Effects of HIV-1 MA domain on gag multimerization and viral assembly in mice.

用HIV-1对小鼠进行类似于自然HIV-1感染的稳健致病性感染的实现 将极大地促进对HIV-1体内生物学的理解，艾滋病疫苗的开发， 新疗法的测试。在细胞系中进行的早期研究表明， HIV-1在病毒生命周期的几个阶段在小鼠细胞中复制，但最近的研究表明， 原代小鼠细胞和小鼠体内的HIV-1感染原则上是可能的。这项研究表明， 限制HIV-1在小鼠细胞中复制的主要障碍是病毒Gag装配中的缺陷 减少Gag与质膜结合和病毒颗粒输出的多蛋白。研究 在这种缺陷中暗示了Gag内MA蛋白的膜结合功能;然而， 在Gag能够结合到膜之前需要多聚化步骤。这个项目的总体目标是 奖学金申请是继续研究一种新的缺陷，在胞质Gag-Gag相互作用， 可能影响Gag膜结合、组装和病毒体输出的小鼠细胞。初步结果 使用FRET和分馏技术表明，Gag中MA的存在阻碍了早期Gag 小鼠细胞中的多聚化步骤。这些结果支持了这样的假设，即小鼠对HIV的限制- 1组装是由于在膜结合之前抑制了Gag-Gag多聚化，这是由于 MA与小鼠细胞中存在的因子之间的相互作用。区域的鉴定和诱变 在这种限制中涉及的MA可能改善小鼠细胞中Gag多聚化，促进病毒体输出， 并提高HIV-1在小鼠体内的复制。这一假设将在三个具体目标进行检验：1）研究 使用FRET和功能测定法在小鼠细胞中进行Gag-Gag相互作用，以确定MA和NC的作用 2）利用细胞免疫荧光染色和流式细胞术鉴定HIV-1在小鼠细胞中的组装位点。 融合技术;和3）通过构建鉴定具有增强的多聚化的MA突变体， MA缺失和丙氨酸扫描突变体的功能测试，并测试这些突变体是否克隆 转化为全长病毒能够支持HIV-1的完整复制周期。拟议的研究将采用 病毒学和分子生物学方法;流式细胞术，共聚焦显微镜，FRET和 免疫荧光技术;和原代巨噬细胞和星形胶质细胞培养物。 这项研究的目的是了解和规避小鼠细胞中HIV-1复制的障碍， 并可能有助于在小鼠中建立一个强大的HIV-1感染模型。这样的模式将是 这是在寻找HIV-1感染的疫苗和治疗方法方面迈出的巨大一步。

The achievement of a robust pathogenic infection of mice with HIV-1 resembling natural HIV-1 infection in people would greatly facilitate understanding of HIV-1 biology in vivo, development of AIDS vaccines, and testing of new therapeutics. Early studies conducted in cell lines indicated that there are major blocks to HIV-1 replication in mouse cells at several points in the viral life cycle, but recent research suggests that HIV-1 infection of primary mouse cells and mice in vivo is in principle possible. This research indicates that the major impediment limiting HIV-1 replication in mouse cells is a defect in the assembly of the viral Gag polyprotein that reduces Gag binding to the plasma membrane and viral particle export. Research has implicated the membrane-binding function of the MA protein within Gag in this defect; however, early multimerization steps are required before Gag is able to bind to membranes at all. The overall goal of this fellowship application is to continue the investigation of a novel defect in cytosolic Gag-Gag interaction in mouse cells that may influence Gag membrane binding, assembly and virion export. Preliminary results using FRET and fractionation techniques indicate that the presence of MA in Gag impedes early Gag multimerization steps in mouse cells. These results support the hypothesis that the murine restriction of HIV- 1 assembly is due to inhibition of Gag-Gag multimerization prior to membrane binding, as a result of interactions between MA and factors present in mouse cells. Identification and mutagenesis of the regions in MA involved in this restriction may improve Gag multimerization in mouse cells, facilitate virion export, and improve HIV-1 replication in mice. This hypothesis will be tested in three Specific Aims: 1) To study Gag-Gag interactions in mouse cells using FRET and functional assays to pinpoint the roles of MA and NC in Gag multimerization; 2) To identify the sites of HIV-1 assembly in mouse cells, using staining and cell fusion techniques; and 3) To identify MA mutants with enhanced multimerization by construction and functional testing of MA deletion and alanine scanning mutants, and to test whether such mutants cloned into full-length virus are able to support the full replication cycle of HIV-1. The proposed studies will employ virological and molecular biology methods; flow cytometry, confocal microscopy, FRET and immunofluorescence techniques; and primary macrophage and astrocyte cultures. This research is aimed at understanding and circumventing the block to HIV-1 replication in mouse cells, and may help in the development of a powerful model of HIV-1 infection in mice. Such a model would be an enormous step forward in the search for vaccines and therapeutics for HIV-1 infection.