Hiv 1 Gag And Env Proteins In Virus Assembly And Infecti

病毒Gag蛋白控制着HIV-1复制周期的许多方面。Gag前体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。C型逆转录病毒和慢病毒（包括HIV-1）的组装发生在质膜上。已经确定这个过程是由Gag蛋白促进的；然而，大会具体针对PM的机制仍有待确定。最近的研究表明，PM含有具有不同蛋白质和脂质组成的微结构域。一类微结构域脂筏富含鞘脂和胆固醇，可作为抗洗涤剂膜（DRM）分离。木筏已被证明在各种生物过程中发挥重要作用，通常作为参与共同途径的蛋白质的靶位。去年，我们报道了大部分膜结合Gag在DRM中被恢复（Ono和Freed， PNAS, 2001）。为了解决Gag-raft关联的特异性，我们检查了这种关联的动力学。我们发现Gag的DRM结合比Gag与膜的结合更慢，这表明HIV-1 Gag在与膜结合后特异性靶向PM筏。我们观察到木筏结合的决定因素映射到Gag的n端，但Gag-Gag相互作用促进或稳定木筏结合。为了解决Gag-raft关联的生理相关性，我们分析了胆固醇消耗对hiv -1产生细胞的影响，胆固醇消耗会破坏raft结构。我们发现胆固醇的消耗显著地、特异性地减少了病毒的释放。此外，从胆固醇耗尽的细胞中释放的病毒显示出较低的传染性。有趣的是，在p6晚结构域基序中表达Gag突变的细胞没有显示出病毒释放的减少。这些结果确定Gag与PM筏的关联是HIV-1产生的重要步骤，并提示筏与病毒组装后期步骤之间可能存在联系。最近的工作旨在确定木筏破坏损害病毒产生的机制。这些结果还表明，胆固醇消耗阻止Gag与膜的有效结合。HIV-1的p6结构域位于Gag前体蛋白Pr55Gag的c端。先前的研究表明，p6在表达病毒的HeLa细胞的HIV-1颗粒出芽中起关键作用。我们对p6的n端进行了详细的突变分析，以确定有效释放病毒所需的序列。通过检查p6突变在生物和生化分析中的影响以及电子显微镜，我们确定了p6在一组细胞系以及外周血单核细胞（PBMC）和单核细胞源性巨噬细胞（MDM）中的颗粒释放中的作用。Demirov et al.， J. Virol. 2002)的结果表明：i)位于p6 n端附近的高度保守的P-T/S-A-P基序对变化非常敏感；即使这个序列中的保守突变也会在HeLa细胞中引起严重的病毒释放缺陷。ii) P-T/S-A-P基序外的单和双氨基酸替换对颗粒释放无显著影响。iii)在P-T-/S-A-P基序之外引入一个或两个终止密码子可以阻断病毒粒子的释放，而截断P-T/S-A-P基序之外的四个基对HeLa细胞中的颗粒产生没有影响。iv)尽管p6突变对t细胞系中病毒复制的影响依赖于细胞类型，即使在p6突变阻断病毒复制的t细胞系中，这些变化对颗粒释放的影响很小或没有影响。v)在t细胞系中产生的p6突变粒子在病毒粒子-病毒粒子分离方面表现出缺陷，导致产生病毒粒子的系链。(6) HeLa细胞与T细胞系之间产生的瞬时异核体在颗粒释放中对p6的需求表现出T细胞样表型，表明抑制释放缺陷表型的活性在该细胞系统中占主导地位。vii)最后，在原发性MDM中，p6突变导致颗粒释放和病毒复制出现明显缺陷。这些结果揭示了p6在病毒颗粒释放过程中具有很强的细胞类型依赖性。多种证据表明逆转录病毒L结构域功能与宿主泛素化机制之间存在联系。最近，研究表明肿瘤易感基因101 （TSG101）的产物，在其n端包含一个与泛素偶联（E2）酶高度相关的结构域，以p6依赖的方式结合HIV-1 Gag。我们研究了过表达TSG101的n端区域对HIV-1颗粒产生的影响。有趣的是，我们观察到这个结构域（称为TSG-5'）有效地抑制病毒的产生（Demirov等人，PNAS 2002）。通过电子显微镜检查共表达HIV-1 Gag和TSG-5'的细胞，发现病毒出芽的缺陷与p6l结构域突变体非常相似。此外，TSG-5'的作用依赖于完整的p6l结构域；由缺乏p6 PTAP基序的Gag突变体产生的病毒样颗粒的组装和释放不受TSG-5'的显著影响。此外，小鼠白血病病毒和Mason-Pfizer猴病毒的组装和释放对TSG-5′不敏感。TSG-5'被整合到病毒粒子中，证实了Gag/TSG101在病毒产生细胞中的相互作用。使p6l结构域失活的突变会阻断TSG-5'的结合。这些数据证明了TSG101的e2样结构域与HIV-1 L结构域功能之间的联系，并提出了TSG101衍生物可以通过阻断病毒出芽作为HIV-1复制的有效特异性抑制剂的可能性。为了明确TSG-5‘抑制病毒释放的机制，我们研究了TSG-5’/Gag相互作用在病毒释放抑制中的重要性。我们观察到，TSG-5'中的突变阻止Gag与这种截断的蛋白质相互作用，从而消除了其破坏颗粒释放的能力。这些结果表明，与Gag的直接结合，而不是破坏细胞分选机制，是TSG-5'阻止出芽的能力的原因。我们还研究了过表达较长形式的TSG101（包括全长蛋白）对病毒出芽的影响。我们观察到过表达其他截断突变体以及全长蛋白会破坏出芽。有趣的是，全长TSG101在过表达时抑制病毒释放的能力并不需要与Gag结合。相反，通过共聚焦显微镜实验表明，过表达全长蛋白会干扰细胞内体分选机制。为了补充我们对HIV-1的研究，我们正在研究TSG101和其他宿主因子在多种逆转录病毒出芽中的作用，包括小鼠白血病病毒、人T细胞白血病病毒、牛白血病病毒、Mason-Pfizer猴病毒和马传染性贫血病毒。

The viral Gag proteins control many aspects of the HIV-1 replication cycle. The Gag precursor 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. The assembly of type C retroviruses and lentiviruses, including HIV-1, takes place at the plasma membrane (PM). It is well established that this process is promoted by Gag proteins; however, the mechanisms by which assembly is specifically targeted to the PM remain to be determined. Recent studies have suggested that the PM contains microdomains with distinct protein and lipid compositions. One type of microdomain, the lipid raft, is enriched in sphingolipids and cholesterol and can be isolated as detergent-resistant membrane (DRM). Rafts have been shown to play essential roles in a variety of biological processes, often by acting as a target site for proteins involved