An inducible and cell specific transgenic mouse model to study the HIV-1 antisense protein ASP

项目总结 HIV-1前病毒基因组的负链包含一个高度保守的开放阅读框架(ORF)， 与跨越gp120/gp41边界的env基因重叠。该ORF编码一种反义蛋白(ASP) ~189个残基，富含疏水氨基酸，与细胞膜有关。ASP的表达方式是 由位于3‘端长重复序列的TAT非依赖性负义启动子(NSP)驱动。这个 HIV-1感染者对天冬氨酸蛋白的体液和细胞免疫反应 提供了在体内感染HIV-1期间表达天冬氨酸的证据。 ASP ORF只在属于大流行组M分支的HIV-1毒株中发现，并且 每个分支中具有完整天冬氨酸转氨酶ORF的菌株的百分比与全球范围内的流行相关 那支树枝。相反，ASP ORF在所有其他人类和非人类灵长类慢病毒中都不存在： O、N和P组的非大流行HIV-1毒株、HIV-2毒株和所有物种的SIV毒株。这表明一种 天冬氨酸在病毒传播中可能的辅助作用。事实上，我们的研究表明，天冬氨酸在 非生产性感染细胞的细胞核，以及生产性感染细胞的细胞表面。此外，在 在病毒萌发和释放过程中，天冬氨酸存在于无细胞HIV-1病毒粒子的包膜上。我们还发现敲门声 Out ASP的表达减少了HIV-1在淋巴系和髓系原代人类细胞中的复制。然而， 尽管越来越多的证据表明，天冬氨酸在体内表达，并促进病毒复制，但其功能和 目前，天冬氨酸在HIV-1感染中的作用机制和发病机制尚不清楚。 阐明HIV-1蛋白在病毒复制、促进HIV相关病理过程中的作用， 而在改变正常细胞生理学方面，往往依赖于转基因(TG)小鼠模型的发展。这个 这一应用的中心假设是，开发一种ASP TG小鼠模型将有助于研究 ASP在病毒生命周期中发挥的作用，它将进一步促进我们对HIV-1的认识，并可能导致 到新的治疗干预措施。我们提出了两个具体目标。特定目标1寻求建立一只转基因小鼠 模型研究3‘LTR驱动的、可诱导的天冬氨酸在HIV-1感染的天然靶点--CD4+细胞中的表达。 我们已经开发出一种能够实现这些目标的结构，我们已经在体外进行了实验。 以测试其功能。除了建立TG模型外，这个特定的目的将证实ASP是 诱导后在体内的CD4+细胞中表达。具体目标2试图确定主要的表型特征- 天冬氨酸转氨酶转基因小鼠的免疫功能。在这个特定的目标下，我们将进行一系列的形态，解剖，临床， 行为学和免疫学分析，将使我们能够充分描述这种新的TG小鼠模型，并 识别任何病理或异常情况。 在这些研究的结论中，我们将建立并表征一个新的研究工具，在未来 研究将被用来检验关于天冬氨酸在HIV-1复制、传播和发病机制中的作用的新假说。

PROJECT SUMMARY The negative strand of the HIV-1 proviral genome contains a highly conserved open reading frame (ORF) that overlaps the env gene straddling the gp120/gp41 boundary. This ORF encodes an antisense protein (ASP) of ~189 residues, rich in hydrophobic amino acids, and associated with cellular membranes. Expression of ASP is driven by a Tat-independent negative sense promoter (NSP) located in the 3’ long terminal repeat (LTR). The presence of humoral and cellular immune responses against the ASP protein in people living with HIV-1 (PLWH) provide evidence that ASP is expressed during HIV-1 infection in vivo. The ASP ORF is found exclusively in HIV-1 strains that belong to clades of the pandemic group M, and the percentage of strains in each clade that have an intact ASP ORF correlates with the worldwide prevalence of the clade. On the contrary, the ASP ORF is absent in all other human and non-human primate lentiviruses: the non-pandemic HIV-1 strains in groups O, N and P, HIV-2 strains, and SIV strains of all species. This suggests a possible accessory role of ASP in virus spread. Indeed, our studies have shown that ASP is expressed in the nucleus of non-productively infected cells, and on the cell surface of productively infected cells. In addition, upon viral budding and release, ASP is present on the envelope of cell-free HIV-1 virions. We also found that knocking out ASP expression reduces HIV-1 replication both in lymphoid and myeloid primary human cells. However, despite mounting evidence that ASP is expressed in vivo, and that it promotes viral replication, the function and the mechanism of action of ASP in HIV-1 infection and pathogenesis remain unknown. The elucidation of the role that HIV-1 proteins play in viral replication, in promoting HIV-associated pathologies, and in altering normal cell physiology has often relied on the development of transgenic (Tg) mouse models. The central hypothesis of this application is that development of an ASP Tg mouse model will facilitate the study of the function that ASP plays in the virus lifecycle, it will further advance our knowledge of HIV-1, and it may lead to novel therapeutic interventions. We propose two specific aims. Specific Aim 1 seeks to establish a Tg mouse model to study 3’LTR-driven, inducible expression of ASP in CD4+ cells, the natural targets of HIV-1 infection. We have developed a construct that will allow us to achieve those goals, and we have performed in vitro exper- iments to test its function. In addition to establishing the Tg model, this Specific Aim will confirm that ASP is expressed in CD4+ cells in vivo after induction. Specific Aim 2 seeks to identify the main phenotypic character- istics of ASP Tg mice. Under this Specific Aim, we will conduct a series of morphologic, anatomical, clinical, behavioral, and immunological analyses that will allow us to fully characterize this new Tg mouse model and to identify any pathologies or abnormalities. At the conclusion of these studies, we will have established and characterized a new research tool that in future studies will be used to test new hypotheses about the role of ASP in HIV-1 replication, spread, and pathogenesis.