Live imaging of virus assembly and release by simultaneous, correlative topographical and fluorescence confocal microscopy

病毒对人类健康和经济造成重大负担。特别是人类免疫缺陷病毒（HIV）感染是世界范围内主要的健康挑战之一。病毒是一种很小的传染因子，只能在其他生物体的活细胞内通过复杂的多步骤过程进行繁殖。因为所有的生物细胞都覆盖着一层细胞膜，将细胞内空间与外界隔开，所以所有的病毒在复制之前都必须穿过这层屏障。同样，对于许多病毒，如艾滋病毒，细胞膜是组装和释放到细胞外空间的地方，这是病毒传播所必需的。在埃博拉病毒有可能大流行的时候，在艾滋病毒疫苗仍然没有早期前景的时候，了解病毒组装和与细胞相互作用的基本机制就变得更加重要了。从基本分子组装机制的角度研究病毒出芽动力学具有重要意义，有助于估计抗病毒药物靶向释放和传播的最佳时间窗口。尽管艾滋病毒已被广泛研究，并且由于构成病毒颗粒的成分数量较少，它是一个很好的模型，但能够研究病毒在活细胞中的组装和释放的方法缺乏。大多数高倍率显微镜，如电子显微镜。不能直接“看到”生物体，生物细胞必须干燥并涂上一层薄薄的金属层才能成像。其他显微镜技术使用间接方法，通过对附着在病毒结构成分上的荧光标记成像来可视化病毒。然而，这种荧光标签的大小与它们所标记的分子相当，并且可以影响病毒的组装。我们开创了一种替代成像技术，称为扫描离子电导显微镜（SICM），现在允许在活细胞表面的微小结构的可视化，同时保持细胞在其天然环境中。我们现在计划开发一种基于SICM和荧光成像相结合的新方法，以超分辨率研究病毒在活细胞中的组装。然后，我们将使用我们的方法来回答仍然存在高度争议的问题，例如单个病毒颗粒的组装速度以及组装速度如何依赖于关键病毒蛋白质以及周围细胞内环境中的蛋白质。新方法的研究和发展旨在发现新的现象，并进一步提高对病毒组装和释放的基本机制的认识。这一信息对于理解病毒传播和开发针对病毒生命周期中这些可达步骤的新型抗病毒药物至关重要。虽然很难预测对公共和第三部门的直接好处，但这项研究和方法将引起病毒学、转化医学研究人员的兴趣，我们希望这将导致更好的有针对性的卫生保健和治疗方法，我们预计这将对英国内外的人类健康和财富都非常有益。目前，英国在SICM方面处于世界领先地位。这一领域受到了日本、韩国和美国的研究人员和工业界的极大关注，特别是在过去两年中，SICM的潜力终于得到了承认。保持这一领先地位对英国的研究声誉和经济至关重要，我决心通过开展尖端的SICM支持研究和进一步开发SICM仪器来帮助实现这一目标。所提出的方法的发展将进一步加强英国SICM的地位，我很自豪能在一个代表世界领先的超分辨率地形实时成像研究的实验室工作。我相信这是对英国科学和经济的长期贡献。

Viruses pose a major human health and economic burden. Human immunodeficiency virus (HIV) infection in particular is one of the predominant health challenges worldwide. A virus is a small infectious agent that reproduces only inside the living cells of other organisms through complex and multistep processes. Because all biological cells are covered with a cell membrane that separates the intracellular space from the outside world, all viruses have to cross this barrier before they can replicate. Similarly, for many viruses, such as HIV, the cell membrane is the place of assembly and release into extracellular space necessary for dissemination of the virus.At a time when the ebola virus is threatening a pandemic and where there is still no early prospect of an HIV vaccine it has become even more important to understand the basic mechanisms by which viruses are assembled and interact with cells. The study of virus budding dynamics is important from the point of basic molecular mechanisms of assembly and will help to estimate the best time window for antiviral drugs targeting release and transmission. Although, HIV has been extensively studied and is an excellent model due to the low number of components that make up virus particles, there is a paucity of methods capable of studying virus assembly and release in living cells . Most high magnification microscopes, such as electron microscopes. cannot directly 'see' living organisms, and biological cells have to be dried and coated with a thin metal layer to be imaged. Other microscopy techniques use an indirect approach and visualise viruses by imaging fluorescent tags attached to virus structural components. However, such fluorescent tags are comparable in size with the molecules they label and can affect virus assembly. We have pioneered an alternative imaging technique called Scanning Ion Conductance Microscopy (SICM) that now allows the visualisation of minute structures at the surface of living cells whilst keeping cells in their native environment. We now plan to develop a new approach based on a combination of SICM and fluorescence imaging to study virus assembly in living cells with super resolution. We will then use our method to answer questions that remain highly controversial such as the rate of assembly of individual virus particles and how the assembly rate depend on key virus proteins and also proteins in the surrounding intracellular environment.The proposed study and development of the new methodology aims to discover new phenomena, and to further improve the understanding of the basic mechanisms of virus assembly and release. This information is crucial for understanding virus transmission and for the development of novel antiviral agents targeting these accessible steps in the viral life cycle. Although it is difficult to anticipate immediate benefits to public and third sector, this research and methodology will be of interest to researchers in virology, translational medicine and we hope will result in better targetted health care and therapeutic approaches that we anticipate could be highly beneficial to human health and wealth both within and outside the UK.Currently, UK is the world leader in SICM. This field has received a great deal of attention from researchers and industry in Japan, Korea and the United States, particularly during the last two years when SICM's potential has finally been recognised. It is important for the UK research reputation and economy to maintain this leading role, and I am determined to help ito achieve this by conducting cutting edge SICM-enabled research and further development of the SICM instrumentation. The development of the proposed method will further strengthen the position of UK SICM and I am proud to be working in a laboratory which represents world leading research in super resolution topographical live imaging. I believe that this is a valuable contribution to long term UK science and economy.