Retrovirus-Gene Regulation

逆转录病毒基因调控

• 批准号: 6433170
• 负责人: DAVID DERSE
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 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/6433170
• 关键词: Lentivirus; Retroviridae; T lymphocyte; cell transformation; crosslink; equine infectious anemia virus; gene targeting; genetic regulation; genetic transcription; human T cell lymphotropic virus type 1; human immunodeficiency virus 1; open reading frames; protein structure function; provirus; recombinant virus; simian immunodeficiency virus; tissue /cell culture; virus genetics; virus infection mechanism; virus protein

We are studying the cooperative interactions between cellular and retrovirus-encoded proteins that together regulate RNA synthesis, pre-mRNA processing, and mRNA transport. These interactions are essential for virus replication and for the alterations of intracellular functions that underlie viral pathogenesis. Our research is primarily directed towards identifying the gene products and mechanisms essential for human T-cell leukemia virus type I (HTLV-I) infectivity and oncogenic transformation. We have developed an infectious molecular clone of HTLV-I and have established cell culture systems to monitor virus infectivity, replication and T-cell transformation. We have recently devoloped a single-round infection assay for HTLV-I and are currently examining determinants of virus entry and replication in cell culture. The mechanisms and gene products involved in HTLV-I replication are poorly characterized due to the poor infectivity and inefficient replication of the virus in vitro. In order to define the HTLV-I replication process we have begun to apply quantitative, real-time PCR methods to study replication products in infected cells. We are also examining HTLV-I virion assembly and maturation events to determine their roles in the infectivity process. Primary human T-cells have been infected and immortalized with cloned HTLV-I and with lentivirus vectors that encode HTLV-I regulatory genes in order to examine how cellular signaling networks are reprogrammed by virus gene products. We have begun to examine HTLV-I induced alterations in cellular gene expression profiles in primary human T-cells early after infection. In this regard, we are also investigating HTLV-I Tax-mediated activation of cellular genes controlled by serum response factor (SRF). We found that transcriptional activation mediated by SRF requires the participation of the cellular cofactors Sap1a and CBP. We are investigating how Tax alters the composition and assembly of these enhancer complexes in vitro. We are also asking how Tax modification of the SRF pathway affects the transformation process. We are also comparing the functions and molecular mechanisms of the Rev proteins encoded by various lentiviruses, including human immunodeficiency virus (HIV-1) and its relatives. This research is focused on a comparative analysis of the components and pathways that regulate retroviral mRNA splicing and transport. These processes are mediated by interactions among the viral Rev proteins, cellular factors, and cis-acting RNA elements. A comparison of HIV-1 and equine infectious anemia virus (EIAV) Rev proteins revealed that the latter mediates alternative splicing and nuclear export of viral pre-mRNA. The distantly related Rev proteins share many features in common but also display distinct differences in the ways in which they interact with RNA. We have characterized the primary RNA element bound by EIAV Rev and showed that it is quite different compared to that of other retroviruses. We have also exploited the EIAV system to identify cellular splicing factors that cooperate with Rev in its post-transcriptional regulation of virus gene expression. We have developed an in vitro RNA splicing system to examine the mechanism of Rev-induced alternative splicing. (Project transferred to BRL)AIDS TITLE: Structure and Function of Lentivirus Tat and Rev Proteins.

我们正在研究细胞和逆转录病毒编码蛋白之间的协同相互作用，这些蛋白共同调节 RNA 合成、前 mRNA 加工和 mRNA 运输。这些相互作用对于病毒复制和病毒发病机制的细胞内功能的改变至关重要。我们的研究主要旨在确定人类 T 细胞白血病病毒 I 型 (HTLV-I) 感染性和致癌转化所必需的基因产物和机制。我们开发了HTLV-I的感染性分子克隆，并建立了细胞培养系统来监测病毒感染性、复制和T细胞转化。我们最近开发了 HTLV-I 的单轮感染测定，目前正在研究病毒进入细胞培养物和复制的决定因素。由于HTLV-I病毒在体外的传染性差且复制效率低，因此对HTLV-I复制所涉及的机制和基因产物的表征很差。为了定义 HTLV-I 复制过程，我们已开始应用定量、实时 PCR 方法来研究受感染细胞中的复制产物。我们还在研究 HTLV-I 病毒体组装和成熟事件，以确定它们在感染过程中的作用。原代人 T 细胞已被克隆的 HTLV-I 和编码 HTLV-I 调节基因的慢病毒载体感染并永生化，以检查病毒基因产物如何重新编程细胞信号网络。我们已经开始检查感染后早期 HTLV-I 诱导的原代人类 T 细胞中细胞基因表达谱的变化。在这方面，我们还在研究 HTLV-I Tax 介导的由血清反应因子 (SRF) 控制的细胞基因激活。我们发现SRF介导的转录激活需要细胞辅因子Sap1a和CBP的参与。我们正在研究 Tax 如何在体外改变这些增强子复合物的组成和组装。我们还询问 SRF 途径的税收修改如何影响转型过程。 我们还比较了各种慢病毒（包括人类免疫缺陷病毒（HIV-1）及其相关病毒）编码的 Rev 蛋白的功能和分子机制。这项研究的重点是对调节逆转录病毒 mRNA 剪接和运输的成分和途径进行比较分析。这些过程是由病毒 Rev 蛋白、细胞因子和顺式作用 RNA 元件之间的相互作用介导的。 HIV-1 和马传染性贫血病毒 (EIAV) Rev 蛋白的比较表明，后者介导病毒前 mRNA 的选择性剪接和核输出。关系较远的 Rev 蛋白具有许多共同特征，但在与 RNA 相互作用的方式上也表现出明显的差异。我们对 EIAV Rev 结合的主要 RNA 元件进行了表征，并表明它与其他逆转录病毒相比有很大不同。我们还利用 EIAV 系统来识别与 Rev 病毒基因表达转录后调控相配合的细胞剪接因子。我们开发了一种体外 RNA 剪接系统来检查 Rev 诱导的选择性剪接机制。 （项目转移至BRL）艾滋病标题：慢病毒Tat和Rev蛋白的结构和功能。