Entry Mechanisms used by a model retrovirus

描述（由申请方提供）：包膜病毒通过病毒包膜蛋白介导的病毒膜和细胞膜之间的融合将其核衣壳存款到宿主细胞中。因此，了解病毒融合的机制对于开发预防感染的新策略是重要的。然而，病毒蛋白介导的融合的分子细节尚未完全阐明。病毒利用两种主要的进入途径来建立感染：病毒融合蛋白在与细胞表面的受体结合后或在进入酸性内体后被激活。禽肉瘤和白血病病毒（ASLV）是研究不同病毒使用的感染途径的极好模型，因为其进入涉及连续的受体依赖性和低pH依赖性步骤，最终与酸性内体融合。单病毒成像是一种强大的技术，可以跟踪细胞内的单个病毒体。通过单个ASLV融合的时间分辨成像，我们的小组先前已经可视化了脂质和内容物转移步骤，证明了融合通过半融合（脂质转移）、小孔形成和孔扩大（内容物释放）的关键中间阶段的进展。我们的初步数据表明，新生融合孔的命运是由融合位点决定的：ASLV在细胞表面形成的孔没有完全扩大，而与内体的限制膜形成的孔有效扩张，允许病毒内容物释放到胞质溶胶中。融合孔扩张倾向的巨大差异导致了这样的假设，即病毒已经适应在指定位点形成相对较小的孔，依赖于细胞内吞机制来扩大这些孔并启动感染。为了验证这一假设，我们将：（i）定义ASLV进入途径，并确定融合发生的首选酸性细胞器;（ii）开发工具来检测小融合孔的形成，并测量它们在细胞表面和不同内吞隔室中的扩张功效;以及（iii）通过测试是否需要多种融合蛋白的协同作用来促进病毒内容物释放，确定病毒蛋白是否有助于孔扩大进入细胞质。这些研究有望为ASLV融合机制提供新的见解，并阐明控制孔扩张的因素。使用ASLV模型获得的数据将为理解通过内吞途径进入其宿主细胞的人类病毒的进入过程提供概念框架。包膜病毒通过将其基因组沉积到宿主细胞中来启动感染。这一过程涉及病毒核心周围的膜与细胞膜的融合。膜融合是一个复杂的多步骤反应，由专门的病毒融合蛋白介导。为了阐明这一过程，我们将使用荧光显微镜观察单个禽肉瘤和白血病病毒粒子与宿主细胞的融合，从其开始（病毒和细胞膜的局部合并）到完成（病毒基因组释放到细胞质中）。这些研究将有助于确定人类病毒的进入机制，并提出预防感染的新策略。

DESCRIPTION (provided by applicant): Enveloped viruses deposit their nucleocapsid into a host cell through the viral envelope protein-mediated fusion between viral and cellular membranes. Thus understanding the mechanism of viral fusion is important for developing novel strategies to prevent infection. However, the molecular details of viral protein-mediated fusion have not been fully elucidated. Two principal entry pathways are used by viruses to establish infection: viral fusion proteins are either activated upon binding to receptors at the cell surface or upon entering acidic endosomes. The avian sarcoma and leukosis virus (ASLV) is an excellent model to study infectious pathways used by disparate viruses, because its entry involves sequential receptor-dependent and low pH-dependent steps that culminate in fusion with acidic endosomes. Single virus imaging is a powerful technique that permits tracking individual virions inside a cell. Through time-resolved imaging of single ASLV fusion, our group has previously visualized the lipid and content transfer steps, demonstrating the progression of fusion through key intermediate stages of hemifusion (lipid transfer), small pore formation and pore enlargement (content release). Our preliminary data show that the fate of nascent fusion pores is determined by the site of fusion: pores formed by ASLV at the cell surface do not fully enlarge, whereas pores formed with the limiting membrane of an endosome dilate efficiently, permitting the release of viral content into the cytosol. This vast difference in the propensity of fusion pores to dilate has led to the hypothesis that viruses have adapted to form relatively small pores at designated sites, relying on cellular endocytic machinery to enlarge these pores and initiate infection. To test this hypothesis, we will: (i) define the ASLV entry pathways and determine the preferred acidic organelles in which fusion occurs; (ii) develop tools to detect the formation of small fusion pores and measure the efficacy of their dilation at the cell surface and in different endocytic compartments; and (iii) determine whether viral proteins can contribute to pore enlargement by testing whether the concerted action of multiple fusion proteins is required to promote viral content release into the cytosol. The proposed studies are expected to provide new insights into the mechanism of ASLV fusion and to elucidate the factors governing pore dilation. Data obtained using the ASLV model would provide a conceptual framework for understanding the entry process of human viruses that enter their host cells via the endocytic pathway. Enveloped viruses initiate infection by depositing their genome into a host cell. This process involves fusion of a membrane surrounding the viral core to a cell membrane. Membrane fusion is a complex multi-step reaction mediated by specialized viral fusion proteins. To elucidate this process, we will visualize fusion of single avian sarcoma and leukosis virions with a host cell from its initiation (a local merger of viral and cellular membranes) to completion (release of the viral genome into the cytosol), using fluorescence microscopy. These studies will help to define the entry mechanisms of human viruses and will suggest new strategies to prevent infection.