HIV/AIDS Intravirion Reverse Transcription and Virucides

描述（由申请人提供）：灵长类慢病毒（如HIV-1）通过性传播的分子机制尚未完全阐明。我们的实验室已经证明，慢病毒的内源性逆转录可以在感染靶细胞之前在完整的病毒体内发生。这是一个生物化学活性过程，是逆转录病毒生命周期中的一个新阶段，并被HIV-1或SIV病毒粒子所处的微环境改变。刺激病毒粒子颗粒内的内源性逆转录，而不对病毒包膜进行非生理性透化，被称为“天然内源性逆转录”（NERT）。这种分子机制已被证明刺激HIV-1感染的初始静止的PBMC，以及非增殖的巨噬细胞。因此，这一过程可能在增加HIV-1的性传播方面很重要，因为某些生殖器分泌物已被证明能刺激HIV-1病毒粒子内的NERT。我们还证明了核苷类似物逆转录酶抑制剂（NRTI）和非核苷类似物RT抑制剂（NNRTI）的三磷酸化衍生物可以通过病毒粒子表面的孔直接进入慢病毒粒子，并通过抑制NERT降低病毒感染性。在这个提议中，我们将直接研究作为分子杀病毒剂靶点的病毒内逆转录。在第一个具体目标中，将探索开发“分子杀病毒剂”的能力，靶向病毒粒子内逆转录。将使用病毒内逆转录抑制剂与NNRTI（用于HIV-1和SHIV-RT）和三磷酸化NRTI（用于HIV-1、SIV和SHIV-RT）进行SIV/HIV-1和SHIV-RT构建体的体外分析。研究还将包括分析病毒内逆转录、细胞内逆转录和病毒感染性的变化。然后将显示前景的药剂适当地预配制，进行毒性分析，并配制用于体内用途作为杀病毒制剂，并在特定目的II中重新分析抗病毒活性。所提出的实验将为未来开发猕猴传播模型以分析这种体内杀病毒方法奠定基础。这些研究还旨在将病毒内逆转录的分析从实验室转向改变人类慢病毒性传播的临床实用性。

DESCRIPTION (provided by applicant): Molecular mechanisms by which primate lentiviruses, such as HIV-1, are sexually transmitted, have yet to be fully elucidated. It has been demonstrated in our laboratories that endogenous reverse transcription of lentiviruses can occur within the intact virion, before infection of target cells. This is a biochemically-active process, a new stage in the retroviral life-cycle, and is altered by the microenvironment to which HIV-1 or SIV virions are subjected. Stimulation of endogenous reverse transcription within virion particles, without non-physiological permeabilization of the viral envelope, has been entitled "natural endogenous reverse transcription" (NERT). This molecular mechanism has been shown to stimulate HIV-1 infection in initially-quiescent PBMCs, as well as non-proliferating macrophages. As such, this process may be important in augmenting the sexual transmission of HIV-1, as certain genital secretions have been shown to stimulate NERT within HIV-1 virion particles. We have also demonstrated that triphosphorylated derivatives of nucleoside-analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside-analogue RT inhibitors (NNRTIs) can directly enter lentivirions via pores in the virion particles' surface, and decrease viral infectivity by inhibiting NERT. In this proposal, we will directly investigate intravirion reverse transcription as a target for molecular virucides. In the first specific aim, the ability to develop "molecular virucides" will be explored, targeting intravirion reverse transcription. In vitro analyses of SIV/HIV-1 and SHIV-RT constructs will be performed using inhibitory agents of intravirion reverse transcription with NNRTIs (for HIV-1 and SHIV-RT) and triphosphorylated NRTIs (for HIV-1, SIV, and SHIV-RT). Studies will also include analysis of changes in intravirion reverse transcription, intracellular reverse transcription, and viral infectivity. Agents showing promise will then be properly pre-formulated, with toxicity analyses, and formulated for in vivo utility as virucidal preparations and re-analyzed for antiviral activity in specific aim II. The proposed experiments will set the groundwork for future development of a macaque transmission model to analyze this virucide approach in vivo. These studies are also designed to move analysis of intravirion reverse transcription from the bench towards clinical utility in altering sexual transmission of human lentiviruses.