Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MMXVI (Minimal Model for Pox-Virus Infection)

Teaser

The initial contact between a virus particle and its host is a crucial step during infection. The process depends on nanoscale structural changes that due to technical limitations remain a major challenge in infection biology. Methods such as fluorescence or electron...

Summary

The initial contact between a virus particle and its host is a crucial step during infection. The process depends on nanoscale structural changes that due to technical limitations remain a major challenge in infection biology. Methods such as fluorescence or electron microscopy provide either the required molecular specificity or resolution to investigate such changes. Recent developments in super-resolution microscopy allow the visualization of cellular and viral structures at resolutions in the range of tens of nanometers and therefore provide new tools to address the dynamics of virus architectural changes during the first stages of infection.
The focus of the project was investigating protein structure-function relationships within the prototypic poxvirus, vaccinia. However, the model system and imaging tools are broadly applicable to other biological systems such as different viruses. A better understanding of virus host interactions benefits society in general as it may serve as a foundation to new antiviral strategies.
The overall objectives were to develop and implement a novel minimal model of virus infection based on cell-derived membrane blebs to investigate virus host interactions using vaccinia. The project addressed the nanoscale virus architecture, particularly at the membrane level, by combining single-molecule techniques with membrane blebs. Furthermore, the dynamics of protein organization in the viral membrane were investigated. Based on our findings, we propose that polarization and clustering of the entry fusion compley of vaccinia virus is critical for efficient virus-cell fusion and entry of the viral particle. The nanoscale organization of the poxvirus membrane suggests that virion protein architecture is critical to virus function. In conclusion, the organization of the vaccinia virus membrane into functionally distinct domains may have evolved as a mechanism to maximise virion binding and fusion efficiency for productive infection.

Work performed

Cell derived membrane blebs were generated from different cell lines including HeLa, BSC-40, and U-2 OS. A robust protocol for purification of blebs based on filtration was established and blebs characterized by fluorescence microscopy. Super-resolution microscopy was optimized on purified virus particles and the distribution of different binding and fusion proteins mapped. In particular, the polarization of several viral proteins that together form the so-called entry fusion complex (EFC) was discovered (Gray et al. 2019). Clustering of the EFC at the virion’s tips is required for efficient fusion. Vaccinia virus predominantly binds on its side to the cellular surface and blebs. However, fusion occurs almost exclusively at the tips where the EFCs are concentrated. Binding is facilitated by different binding proteins in the viral membrane that are enriched on the side of the viral particle. The underlying paradigm is that structural features of the viral particle drive its function. The initial interaction between virus and host is therefore not only dependent on the cellular surface but also the organization of the viral membrane. Working towards the development of improved super-resolution imaging methods, quality control metrics were implemented (Culley et al. 2018) and approaches for the correction of aberrations such as drift developed (Balinovic et al. 2019). The effect of chemical fixation on membrane organization and cytoskeleton of biological samples are described in Pereira et al. (2019). Detailed instructions for imaging of vaccinia virus by super-resolution microscopy will be made available as a book chapter (Gray, Albrecht 2019).

References:

• Gray, R.D.M.*, Albrecht, D.*, Beerli, C., Huttunen, M., Cohen, G.H., White, I.J., Burden, J. J., Henriques, R., Mercer, J., 2019. Nanoscale polarization of the entry fusion complex of vaccinia virus drives efficient fusion. Nat. Microbio. doi: 10.1038/s41564-019-0488-4 (preprint on bioRxiv: doi:10.1101/360073)
• Culley, S., Albrecht, D., Jacobs, C., Pereira, P.M., Leterrier, C., Mercer, J., Henriques, R., 2018. Quantitative mapping and minimization of super-resolution optical imaging artifacts. Nat. Methods doi:10.1038/nmeth.4605 (preprint on bioRxiv: doi: 10.1101/158279)
• Pereira, P.M.*, Albrecht, D.*, Jacobs, C., Marsh, M., Mercer, J., and Henriques, R., 2019. Fix your membrane receptor imaging: Actin cytoskeleton and CD4 membrane organization disruption by chemical fixation. Front. Immunol. doi: 10.3389/fimmu.2019.00675 (preprint on bioRxiv: doi:10.1101/450635
• Balinovic, A., Albrecht, D., & Endesfelder, U., 2019. Spectrally red-shifted fluorescent fiducial markers for optimal drift correction in localization microscopy. Journal of Physics D: Applied Physics. doi: 10.1088/1361-6463/ab0862
• Laine, R.F., Tosheva, K.L., Gustafsson, N., Gray, R.D.M., Almada, P., Albrecht, D., Risa, G.T., Hurtig, F., Lindås, A., Baum, B., Mercer, J., Leterrier, C., Pereira, P.M., Culley, S., Henriques, R., 2019. NanoJ: a high-performance open-source super-resolution microscopy toolbox. Journal of Physics D: Applied Physics. doi: 10.1088/1361-6463/ab0261 (preprint on bioRxiv: doi: 10.1101/432674)

* authors contributed equally

Final results

The project required the development of new techniques for sample preparation and imaging and as a result advances in both areas were achieved. Fluorescence based super-resolution microscopy was combined with electron microscopy for correlative imaging to map protein distribution on ultrastructural details. Thus, the project brought together experts from different fields including virology, fluorescence microscopy, electron microscopy, and image analysis. A potential impact of the findings is specifically targeting the formation of polarized viral particles as part of a strategy to reduce viral infectivity. Additionally, the bleb based model system and super-resolution imaging techniques have an impact on a wider scientific community as they will be made available for further research. Thus, the work carried out serves as a platform for further research in the area of virus host interactions.

Website & more info

More info: https://www.ucl.ac.uk/lmcb/users/david-albrecht-0.