in a common pathway. Last year, we reported that a large portion of membrane-bound Gag was recovered in DRM (Ono and Freed, PNAS, 2001). To address the specificity of Gag-raft association, we examined the kinetics of this association. We found that DRM association of Gag occurred more slowly than binding of Gag to membranes, suggesting that HIV-1 Gag is specifically targeted to PM rafts after it binds membrane. We observed that the determinant for raft association maps to the N-terminus of Gag, but that Gag-Gag interactions facilitate or stabilize raft association. To address the physiological relevance of Gag-raft association, we analyzed the impact of cholesterol depletion, which disrupts raft structure, in HIV-1-producing cells. We found that cholesterol depletion markedly and specifically reduces virus release. Moreover, virus released from cholesterol-depleted cells displays reduced infectivity. Interestingly, cells expressing a mutant Gag with substitutions in the p6 late domain motif show no reduction in virus release. These results identify the association of Gag with PM rafts as an important step in HIV-1 production, and suggest possible links between rafts and late steps in virus assembly. Recent work has been aimed at defining the mechanism by which raft disruption impairs virus production. These results also suggest that cholesterol depletion prevents the efficient binding of Gag to membrane. The p6 domain of HIV-1 is located at the C-terminus of the Gag precursor protein Pr55Gag. Previous studies indicated that p6 plays a critical role in HIV-1 particle budding from virus-expressing HeLa cells. We performed a detailed mutational analysis of the N-terminus of p6 to define sequences required for efficient virus release. By examining the effects of p6 mutation in biological and biochemical analyses and by electron microscopy, we determined the role of p6 in particle release in a panel of cell lines as well as in peripheral blood mononuclear cells (PBMC) and monocyte-derived macrophages (MDM). The results (Demirov et al., J. Virol. 2002) indicate that: i) the highly conserved P-T/S-A-P motif located near the N-terminus of p6 is remarkably sensitive to change; even conservative mutations in this sequence induce profound virus-release defects in HeLa cells. ii) Single and double amino acid substitutions outside the P-T/S-A-P motif have no significant effect on particle release. iii) The introduction of stop codons one or two residues beyond the P-T-/S-A-P motif blocks virion release, whereas truncation four residues beyond P-T/S-A-P has no effect on particle production in HeLa cells. iv) Though the effects of p6 mutation on virus replication in T-cell lines are cell-type dependent, even in T-cell lines in which p6 mutations block virus replication, these changes have little or no effect on particle release. v) p6-mutant particles produced in T-cell lines exhibit a defect in virion-virion detachment, resulting in the production of tethered chains of virions. vi) Transient heterokaryons produced between HeLa cells and a T-cell line display a T cell-like phenotype with respect to the requirement