Molecular Analysis Of Retroviral Genes And Their Products

逆转录病毒基因及其产物的分子分析

基本信息

批准号：
10692039
负责人：
KLAUS STREBEL
金额：
$ 164.02万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
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/10692039
关键词：
Adopted Affect Antiviral Therapy Binding Sites Biological Biological Assay CSF1 gene Cell Lineage Cells Collaborations Complex Dendritic Cells Development Elements Endothelial Cells Environment Equilibrium Event Exhibits Family Feedback Gel Gene Expression Genes Genetic Transcription Goals HIV HIV-1 HIV-2 Human Individual Infection Integration Host Factors Investigation Lead Light Link Liver Lymphatic Mediating Modeling Molecular Molecular Analysis Mutation Myelogenous Pathway interactions Phenotype Portugal Primate Lentiviruses Process Property Proteins Reporting Research Role Sampling Surface Time Tissues Viral Viral Genes Viral Physiology Virion Virus Virus Latency Virus Replication Work antagonist experimental study interest macrophage mannose receptor member monocyte novel promoter protein function receptor receptor-mediated signaling transcription factor vif Gene Products virus host interaction

项目摘要

HIV-1 encodes genes that are crucial for replication in primary cells, exerting functions not provided by the host. Gag, Pol, and Env products represent the main virion components, while Tat and Rev regulate intracellular transcriptional and post-transcriptional events for the controlled expression of viral genes. Of particular interest to us are the HIV accessory proteins Vif, Vpr, Vpu, Vpx, and Nef, which are unique to primate lentiviruses. There is now strong evidence that these proteins operate in conjunction with specific host factors. They do not have enzymatic activity but instead function primarily if not exclusively as molecular adaptors to link viral or cellular factors to pre-existing cellular pathways. Over the past decade, our research focus has shifted more and more towards the characterization of host factors and their roles in virus replication. One of the factors we recently identified is human mannose receptor I (hMRC1), a protein expressed on the surface of most tissue macrophages, dendritic cells, and select lymphatic or liver endothelial cells. We reported that hMRC1 inhibits the detachment of progeny viruses from infected macrophages in a manner that is phenotypically similar to the effect exerted by another host factor, BST-2, but is mechanistically distinct. Our continued investigations revealed that hMRC1 can also affect viral infectivity. Interestingly, while the effect of hMRC1 on virus detachment is not virus isolate specific, its effect on viral infectivity appears to affect primarily R5-tropic viruses. Experiments are ongoing to understand the mechanistic basis. We also continued our work on characterizing the mechanism by which HIV-1 infection of macrophages reduces the expression of hMRC1. We have now clear evidence that HIV-1 Tat is a main contributor to this process. We identified a complex interplay between HIV-1 Tat and the myeloid-specific transcription factor PU.1 that regulates the mannose receptor promoter. Interestingly, while Tat activates HIV-1 gene expression through a positive feedback loop, PU.1 is involved in a negative feedback loop that acts on the HIV-1 LTR promoter and inhibits viral gene expression, including Tat. Tat, on the other hand, inhibits the activity of PU.1. Thus, we found that there is a complex equilibrium between viral and host gene expression in HIV-infected macrophages. Indeed, disturbing this equilibrium has opposing effects on HIV-1 and hMRC1 gene expression. Our work has important implications on understanding how HIV-1 gene expression is regulated and we are conducting experiments to see if the regulatory feedback loops identified in our study contribute to the still poorly understood phenomenon of viral latency. In addition, we have worked on the detailed characterization of promoter elements responsible for PU.1 responsiveness using electrophoretic gel mobility assays (EMSA). We found that the hMRC1 promoter contains two 5 bp PU.1 binding sites. Deletion or mutations of one or both PU.1 binding sites severely hampers PU.1 responsiveness. Finally, transfer of the PU.1 binding site to a heterologous promoter rendered that promoter PU.1 responsive. Binding sites for PU.1 can be found in multiple cellular promoters, incl. the promoter for the MCSF receptor. MCSF receptor mediated signaling is crucial for the survival, function, proliferation, and differentiation of myeloid lineage cells, including monocytes/macrophages. Experiments are ongoing to see if and how expression of the MCSF receptor is affected by PU.1 and, consequently, affected by HIV-1 infection. Aside from our work on PU.1 mediated gene expression in HIV-infected macrophages, we continued research on Vif, Vpu, Vpr, and Vpx to understand their importance for HIV-1 and HIV-2 replication. In collaboration with the Barahona lab in Portugal, we investigated the contribution of each member of the APOBEC3 (A3) family for the restriction of HIV-2. We found that A3G strongly restricted both HIV-1 and HIV-2 while A3C restricts neither HIV-1 nor HIV-2. Importantly, A3B exhibited potent antiviral activity against Vif-deficient HIV-2 but its effect was negligible against Vif-deficient HIV-1. Whereas A3B appears to be packaged with similar efficiency into both viruses in the absence of Vif, HIV-2 and HIV-1 differ in their sensitivity to A3B. HIV-2 Vif targets A3B by reducing its cellular levels and inhibiting its packaging into virions whereas HIV-1 Vif did not evolve to antagonize A3B. Our observations support the hypothesis that during wild-type HIV-1 and HIV-2 infections, HIV-1 and HIV-2 are both capable of replicating in host cells expressing A3B using different mechanisms of antagonism that likely resulted from distinct adaptation over evolutionary time. It is interesting to note that in a previous report, A3B was found to strongly restrict HIV-1. Experiments are ongoing to shed light on this apparent discrepancy. With regard to Vpr and Vpx, we have started a collaboration with the Taveira lab in Portugal who have provided us with samples from multiple HIV-2 infected individuals. We have cloned the vpr and vpx genes from these individuals and experiments are ongoing to study their functional properties in comparison to the functional properties of lab-adapted isolates. Importantly, since HIV-2 encodes both Vpr and Vpx, we will compare matched pairs of Vpr and Vpx from primary HIV-2 isolates to determine if Vpr and Vpx can potentially adopt redundant biological activities.

