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

HIV-1 小鼠获得类似于自然 HIV-1 感染的强致病性感染 人类体内的病毒将极大地促进对 HIV-1 体内生物学的理解、艾滋病疫苗的开发以及 测试新疗法。在细胞系中进行的早期研究表明，存在主要障碍 HIV-1 在病毒生命周期的几个阶段在小鼠细胞中进行复制，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 的组装缺陷 减少 Gag 与质膜结合和病毒颗粒输出的多蛋白。研究有 表明该缺陷与 Gag 内的 MA 蛋白的膜结合功能有关；然而，早 在 Gag 能够与膜结合之前，需要进行多聚化步骤。本次活动的总体目标 奖学金申请的目的是继续研究细胞质 Gag-Gag 相互作用的新缺陷 可能影响 Gag 膜结合、组装和病毒颗粒输出的小鼠细胞。初步结果 使用 FRET 和分级分离技术表明，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：小鼠对 HIV 的限制 1 组装是由于在膜结合之前抑制 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 MA 中参与这种限制的可能会改善小鼠细胞中的 Gag 多聚化，促进病毒粒子输出， 并改善小鼠体内的 HIV-1 复制。该假设将通过三个具体目标进行检验：1）研究 使用 FRET 和功能检测确定小鼠细胞中的 Gag-Gag 相互作用以确定 MA 和 NC 的作用 在 Gag 多聚化中； 2) 使用染色和细胞鉴定小鼠细胞中 HIV-1 组装的位点 融合技术； 3) 通过构建和鉴定具有增强多聚化的 MA 突变体 MA缺失和丙氨酸扫描突变体的功能测试，并测试此类突变体是否被克隆 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；以及原代巨噬细胞和星形胶质细胞培养物。 这项研究旨在了解并规避小鼠细胞中 HIV-1 复制的阻碍， 并可能有助于在小鼠中开发强大的 HIV-1 感染模型。这样的模型将是 在寻找 HIV-1 感染的疫苗和治疗方法方面迈出了一大步。

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.