HIV 1 GAG AND ENV PROTEINS IN VIRUS ASSEMBLY AND INFECTION

病毒Gag蛋白控制着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内的组装，并通过假定的与跨膜包膜（Env）糖蛋白gp41的相互作用，指导Env整合到病毒颗粒中。感染后，Gag蛋白在脱壳和协助逆转录过程中发挥核心作用。为了表征HIV-1 Gag基质（MA）蛋白的功能，我们在MA中引入了80多个单氨基酸和双氨基酸突变。生物和生化分析确定了在病毒生命周期的不同阶段显示缺陷的突变类别。我们还在Gag前体蛋白中引入了许多过早终止密码子，以产生一系列截断的Gag蛋白。分析这些截断的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Gag中负责膜结合和病毒组装的结构域的信息。我们观察到MA的n端附近的残基在Gag与膜的结合中起关键作用，并且观察到截断的Gag蛋白含有MA和衣壳（CA）结合膜的c端结构域，其效率与全长Gag一样高。MA氨基酸20的突变在病毒生命早期造成了缺陷。在内源性RT实验中，病毒DNA合成也减少，含有MA残基20取代的Gag蛋白相对于野生型表现出更高的膜结合能力。残基20突变体的病毒逆转录物保持了20位的变化，并在MA残基73和82处获得了第二位点的变化。在内源性RT试验中，逆转录病毒的活性恢复到接近野生型的水平。有趣的是，当病毒粒子与小鼠白血病病毒Env糖蛋白假型时，MA的逆转变化阻断了HIV-1蛋白酶切割小鼠白血病病毒跨膜Env蛋白的细胞质结构域的能力，从而消除了病毒的传染性。慢病毒，包括HIV-1，具有跨膜Env糖蛋白和异常长的细胞质尾部。我们观察到，在病毒复制过程中，对长细胞质尾部有很强的细胞类型依赖性；一些细胞系对gp41细胞质尾部的大缺失是允许的，而另一些细胞系则是非允许的。由于缺乏gp41细胞质尾部的突变体有效地感染了受纳和非受纳细胞类型，因此在病毒进入的水平上不存在这一要求。利用水疱性口炎病毒G蛋白假型，我们开发了一个高瞬时表达系统，使我们能够生化评估Env在非允许细胞类型中的结合。这一分析表明，非允许细胞中的阻滞是在Env掺入水平上施加的。这些结果表明，长gp41细胞质尾巴以细胞类型依赖的方式在Env掺入过程中起关键作用，这意味着宿主因子在Env掺入过程中起作用。HIV-1 gp41细胞质结构域的一个突出结构特征是在细胞质结构域的中心（螺旋2）和c端（螺旋1）部分存在两个预测的α -螺旋。为了描述这些结构域，我们在两个α -螺旋结构域中引入了许多截断、缺失和单、双氨基酸替代突变。评估了对Env表达、Env并入病毒粒子、单周期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，gp41细胞质尾部螺旋结构域在Env掺入和病毒感染中起重要作用。分离并鉴定了几种α -螺旋2突变体的病毒复归体。有趣的是，其中一个可逆变化与MA相关；MA的第二位点改变弥补了gp41突变造成的Env结合缺陷。这些结果支持在Env结合过程中MA/gp41相互作用的存在。- Gag，基质，包膜糖蛋白，HIV-1，逆转录病毒，病毒组装，膜结合，gp41， 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.