Molecular Analysis Of Retroviral Genes And Their Product

逆转录病毒基因及其产物的分子分析

• 批准号: 6669560
• 负责人: KLAUS STREBEL
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6669560
• 关键词: CD4 molecule; clinical research; confocal scanning microscopy; endoplasmic reticulum; functional /structural genomics; human immunodeficiency virus 1; human tissue; membrane proteins; mutant; phosphorylation; protein degradation; protein structure function; tissue /cell culture; virion; virus genetics; virus infection mechanism; virus protein

The purpose of this project is to investigate the biological and biochemical functions of HIV accessory proteins, in particular Vif and Vpu, and to understand their precise role in virus replication and in virus-host interaction. One of our goals is to characterize cellular factors involved in Vif or Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein degradation from the ER and into mechanisms involving late stages of virus production, in particular the involvement of lipid rafts in the secretory pathway. As a result of our experiments on Vif, we not only expect to gain insights into the function of this viral factor but we expect to learn more about the role of the cytoskeleton in virus replication and about the interactions of various cellular and viral factors during viral assembly and virus maturation. We hope that our research will provide a basis for the assessment of viral proteins as potential antiviral targets. The vpu gene is unique to HIV-1 and encodes a small integral membrane protein. Vpu regulates virus release from the cell surface and degradation of CD4 in the endoplasmic reticulum. These two biological activities of Vpu are based on two independent and distinct molecular mechanisms that can be attributed to separable structural domains of Vpu. Vpu-regulated virus release is sensitive to changes in the transmembrane (TM) domain of Vpu and is correlated with an ion channel activity of Vpu. CD4 degradation, on the other hand, involves a direct interaction of the Vpu and CD4 cytoplasmic domains. We demonstrated in the past that Vpu-mediated CD4 degradation involves the ubiquitin-dependent proteasome pathway and requires an interaction with a novel cellular protein, betaTrCP, which is a component of an E3 ubiquitin ligase complex. Unlike normal cellular substrates of TrCP, such as the NF-kB inhibitor I-kB, Vpu is not targeted for degradation by proteasomes. In fact, we found that Vpu has a dominant negative effect on cellular TrCP function by acting as a competitive inhibitor of TrCP. Consistent with its effect on I-kB-alpha, Vpu substantially repressed the HIV-induced activation of NF-kB (Bour et al, 2001). Since NF-kB is a key regulator of cytokine expression as well as the expression of anti-apoptotic genes, we have explored the possible involvement of Vpu in HIV-1-induced apoptosis. We found that in HIV-1-infected CD4+ T cells Vpu contributed significantly to the induction of apoptosis. Using an inducible expression system we found that the effect of Vpu on apoptosis was direct and did not require the coexpression of other viral proteins. Analysis of cellular factors involved in the induction of apoptosis demonstrated that Vpu down-modulated the NF-kB-dependent expression of anti-apoptotic genes such as Bcl-xL and A1/Bfl-1. Concomitantly, Vpu expression resulted in increased levels of active caspase-3. These effects of Vpu involved