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: 6808649
• 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/6808649
• 关键词: CD4 molecule; confocal scanning microscopy; cytoskeleton; functional /structural genomics; gene expression; host organism interaction; human immunodeficiency virus 1; human tissue; immunoprecipitation; membrane proteins; molecular biology; molecular genetics; polymerase chain reaction; protein degradation; protein protein interaction; protein structure function; site directed mutagenesis; tissue /cell culture; virion; virus cytopathogenic effect; virus genetics; virus protein; virus replication; western blottings

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 and Vpu function. From our studies on Vpu we expect to gain insights into general principles of protein-protein interactions and into mechanisms involving late stages of virus production such as the involvement of lipid rafts in the secretory pathway. Our studies on Vif will provide insights into the function of this viral factor and teach us more about the role of the cytoskeleton in virus replication, about the interactions of cellular and viral factors during viral assembly or maturation, and about the role of host factors in restricting viral replication. Results from our research will enable us to assess viral accessory proteins with respect to their potential as novel 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. However, the precise mechanism of Vpu-mediated virus release remains unclear. In the course of our studies we found that the requirement of Vpu for efficient virus release is host cell dependent. In permissive cell types, virus release was equally efficient in the presence or absence of Vpu while in non-permissive cell types virus release was significantly impaired in the absence of Vpu. This suggested the involvement of host factors in Vpu function. Biochemical analyses demonstrated that Vpu is associated with cholesterol-rich lipid rafts in the plasma membrane. We found that addition of cholesterol increased the release of Vpu-defective virus from non-permissive cells but had no effect on virus release from permissive cells. Our results suggest that Vpu modulates structures at the cell surface to facilitate shedding of virus particles. It remains to be shown whether Vpu overcomes the inhibitory effects of a cellular factor in non-permissive cells or whether permissive cells are Vpu-independent because of the presence of a cellular Vpu-like factor. Experiments are ongoing to address this issue. 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 remained largely obscure. It is generally accepted, however, 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 remained inconclusive. About 40 to 100 molecules of Vif are packaged into virions. The majority of Vif, however, remains cell associated. Packaging of Vif protein into virus particles is mediated through an interaction with viral genomic RNA and results in the association of Vif with the nucleoprotein complex. Despite the specificity of this process, calculations of the amount of Vif packaged have produced vastly different results. We compared the packaging efficiency of Vif into virions derived from acutely and chronically infected H9 cells and found that Vif was efficiently packaged into virions from acutely infected cells (60-100 copies per virion) while packaging into virions from chronically infected H9 cells was near the limit of detection (4-6 copies of Vif per virion). Superinfection by an exogenous Vif-defective virus did not rescue packaging of endogenous Vif expressed in the chronically infected culture. In contrast, exogenous Vif expressed by superinfection of wild type virus was readily packaged (30-40 copies per virion). Biochemical analyses suggest that the differences in the relative packaging efficiencies were due to the accumulation of endogenously expressed Vif in a packaging-incompetent insoluble form. The packaging efficiency was correlated with the level of soluble Vif, which was elevated in cells producing exogenous Vif. The accumulation of endogenously expressed Vif in a detergent-insoluble form is at least in part due to the rapid turnover of soluble Vif and the higher stability of insoluble Vif. Despite its low packaging efficiency, endogenously expressed Vif was sufficient to direct the production of viruses with almost wild type infectivity. The results from this study provide novel insights into the biochemical properties of Vif and offer an explanation for the reported differences regarding Vif packaging . As packaging of Vif requires viral genomic RNA, it is likely that Vif recognizes specific sequences in the viral genome similar to those recognized by Tat and Rev proteins. We have continued our analysis of the sequences on the viral genomic RNA required for efficient packaging of Vif. To this end, we have constructed a series of deletion mutants and analyzed each construct for its ability to support packaging of Vif into a helper virus. In the process, we have narrowed down the sequence motif required for Vif packaging to an approximately 600-nucleotide region located near the 5'-end of the viral genome and encompassing a series of known RNA structures, including the TAR element, the primer binding site, and RNA dimerization signals. Final experiments are being performed to control various experimental parameters. We hope to complete this project within the next year. Replication of HIV-1 in most primary cells and some immortalized T cell lines is critically dependent on the activity of Vif. Vif has the ability to counteract a cellular inhibitor, recently identified as CEM15, that blocks replication of Vif-defective HIV-1 variants. CEM15 is identical to APOBEC3G and belongs to a family of proteins involved in RNA and DNA deamination. Recent evidence suggests that APOBEC3G targets nascent viral minus strand cDNA, introducing cytidine to uridine changes throughout the genome. These changes are believed to either destabilize the viral genome by subjecting it to the activity of uracil DNA glycosylase or to induce hypermutation of the viral genome, resulting in the impairment of viral fitness. We cloned APOBEC3G from a human kidney cDNA library and confirmed that the protein acts as a potent inhibitor of HIV replication and is sensitive to the activity of Vif. We found that wild type Vif blocks packaging of APOBEC3G into virus particles in a dose-dependent manner. In contrast, biologically inactive variants carrying in-frame deletions in various regions of Vif or mutation of two highly conserved cysteine residues did not inhibit packaging of APOBEC3G. Interestingly, expression of APOBEC3G in the presence of wild type Vif not only affected viral packaging but also reduced its intracellular expression level. This effect was not seen in the presence of biologically inactive Vif variants. Pulse/chase analyses did not reveal a significant difference in the stability of APOBEC3G in the presence or absence of Vif. However, in the presence of Vif, the rate of synthesis of APOBEC3G was slightly reduced. The reduction of intracellular APOBEC3G in the presence of Vif does not fully account for the Vif-induced reduction of virus-associated APOBEC3G suggesting that Vif may function at several levels to prevent packaging of APOBEC3G into virus particles.

