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

项目摘要 HIV-1前病毒基因组的负链含有高度保守的开放阅读框（ORF）， 与跨越gp 120/gp 41边界的env基因重叠。该ORF编码反义蛋白（ASP）， ~189个残基，富含疏水性氨基酸，与细胞膜相关。ASP的表达是 由位于3 ′长末端重复序列（LTR）中的Tat非依赖性负义启动子（NSP）驱动。的 HIV-1感染者（PLWH）中存在针对ASP蛋白的体液和细胞免疫应答 提供了ASP在体内HIV-1感染期间表达的证据。 ASP ORF仅在属于流行性M组进化枝的HIV-1株中发现， 每个进化枝中具有完整ASP ORF的菌株的百分比与以下疾病的全球流行率相关： 进化枝相反，ASP ORF在所有其他人和非人灵长类慢病毒中不存在： O、N和P组中的非流行性HIV-1毒株，HIV-2毒株和所有物种的SIV毒株。这表明 ASP在病毒传播中可能起辅助作用。事实上，我们的研究表明，ASP表达在 非生产性感染细胞的细胞核，和生产性感染细胞的细胞表面。此外，在 在病毒出芽和释放过程中，ASP存在于无细胞HIV-1病毒体的包膜上。我们还发现敲门 ASP的表达减少了HIV-1在淋巴和骨髓原代人类细胞中的复制。然而，在这方面， 尽管有越来越多的证据表明ASP在体内表达，并且它促进病毒复制，但其功能和 ASP在HIV-1感染中的作用机制和发病机制尚不清楚。 阐明了HIV-1蛋白在病毒复制、促进HIV相关病理中的作用， 并且在改变正常细胞生理学方面通常依赖于转基因（Tg）小鼠模型的开发。的 本申请的中心假设是ASP Tg小鼠模型的开发将促进 ASP在病毒生命周期中发挥的作用，它将进一步推进我们对HIV-1的认识， to novel新therapeutic治疗interventions干预.我们提出两个具体目标。具体目标1旨在建立Tg小鼠 模型来研究3 'LTR驱动的ASP在CD 4+细胞（HIV-1感染的天然靶标）中的诱导表达。 我们已经开发了一种结构，使我们能够实现这些目标，我们已经进行了体外实验， 测试其功能。除了建立Tg模型外，本具体目标还将确认ASP是 诱导后在体内CD 4+细胞中表达。具体目标2旨在确定主要的表型特征- ASP Tg小鼠的免疫学。在这个特定的目标下，我们将进行一系列的形态，解剖，临床， 行为和免疫学分析，这将使我们能够充分表征这种新的Tg小鼠模型， 发现任何病理或异常 在这些研究的结论，我们将建立和特点的新的研究工具，在未来 研究将被用来测试关于ASP在HIV-1复制、传播和发病机制中的作用的新假设。

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.