag targeting.