HIV-1编码对于在原代细胞中复制至关重要的基因，发挥宿主未提供的功能。 GAG，POL和ENV产品代表主要的病毒粒子成分，而TAT和REV调节了病毒基因的受控表达的细胞内转录和转录后事件。我们特别感兴趣的是HIV附件VIF，VPR，VPU，VPX和NEF，这是灵长类动病毒所独有的。现在有强有力的证据表明，这些蛋白质与特定宿主因素结合起作用。它们没有酶活性，而是主要起作用，即使不是仅仅是将病毒或细胞因子与预先存在的细胞途径联系起来的分子适配器。在过去的十年中，我们的研究重点越来越多地转向宿主因素的表征及其在病毒复制中的作用。我们最近确定的因素之一是人类甘露糖受体I（HMRC1），一种在大多数组织巨噬细胞，树突状细胞和选择淋巴或肝内皮细胞表面表达的蛋白质。我们报告说，HMRC1以表型在表型相似的方式抑制了后代病毒与感染巨噬细胞的脱离，而另一个宿主因子BST-2造成的效果，但机械上是不同的。我们的持续研究表明，HMRC1也会影响病毒感染力。有趣的是，虽然HMRC1对病毒脱离的影响不是病毒孤立的特异性，但其对病毒感染性的影响似乎主要影响R5循环病毒。实验正在进行了解机理基础。我们还继续致力于表征巨噬细胞HIV-1感染降低HMRC1表达的机制。现在，我们有明确的证据表明HIV-1 TAT是这一过程的主要贡献者。我们确定了HIV-1 TAT与调节甘露糖受体启动子的髓样特异性转录因子PU.1之间的复杂相互作用。有趣的是，尽管TAT通过正反馈循环激活HIV-1基因表达，但PU.1参与了作用于HIV-1 LTR启动子的负反馈回路，并抑制包括TAT在内的病毒基因表达。另一方面，tat抑制了pu.1的活性。因此，我们发现在感染HIV的巨噬细胞中，病毒和宿主基因表达之间存在复杂的平衡。实际上，干扰这种平衡对HIV-1和HMRC1基因表达具有相反的影响。我们的工作对理解HIV-1基因表达的调节具有重要意义，并且我们正在进行实验，以查看我们研究中确定的调节反馈回路是否有助于仍然知之甚少的病毒潜伏期现象。此外，我们还研究了使用电泳凝胶迁移率分析（EMSA）的负责PU.1响应性的启动子元素的详细表征。我们发现HMRC1启动子包含两个5 BP PU.1结合位点。一个或两个PU的删除或突变1结合位点严重阻碍PU.1响应能力。最后，将PU.1结合位点转移到异源启动子中，使启动子PU.1响应能力。 PU.1的结合位点可以在多个细胞启动子中找到。 MCSF受体的启动子。 MCSF受体介导的信号传导对于髓样谱系细胞的存活，功能，增殖和分化至关重要，包括单核细胞/巨噬细胞。正在进行实验，以了解MCSF受体的表达是否以及如何受到HIV-1感染影响。除了我们在hiv感染的巨噬细胞中介导的基因表达的工作外，我们继续研究VIF，VPU，VPR和VPX，以了解它们对HIV-1和HIV-2复制的重要性。在与葡萄牙的Barahona实验室合作的情况下，我们调查了APOBEC3（A3）家族的每个成员对HIV-2的限制的贡献。我们发现A3G严重限制了HIV-1和HIV-2，而A3C既不限制HIV-1也不限制HIV-2。重要的是，A3b对缺乏VIF的HIV-2表现出有效的抗病毒活性，但其对缺陷VIF缺陷HIV-1的作用可忽略不计。而在没有VIF，HIV-2和HIV-1的情况下，A3B似乎以相似的效率包装到两种病毒中，它们对A3B的敏感性有所不同。 HIV-2 VIF通过降低其细胞水平并抑制其包装成病毒体来靶向A3B，而HIV-1 VIF并未演变为拮抗A3B。我们的观察结果支持以下假设：在野生型HIV-1和HIV-2感染期间，HIV-1和HIV-2都能够在表达A3B的宿主细胞中使用不同的拮抗作用机制复制，这可能是由于进化时期的明显适应性而导致的。有趣的是，在先前的报告中，发现A3B严重限制了HIV-1。实验正在阐明这种明显的差异。关于VPR和VPX，我们已经与葡萄牙的Taveira Lab合作，为我们提供了来自多个HIV-2感染者的样本。我们已经从这些个体中克隆了VPR和VPX基因，并且与实验室适应的分离株的功能特性相比，实验正在研究其功能特性。重要的是，由于HIV-2都编码VPR和VPX，因此我们将比较原代HIV-2分离株的匹配对VPR和VPX，以确定VPR和VPX是否可以潜在地采用冗余生物学活性。