an interaction with TrCP as evidenced by the fact that mutation of the TrCP binding motif in Vpu abolished its apoptogenic potential. These results suggest that Vpu promotes apoptosis through its inhibition of NF-kB. Vif is a 23-kDa basic protein, which has an important function in regulating infectivity of progeny virions. Despite the severe impact of Vif defects on virus infectivity, its mechanism of action has thus far remained obscure. It is generally accepted that Vif-deficient viruses can attach to and penetrate host cells but are blocked at a post-penetration step early in the infection cycle. Yet, comparison of virion morphology or protein composition between wild type and Vif-defective virions has thus far been inconclusive and produced conflicting results. Several reports have suggested that Vif affects the stability of the viral nucleoprotein complex. In particular, NC and reverse transcriptase were found to be less stably associated with viral cores in the absence of Vif. Nevertheless, Vif is generally believed to function within the virus-producing cell. This assumption is largely based on the observation that relatively small amounts of Vif seem to be packaged, with estimates ranging from less than 1 to 100 molecules of Vif per virion. Furthermore, packaging of Vif into virus particles is generally believed to be non-specific, leading to questions regarding the functional significance of Vif incorporation into virions. We have started an in-depth biochemical analysis of Vif in purified virions derived from permissive or restrictive host cells to investigate the specificity of Vif incorporation into virions. Pulse/chase analysis of single-cycle infected H9 cells did not reveal any Vif-dependent differences in viral protein processing and maturation consistent with recent reports by other investigators. Instead, detergent extraction of purified virions demonstrated an association of Vif with the nucleoprotein complex. Interestingly, HIV-1 variants carrying mutations in the nucleocapsid zinc finger domains abolished Vif packaging. In addition, a viral variant defective in RNA-packaging was significantly impaired in packaging of Vif. Finally, deletion of a putative RNA-binding motif between residues 75-114 in Vif abolished its packaging into virions. Taken together, our results indicate that packaging of Vif into virions is specific and is mediated by an interaction of Vif with the viral genomic RNA. These results were recently published (Khan et al., J. Virol. 75:7252-7265 [2001]). Current experiments aim at the analysis of the specificity of the Vif interaction with viral RNA and at the investigation of its functional relevance. Preliminary data suggest that Vif packaging can be rescued by HIV-based retroviral packaging vectors although the minimal sequences required for Vif packaging have not yet been determined. We have continued our analysis of the post-translational modifications of virion-associated Vif. We found that Vif is subject to proteolytic processing by the HIV-encoded protease (Pr). Pr-dependent processing of Vif was observed both in vivo and in vitro. In vivo processing of Vif was cell type-independent and evident by the appearance of a 7-kDa processing product, which was restricted to cell-free virus preparations. Processing of Vif required an active viral protease and was sensitive to protease inhibitors such as ritonavir. The processing