Entry Mechanisms used by a model retrovirus

描述(申请人提供)：被包裹的病毒通过病毒被膜蛋白介导的病毒和细胞膜之间的融合，将其核衣壳存放到宿主细胞中。因此，了解病毒融合的机制对于开发预防感染的新策略非常重要。然而，病毒蛋白介导的融合的分子细节还没有完全阐明。病毒利用两条主要的进入途径来建立感染：病毒融合蛋白要么在与细胞表面的受体结合时被激活，要么在进入酸性内体时被激活。禽肉瘤和白血病病毒(ASLV)是研究不同病毒感染途径的一个很好的模型，因为它的进入涉及一系列依赖受体和低pH的步骤，最终与酸性内小体融合。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粒子。通过单个ASLV融合的时间分辨成像，我们的团队先前已经可视化了脂肪和内容物转移步骤，展示了融合过程中半融合(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。我们的初步数据显示，新生融合孔的命运由融合部位决定：ASLV在细胞表面形成的孔不会完全扩大，而带有内涵体限制膜的孔有效扩张，允许病毒内容物释放到细胞质中。这种融合毛孔扩张倾向的巨大差异导致了一种假设，即病毒已经适应在指定的位置形成相对较小的毛孔，依靠细胞内吞机械来扩大这些毛孔并启动感染。为了检验这一假说，我们将：(1)确定ASLV进入细胞的途径并确定发生融合的首选酸性细胞器；(2)开发检测融合小孔形成的工具，并测量它们在细胞表面和不同的胞内隔间扩张的效力；以及(3)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释放到细胞质，以确定病毒蛋白是否可促进孔的扩大。这些研究有望为ASLV融合的机制提供新的见解，并阐明影响气孔扩张的因素。使用ASLV模型获得的数据将为理解通过内吞途径进入宿主细胞的人类病毒的进入过程提供一个概念性框架。被包膜的病毒通过将它们的基因组存放在宿主细胞中来启动感染。这一过程包括将病毒核心周围的膜融合到细胞膜上。膜融合是由特殊的病毒融合蛋白介导的复杂的多步骤反应。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从开始(病毒和细胞膜的局部合并)到完成(将病毒基因组释放到细胞质中)的融合。这些研究将有助于确定人类病毒的入侵机制，并将提出预防感染的新战略。

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.