Liu Yuliang, Lee Michael S, Olson Mark A, Harty Ronald N
Department of Pathobiology, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104, USA.
Adv Virol. 2011 Jan 1;2011. doi: 10.1155/2011/341816.
Virus-host interactions play key roles in promoting efficient egress of many RNA viruses, including Ebola virus (EBOV or "e") and Marburg virus (MARV or "m"). Late- (L-) domains conserved in viral matrix proteins recruit specific host proteins, such as Tsg101 and Nedd4, to facilitate the budding process. These interactions serve as attractive targets for the development of broad-spectrum budding inhibitors. A major gap still exists in our understanding of the mechanism of filovirus budding due to the difficulty in detecting virus-host complexes and mapping their trafficking patterns in the natural environment of the cell. To address this gap, we used a bimolecular complementation (BiMC) approach to detect, localize, and follow the trafficking patterns of eVP40-Tsg101 complexes in live mammalian cells. In addition, we used the BiMC approach along with a VLP budding assay to test small molecule inhibitors identified by in silico screening for their ability to block eVP40 PTAP-mediated interactions with Tsg101 and subsequent budding of eVP40 VLPs. We demonstrated the potential broad spectrum activity of a lead candidate inhibitor by demonstrating its ability to block PTAP-dependent binding of HIV-1 Gag to Tsg101 and subsequent egress of HIV-1 Gag VLPs.
病毒与宿主的相互作用在促进包括埃博拉病毒（EBOV或“e”）和马尔堡病毒（MARV或“m”）在内的许多RNA病毒的有效释放过程中发挥着关键作用。病毒基质蛋白中保守的晚期（L-）结构域招募特定的宿主蛋白，如Tsg101和Nedd4，以促进出芽过程。这些相互作用成为开发广谱出芽抑制剂的有吸引力的靶点。由于难以在细胞的自然环境中检测病毒-宿主复合物并绘制其运输模式，我们对丝状病毒出芽机制的理解仍存在重大差距。为了填补这一差距，我们使用了双分子互补（BiMC）方法来检测、定位并跟踪活的哺乳动物细胞中eVP40-Tsg101复合物的运输模式。此外，我们将BiMC方法与病毒样颗粒（VLP）出芽试验相结合，以测试通过计算机筛选鉴定的小分子抑制剂阻断eVP40 PTAP介导的与Tsg101的相互作用以及随后eVP40 VLP出芽的能力。我们通过证明一种先导候选抑制剂能够阻断HIV-1 Gag与Tsg101的PTAP依赖性结合以及随后HIV-1 Gag VLP的释放，展示了其潜在的广谱活性。
