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。在我们的实验室中已经证明，在感染靶细胞之前，内源性慢病毒的逆转录可以在完整的病毒粒子内发生。这是一个具有生物化学活性的过程，是逆转录病毒生命周期的一个新阶段，并受到HIV-1或SIV病毒粒子所处的微环境的改变。刺激病毒粒子内的内源性逆转录，而没有病毒包膜的非生理性渗透，被称为“自然内源性逆转录”（NERT）。这种分子机制已被证明可以刺激初始静止的pbmc以及非增殖巨噬细胞中的HIV-1感染。因此，这一过程在增加HIV-1的性传播中可能是重要的，因为某些生殖器分泌物已被证明可以刺激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（NRTIs）和非核苷类似物逆转录酶抑制剂（NNRTIs）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来降低病毒的传染性。在这个建议中，我们将直接研究病毒内逆转录作为分子杀毒剂的靶标。在第一个具体目标中，将探索开发“分子杀毒剂”的能力，靶向病毒内逆转录。体外分析SIV/HIV-1和SHIV-RT构建体将使用含有nnrti（用于HIV-1和SHIV-RT）和三磷酸化nrti（用于HIV-1、SIV和SHIV-RT）的病毒内逆转录抑制剂进行。研究还将包括分析病毒内逆转录、细胞内逆转录和病毒传染性的变化。然后，有希望的药物将适当地预先配制，进行毒性分析，并配制成体内效用的杀病毒制剂，并重新分析特定目的II的抗病毒活性。提出的实验将为未来猕猴传播模型的发展奠定基础，以在体内分析这种抗病毒方法。这些研究还旨在将病毒内逆转录的分析从实验室转向临床应用，以改变人类慢病毒的性传播。

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.