Defensin mediated inhibition of HIV-1 replication

说明(申请人提供)：防御素是具有保守结构的小阳离子多肽，由多种细胞分泌，具有抗病毒、抗细菌和抗真菌活性。Alpha、Beta和theta防御素都有抗HIV-1的活性；它们加在一起可能构成对HIV-1的先天免疫的重要组成部分。肠道、女性生殖道、口腔和血液中分泌的防御素都可能在对抗HIV-1方面发挥作用。尽管有许多关于防御素抗HIV-1活性的报道，但对其作用机制知之甚少。对这些机制的深入了解将增加我们对HIV-1在体内复制的知识，并可能使我们能够通过使用防御素或防御素类药物来增强天然防御素，这些药物可以全身或局部使用。为了实现这些目标，我们将确定最有效的抗HIV-1防御素，并研究它们的作用机制。我们的具体目标是：1.检测所有已知的人类防御素和选定的非人类防御素的抗HIV-1活性。人白蛋白防御素、β防御素和恒河猴防御素将在大肠杆菌中生产，或通过高效液相化学合成和纯化。防御素将在低盐缓冲液中添加到HIV-1中，或在血清逃逸培养液中添加到细胞中。鬼细胞、PBMC、胎儿胸腺器官培养和淋巴组织培养将用于评估防御素对R5和X4 HI-V-1复制的抑制作用。2.鉴定每种活性防御素抑制HIV-1复制的机制。防御素将在感染前添加到细胞或病毒中，并在感染期间和之后添加到细胞中，以确定作用部位和时间。防御素与病毒和细胞的结合将用放射性标记的防御素进行检测，并在可能的情况下用抗防御素抗体进行确认。病毒复制被抑制的阶段将通过病毒DNA和RNA的定量聚合酶链式反应以及病毒蛋白和传染性的分析来确定。

DESCRIPTION (provided by applicant): Defensins are small cationic peptides with conserved structure, which are secreted by a variety of cells and have anti-viral, anti-bacterial and anti-fungal activity. Alpha, beta and theta defensins all have activity against HIV- 1; together they may constitute an important component of innate immunity to HIV-1. Defensins secreted in the gut, female reproductive tract, oral cavity and blood may all play a role in combating HIV-1. Despite numerous reports of anti-HIV- 1 activity of defensins, little is know about their mechanisms of action. Insight into these mechanisms will increase our knowledge about the replication of HIV- 1 in vivo and may allow us to augment natural defensins with administered defensins or defensin-like drugs that could be used systemically or topically. To achieve these goals we will identify the most potent ant-HIV-1 defensins and investigate their mechanisms of action. Our specific aims are: 1. To assay the anti-HIV-1 activity of all known human and selected non-human defensins. Human alpoha and beta defensins and rhesus macaque 0 defensins will be produced in E. coli or chemically synthesized and purified by HPLC. Defensins will be added to HIV- 1 in low salt buffer or to cells in serum-flee medium. GHOST cells, PBMC, fetal thymic organ culture and lymphoid tissue histoculture, will be used to evaluate defensin mediated inhibition of R5 and X4 HI-V- 1 replication. 2. To characterize the mechanism by which each active defensin inhibits HIV-1 replication. Defensins will be added to cells or virus, before infection and to cells during and after infection to determine the site and time of action. Defensin binding to virus and cells will be assayed with radiolabeled defensins and confirmed with anti-defensin antibodies when possible. The stage at which viral replication is inhibited will be determined by quantitative PCR for viral DNA and RNA and assays of viral proteins and infectivity.