Exploring the Role of Semen Amyloids in Promoting HIV Infection and Fertilization

描述（由申请人提供）：艾滋病毒最常通过性接触传播，精液是这种致命病毒在全球传播的载体。我们的研究表明，精液远不是HIV的被动载体，而是在体外急剧增强HIV感染，我们进一步鉴定和表征了人类精液中的淀粉样原纤维，这些淀粉样原纤维增加了HIV-1与细胞靶标的融合。这些原纤维可以提高HIV感染数个数量级以上，因此可以作为开发局部HIV杀菌剂的良好靶点。本提案的前两个目的集中在更好地表征这些精液原纤维增强HIV传染性及其对生殖器粘膜内细胞的影响的机制。在目标1中，我建议使用神经生物学领域的技术来表征最有效地增强HIV传染性的精液原纤维的形态特征。这一信息将揭示精液中淀粉样蛋白构象的类型，这应该是设计针对精液中宿主因子的特异性抑制剂的目标。在Aim 2中，我将确定精液原纤维是否在宿主细胞中诱导炎症，以及这在促进HIV感染中可能起什么作用。众所周知，炎症通常通过招募易感靶细胞和促进HIV基因转录来促进HIV传播。了解精液原纤维在传播过程中对宿主炎症的影响程度，对于开发有效防止病毒性传播的杀微生物剂至关重要。最后，本研究的目的3是为了更好地了解精液原纤维的基本生理功能。精液原纤维并没有进化为促进HIV感染，可能在人类中具有生物学目的。有趣的是，HIV与细胞靶点的融合与精子与卵子的融合有许多相同的特性，这提高了这些原纤维在受精过程中发挥作用的可能性。为此，我提出体外受精（IVF）和体内人工授精实验，以确定我们所描述的精液纤维是否促进了小鼠配子的融合。了解这些原纤维是否有助于促进受精是开发HIV杀菌剂的重要信息，也可能对生殖领域产生重大影响。在研制有效的艾滋病毒杀菌剂方面已经投入了大量努力。然而，该领域仍然缺乏一种有效预防HIV性传播的药物，部分原因是我们对粘膜HIV传播的分子事件缺乏基本的了解。该建议的重点是更好地了解艾滋病毒传播的一个方面，即自然产生的精液原纤维增强艾滋病毒传染性的作用。虽然该建议仅限于这些原纤维的体外分析，但我们正在恒河猴中平行启动实验，以检查这些原纤维在体内的影响。在K99/R00的指导阶段，我的计划是，如果它得到资助，开发淀粉样蛋白和生殖生物学的新技术技能，并应用这些技能来了解艾滋病毒的传播。这将通过与我的K99导师和咨询委员会密切合作来完成。我的长期目标是推进我们对生殖器粘膜中HIV传播的分子事件的理解，并将这些知识转化为新型HIV预防措施的开发。

DESCRIPTION (provided by applicant): HIV is most frequently transmitted following sexual contact, and semen is the vehicle fueling the global spread of this deadly virus. Far from being a passive vector for HIV, our research revealed that semen drastically enhances HIV infection in vitro, and we have further identified and characterized amyloid fibrils from human semen that increase HIV-1 fusion to its cellular targets. These fibrils can enhance HIV infection by over several orders of magnitude, and therefore serve as good targets for the development of a topical HIV microbicide. The first two aims of this proposal focus on better characterizing the mechanisms by which these semen fibrils enhance HIV infectivity and their influence on cells present within the genital mucosa. In Aim 1, I propose to use techniques from the field of neurobiology to characterize the morphological characteristics of semen fibrils that most effectively enhance HIV infectivity. This information will reveal the types of amyloid conformations in semen that should be targeted in efforts to design specific inhibitors against host factors in semen. In Aim 2, I will determine whether semen fibrils induce inflammation in host cells, and what role this may have in promoting HIV infection. It is known that inflammation generally facilitates HIV transmission by recruiting susceptible target cells and promoting HIV gene transcription. Understanding the extent to which semen fibrils contribute to host inflammation during transmission will be vital for developing microbicides that are effective in preventing sexual transmission of the virus. Lastly, Aim 3 of the proposal focuses on better understanding the fundamental physiological function of semen fibrils. Semen fibrils did not evolve to promote HIV infection, and may have a biological purpose in humans. Intriguingly, HIV fusion to its cellular target shares many properties with the fusion of a spermatozoon to an egg, raising the possibility for a role for these fibrils in fertilization. In this aim, I propose in viro fertilization (IVF) and in vivo artificial insemination experiments to determine if the semen fibris we have characterized promote the fusion of murine gametes. Understanding whether these fibrils serve to promote fertilization is vital information for the development of an HIV microbicide, and could also have a significant impact in the field of reproduction. Substantial efforts have been invested into developing an effective HIV microbicide. However, the field still lacks a drug that is highly effective at preventing the sexual spread of HIV, in part due to our lack of basic understanding of the molecular events surrounding mucosal HIV transmission. This proposal focuses on better understanding one aspect of HIV transmission, namely the effect of naturally-occurring semen fibrils that enhance HIV infectivity. Although the proposal is limited to in vitro analysis of these fibrils, we are initiating experiments in parallel in rhesus macaques to examine the effect of these fibrils in vivo. My plan during the mentored phase of the K99/R00, were it to get funded, is to develop new technical skills in amyloid and reproductive biology and to apply these skills towards understanding HIV transmission. This will be accomplished by working closely with my K99 mentors and advisory committee. My long-term goal is to advance our understanding of the molecular events surrounding HIV transmission in the genital mucosa, and to translate this knowledge into the development of new classes of HIV preventatives.