Mechanisms of Viral Fusion and Inactivation

我们的总体方法是动态地解决病毒包膜糖蛋白介导的膜融合途径中的步骤，并使用各种生化、生物物理、病毒学和分子和细胞生物学技术来揭示这些步骤背后的物理参数。体外研究感染病毒和在细胞中表达的HIV包膜蛋白。我们正在使用与特定脂类相连的基于多肽的进入抑制剂来探索融合反应的细节。我们最近发现，鞘氨醇(脱氢鞘磷脂的前体)的偶联对基于HIV的短肽的抑制潜力具有显着的特异性，这表明鞘氨酸肽是一把双刃剑，脂质和多肽都在抑制HIV进入方面发挥作用。我们已经开发了新的方法来研究基于膜中光致化学反应的融合，使用疏水探针，如碘代氨基甲酸酯。我们在分析模式(识别参与融合的病毒蛋白和受体的结构域)和功能模式(影响病毒蛋白诱导的融合)中都应用了这种方法。使用光反应性疏水探针，我们已经找到了在保持病毒包膜糖蛋白整体结构不变的情况下使其失活的方法。这些研究对抗病毒疗法和疫苗开发具有重要意义。

Our overall approach is to kinetically resolve steps in the pathway of viral envelope glycoprotein-mediated membrane fusion and to uncover physical parameters underlying those steps using a variety of biochemical, biophysical, virological, and molecular and cell biological techniques in vitro studies with infectious virus and HIV envelope proteins expressed in cells. We are using peptide-based entry inhibitors linked to specific lipids to probe details of the fusion reaction. We have recently shown that conjugation of sphinganine (a precursor of dehydrospingomyelin) confers a striking specificity to the inhibitory potential of a short HIV-based peptide suggesting that sphingopeptides act as double edged swords with both lipid and peptide playing a role in the inhibition of HIV entry. We have developed novel methodologies to study fusion based on photo-induced chemical reactions in the membrane using hydrophobic probes such as Iodonaphtylazide. We are applying this methodology both in the analytical mode (identification of domains of viral proteins and of receptors involved in fusion) and the functional mode (affecting viral protein-induced fusion). Using photo-reactive hydrophobic probes we have found ways to inactivate viral envelope glycoproteins while leaving their overall structures intact. These studies have important implications for anti-viral therapies and vaccine development.