for p6 in particle release, indicating that the activity that suppresses the release defect phenotype is dominant in this cell system. vii) Finally, in primary MDM, mutation of p6 results in marked defects in both particle release and virus replication. These results reveal a strong cell-type dependent requirement for p6 in virus particle release. A variety of lines of evidence suggest a connection between retroviral L domains function and the host ubiquitination machinery. Recently, it was demonstrated that the product of tumor susceptibility gene 101 (TSG101), which contains at its N-terminus a domain highly related to ubiquitin conjugating (E2) enzymes, binds HIV-1 Gag in a p6-dependent fashion. We examined the impact of overexpressing the N-terminal region of TSG101 on HIV-1 particle production. Intriguingly, we observe that this domain (referred to as TSG-5') potently inhibits virus production (Demirov et al., PNAS 2002). Examination of cells coexpressing HIV-1 Gag and TSG-5' by electron microscopy reveals a defect in virus budding very reminiscent of that observed with p6 L domain mutants. In addition, the effect of TSG-5' is dependent upon an intact p6 L domain; the assembly and release of virus-like particles produced by Gag mutants lacking a functional p6 PTAP motif is not significantly affected by TSG-5'. Furthermore, assembly and release of murine leukemia virus and Mason-Pfizer monkey virus are insensitive to TSG-5'. TSG-5' is incorporated into virions, confirming the Gag/TSG101 interaction in virus-producing cells. Mutations that inactivate the p6 L domain block TSG-5' incorporation. These data demonstrate a link between the E2-like domain of TSG101 and HIV-1 L domain function, and raise the possibility that TSG101 derivatives could serve as potent and specific inhibitors of HIV-1 replication by blocking virus budding. To define the mechanism by which TSG-5' inhibits virus release, we investigated the importance of TSG-5'/Gag interaction in the virus release inhibition. We observed that a mutation in TSG-5' that prevents Gag from interacting with this truncated protein eliminates its ability to disrupt particle release. These results suggest that direct binding to Gag, rather than disruption of cellular sorting machinery, is responsible for the ability of TSG-5' to block budding. We also investigated the effect of overexpressing longer forms of TSG101, including the full-length protein, on virus budding. We observe that overexpressing other truncated mutants as well as the full-length protein, disrupt budding. Interestingly, the ability of full-legth TSG101 to inhibit virus release when overexpressed does not require binding to Gag. Rather, as indicated by confocal microscopy experiments, overexpressing the full-length protein intereferes with the cellular endosomal sorting machinery. To complement our studies on HIV-1, we are investigating the role that TSG101 and other host factors play in the budding of a diverse array of retroviruses, including murine leukemia virus, human T cell leukemia virus, bovine leukosis virus, Mason-Pfizer monkey virus, and equine infectious anemia virus.