site in Vif was characterized both in vivo and in vitro and mapped to Ala150. Interestingly, the Vif processing site is located in a domain that is highly conserved among HIV-1, HIV-2, and SIV Vif isolates. Mutations at or near the processing site did not affect protein stability or packaging efficiency but had dramatic effects on Vif processing. In general, mutations that markedly increased or decreased the sensitivity of Vif to proteolytic processing severely impaired or completely abolished Vif function. In contrast, mutations at the same site that had little or no effect on processing efficiency also did not influence Vif function. None of the mutants affected the ability of the virus to replicate in permissive cell lines. Our data suggest that mutations in Vif that cause a profound change in the sensitivity to Pr-dependent processing also severely impaired Vif function suggesting that intravirion processing of Vif is important for the production of infectious viruses. This work has been published in the September issue of the Journal of Virology (Khan et al. J. Virol. 76:9112-9123 [2002]).

该项目的目的是研究HIV辅助蛋白，尤其是VIF和VPU的生物学和生化功能，并了解它们在病毒复制和病毒宿主相互作用中的精确作用。我们的目标之一是表征VIF或VPU功能中涉及的细胞因素。从我们对VPU的研究中，我们希望从ER到涉及病毒生产后期的机制中介绍蛋白质降解的一般原理，尤其是脂质筏在分泌途径中的参与。由于我们在VIF上的实验，我们不仅希望能够深入了解该病毒因子的功能，而且我们希望更多地了解细胞骨架在病毒复制中的作用以及在病毒组装和病毒成熟过程中各种细胞和病毒因子的相互作用。我们希望我们的研究能为评估病毒蛋白作为潜在抗病毒靶靶标提供基础。 VPU基因是HIV-1独有的，并编码一个小的积分膜蛋白。 VPU调节病毒从细胞表面释放并在内质网中降解。 VPU的这两种生物学活性基于两个独立和不同的分子机制，这些机制可以归因于VPU的可分离结构结构域。 VPU调节的病毒释放对VPU的跨膜（TM）结构域的变化敏感，并且与VPU的离子通道活性相关。另一方面，CD4降解涉及VPU和CD4细胞质结构域的直接相互作用。过去，我们证明了VPU介导的CD4降解涉及泛素依赖性蛋白酶体途径，并且需要与新型细胞蛋白BetatRCP进行相互作用，BetatRCP是E3泛素蛋白 - 酶酶复合物的组成部分。与TRCP的正常细胞底物（例如NF-KB抑制剂I-KB）不同，VPU不是蛋白酶体降解的目标。实际上，我们发现VPU通过充当TRCP的竞争抑制剂对细胞TRCP功能具有显着的负面影响。与其对I-KB-Alpha的影响一致，VPU实质上抑制了HIV诱导的NF-KB激活（Bour等，2001）。由于NF-KB是细胞因子表达以及抗凋亡基因表达的关键调节剂，因此我们探索了VPU在HIV-1诱导的凋亡中的可能参与。我们发现，在HIV-1感染的CD4+ T细胞中，VPU对凋亡的诱导显着贡献。使用诱导表达系统，我们发现VPU对凋亡的影响是直接的，并且不需要其他病毒蛋白的共表达。对诱导凋亡涉及的细胞因子的分析表明，VPU降低了抗凋亡基因的NF-KB依赖性表达，例如Bcl-XL和A1/BFL-1。同时，VPU表达导致活性caspase-3的水平升高。 VPU的这些效果涉及与TRCP的相互作用，这证明了VPU中TRCP结合基序的突变消除了其凋亡潜力。这些结果表明，VPU通过抑制NF-KB促进凋亡。 VIF是一种23 kDa碱性蛋白，在调节后代病毒体的感染性方面具有重要功能。尽管VIF缺陷对病毒感染性产生了严重影响，但迄今为止，其作用机理仍然晦涩难懂。人们普遍认为，缺陷的病毒可以附着并穿透宿主细胞，但在感染周期的早期渗透后步骤被阻塞。然而，到目前为止，野生型和VIF缺陷病毒体之间的病毒体形态或蛋白质组成的比较尚无定论，并产生了矛盾的结果。几份报告表明，VIF影响病毒核蛋白复合物的稳定性。特别是，在没有VIF的情况下，发现NC和逆转录酶与病毒核的稳定性不大。然而，通常认为VIF在产生病毒的细胞中起作用。该假设在很大程度上是基于观察到的，即相对较少的VIF似乎是包装的，估计值范围从少于1到100个分子的VIF范围。此外，通常认为VIF将VIF包装到病毒颗粒中是非特异性的，这导致有关VIF掺入病毒体中功能意义的问题。我们已经开始对VIF进行深入的生化分析，该分析是源自允许或限制性宿主细胞的纯化病毒体，以研究VIF掺入病毒体的特异性。单周期感染的H9细胞的脉搏/追逐分析并未揭示病毒蛋白加工中的VIF依赖性差异和与其他研究者最近报告一致的成熟。取而代之的是，纯化病毒体的洗涤剂提取表明VIF与核蛋白复合物的缔合。有趣的是，在Nucleocapsid锌指域中携带突变的HIV-1变体废除了VIF包装。此外，在VIF包装中，RNA包装中的病毒变异有缺陷受到显着损害。最后，在VIF中75-114之间75-114之间的假定RNA结合基序删除了其包装成病毒体。综上所述，我们的结果表明，将VIF包装到病毒体中是特异性的，并且是由VIF与病毒基因组RNA的相互作用介导的。这些结果最近发表了（Khan等，J。Virol。75：7252-7265 [2001]）。当前的实验旨在分析VIF与病毒RNA相互作用的特异性以及对其功能相关性的研究。初步数据表明，尽管尚未确定VIF包装所需的最小序列，但基于HIV的逆转录病毒包装向量可以挽救VIF包装。我们继续分析与病毒体相关的VIF的翻译后修饰。我们发现VIF受HIV编码的蛋白酶（PR）的蛋白水解处理。在体内和体外都观察到VIF的PR依赖性处理。 VIF的体内加工是与细胞类型无关的，并且通过7 kDa加工产物的出现，仅限于无细胞病毒制剂。 VIF的加工需要活跃的病毒蛋白酶，并且对蛋白酶抑制剂（如利托那韦）敏感。 VIF中的加工位点在体内和体外都被表征，并映射到ALA150。有趣的是，VIF处理位点位于HIV-1，HIV-2和SIV VIF分离株之间高度保守的域中。加工位点或附近的突变不会影响蛋白质稳定性或包装效率，但对VIF加工产生了巨大影响。通常，突变显着增加或降低了VIF对蛋白水解处理的敏感性严重受损或完全消除了VIF功能。相反，对处理效率几乎没有影响的同一位点的突变也不会影响VIF功能。没有一个突变体影响病毒在宽松细胞系中复制的能力。我们的数据表明，VIF中对PR依赖性处理的敏感性发生深刻变化的突变也严重受损的VIF功能，表明VIF的插入式处理对于产生传染性病毒至关重要。这项工作已发表在《病毒学杂志》的9月号（Khan等人J.Virol。76：9112-9123 [2002]）中。