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

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.

We will establish a new method based on correlative Scanning Ion Conductance Microscopy (SICM) and fluorescence confocal imaging for the real-time analysis of virus particle budding at the cell apical plasma membrane.

SICM generates topographical images of samples immersed in liquid by raster scanning a glass nanopipette that follows the cell surface very closely without touching. Two colour fluorescence confocal attachment will consist of two lasers focused right at the pipette tip and a fluorescence detection system. This will generate fluorescence images which can be spatially and temporally correlated with SICM topography.

We will perform super resolution live imaging studies in assembly and release of non-infectious HIV particles at the top, non adherent membrane of cells by performing the following experiments:

(i) Because Direct visualisation of virus - like particles (VLPs) on living cell membranes is challenging particularly because the particle size is small and the apical cell membrane is often complicated by elaborate and dynamic structures much larger than VLPs, such as microvilli and dorsal ruffles, we will first identify a suitable cell type using SICM time lapse topographical imaging.
(ii) Establish a non-infectious fluorescently labelled HIV model that will produce VLPs with wild type morphology by adjusting the proportion of Gag-GFP to SynGP and the GFP position in Gag, as well as using alternative tagging, such as SNAP. We will confirm VLP size, shape and fluorescence intensity by correlative SICM and confocal imaging.
(iii) Measure the rate of virus particle assembly and release by directly following its 3-D profile.
(iv) Correlate the progression of the topographically resolved structure of the virus bud with the recruitment of fluorescently-tagged structural proteins and host factors such as ESCRT.
(v) Study the interplay between virus assembly sites and specific plasma membrane microdomains including clathrin coated pits.

The proposed research aims to develop novel methodology and to apply it to generate new knowledge that should in the first instance be of interest to researchers in the immediate professional surrounding. Both, PI and PDRA will disseminate our findings through peer-reviewed publications in high profile journals, and talks at national and international research conferences and seminars. My previous publications e.g. in Journal of Cell Biology acknowledged with highlight and presentations at Gordon Research Conference on "Lysosomes and Endocytosis" 2012 and EMBO conference on "Systems Dynamics in Endocytosis" 2013 substantially increased the visibility of SICM - based approaches amongst the cell membrane transport community. Although it is difficult to anticipate immediate benefits to the public and third sector, in the longer term this fundamental research is likely to be of considerable interest to scientists in translational medicine studying virus biogenesis and finally may result in more specific health care and therapeutic approaches that could be highly beneficial to public health and wealth both within and outside the UK.
Being a live imaging technique, combined SICM - two colour fluorescence confocal microscopy generates visually attractive 3-D images of cells, both static and dynamic i.e. time lapse, that can be presented using 3-D vision technologies and used for science, art and design exhibitions in museums, galleries and charities. Recent advances and improved availability of 3-D printing technologies has made it feasible to produce scale up models by directly printing SICM topographical images of surfaces of samples, such as biological cells. We have already established 3-D printing of SICM images in our laboratory using open source RepRapPro Ormerod printer. We will continue to actively participate in Imperial College science festival (http://www3.imperial.ac.uk/festival) as we did in 2012 and 2013.
The post doctoral researcher who will work with me on the project will, in addition to advancing his/her knowledge in viral assembly and release mechanisms, develop professional skills in development and application of unique, cutting edge SICM live imaging technique and SICM - based methodology. Such expertise will be a valuable asset for future career steps taken in either academia or industry.
We expect that our development will result in commercially viable instrumentation and we will continue to consult with Imperial Innovations with regard to patenting and licensing activities. Ionscope Ltd. is one of the scanning probe microscope manufacturers that might be interested in acquiring such a license and is well placed to make our development available to other researches by converting it to more user-friendly, ready to use, commercially available equipment. Taking into account that basic SICM is already commercially available, the proposed method is likely to be of benefit to researchers in fields such as virology, immunology, toxicology, endocrinology and neurology. to name a few.
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 scientific and economic competitiveness.