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和VPU功能中涉及的细胞因素。从我们对VPU的研究中，我们希望能够深入了解蛋白质 - 蛋白质相互作用的一般原理，以及涉及病毒产生晚期的机制，例如脂质筏在分泌途径中的参与。我们对VIF的研究将提供有关该病毒因子功能的见解，并教会我们更多有关细胞骨架在病毒复制中的作用，关于病毒组装或成熟过程中细胞和病毒因子相互作用的作用，以及宿主因素在限制病毒复制中的作用。我们研究的结果将使我们能够根据其作为新型抗病毒靶标的潜力来评估病毒附件蛋白。 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。但是，VPU介导的病毒释放的确切机制尚不清楚。在研究过程中，我们发现VPU对有效病毒释放的需求取决于宿主细胞。在宽松的细胞类型中，在存在或不存在VPU的情况下，病毒释放同样有效，而在没有VPU的情况下，在非抗药性细胞类型的病毒中，释放病毒释放显着损害。这表明宿主因子参与VPU功能。生化分析表明，VPU与质膜中富含胆固醇的脂质筏有关。我们发现，胆固醇的添加增加了非腐败细胞中VPU缺陷病毒的释放，但对从宽容细胞释放病毒没有影响。我们的结果表明，VPU调节细胞表面的结构，以促进病毒颗粒的脱落。 VPU是否克服了非腐败细胞中细胞因子的抑制作用，或者由于存在类似细胞VPU的因子而与允许细胞非依赖的抑制作用尚待证明。实验正在进行解决此问题。 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，但其作用机理仍在很大程度上变得晦涩难懂。然而，人们普遍认为，缺乏VIF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，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仍然尚无定论。将大约40至100个VIF分子包装到病毒体中。但是，大多数VIF仍然与细胞相关。通过与病毒基因组RNA的相互作用，将VIF蛋白包装到病毒颗粒中，并导致VIF与核蛋白复合物的关联。尽管此过程具有特异性，但包装的VIF量的计算仍产生了截然不同的结果。我们比较了VIF的包装效率到源自急性和慢性感染的H9细胞的病毒体中，发现VIF有效地将急性感染细胞的病毒体包装到病毒体中（每个病毒粒子的60-100份），而慢性感染的H9细胞中的病毒粒子则接近检测的极限（4-6个VIF的拷贝）。外源性VIF缺陷病毒的超级感染未挽救在慢性感染培养物中表达的内源性VIF的包装。相比之下，通过野生型病毒超级感染表示的外源VIF很容易打包（每个病毒粒子30-40份）。生化分析表明，相对包装效率的差异是由于内源性表达的VIF在包装不足的不溶性形式中的积累所致。包装效率与可溶性VIF的水平相关，后者在产生外源VIF的细胞中升高。内源性表达的VIF以洗涤剂不溶性形式的积累至少部分是由于可溶性VIF的快速离职和较高的不溶性VIF的稳定性。尽管包装效率低，但内源性表达的VIF足以指导几乎野生型感染性的病毒。这项研究的结果为VIF的生化特性提供了新的见解，并为有关VIF包装的差异提供了解释。 由于VIF的包装需要病毒基因组RNA，因此VIF可能识别病毒基因组中的特定序列，类似于TAT和REV蛋白识别的序列。我们继续对有效包装VIF的病毒基因组RNA序列进行分析。为此，我们已经构建了一系列缺失突变体，并分析了每个构建体的能力，能够将VIF包装到辅助病毒中。在此过程中，我们将VIF包装所需的序列基序缩小到位于病毒基因组5'-End附近的大约600个核苷酸区域，并涵盖了一系列已知的RNA结构，包括TAR元件，引物结合位点和RNA Dimerization信号。正在进行最终实验以控制各种实验参数。我们希望在明年完成该项目。 在大多数原代细胞和某些永生的T细胞系中，HIV-1的复制取决于VIF的活性。 VIF具有应对最近被识别为CEM15的细胞抑制剂的能力，该抑制剂阻止了VIF缺陷性HIV-1变体的复制。 CEM15与APOBEC3G相同，属于参与RNA和DNA脱氨基的蛋白质家族。最近的证据表明，apobec3g靶向新生的病毒减去链cDNA，在整个基因组中引入了尿苷变化。人们认为，这些变化要么通过使尿嘧啶DNA糖基化酶的活性或诱导病毒基因组的过度突破来破坏病毒基因组的稳定性，从而导致病毒适应性受损。我们从人类肾脏cDNA文库中克隆了apobec3g，并确认该蛋白质是HIV复制的有效抑制剂，并且对VIF的活性很敏感。我们发现，野生型VIF以剂量依赖性方式将APOBEC3G包装到病毒颗粒中。相反，在VIF的各个区域或两个高度保守的半胱氨酸残基的突变中携带框内缺失的生物无活性变体不会抑制APOBEC3G的包装。有趣的是，在存在野生型VIF的情况下，Apobec3g的表达不仅影响了病毒包装，而且还降低了其细胞内表达水平。在存在生物学无活跃的VIF变体的情况下，没有看到这种效果。在存在或不存在VIF的情况下，脉冲/追逐分析没有揭示Apobec3g的稳定性有显着差异。但是，在存在VIF的情况下，APOBEC3G的合成速率略有降低。在VIF存在下，细胞内APOBEC3G的还原并不能完全解释VIF诱导的与病毒相关的APOBEC3G的减少，这表明VIF可能在多个水平上起作用以防止将Apobec3g包装到病毒颗粒中。