Regensburg 2013 – wissenschaftliches Programm

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BP: Fachverband Biologische Physik

BP 28: Focus session: Intracellular Spectroscopy

BP 28.1: Topical Talk

Donnerstag, 14. März 2013, 09:30–10:00, H44

Advanced Fluorescence Methods for Investigating the Lifecycle of Viruses — •Don C. Lamb — Department of Chemistry, Ludwig-Maximilians-Universität München, Munich, Germany

Advances in fluorescence spectroscopy and microscopy make it possible to perform quantitative experiments on biological systems. One of the goals within my group is to develop and apply methods to quantify the details of viral entry and assembly in live cells. To look at the early interactions of the structural HIV protein Gag in the cytosol, we combined pulsed interleaved excitation (PIE) with Raster Image Correlation Spectroscopy. Gag was labeled using fluorescent fusion proteins and PIE-RICS measurements were performed. A slower then expected diffusion of the Gag protein in the cytosol was observed even though no significant oligomerization of Gag was detected. To investigate the origin of the slow diffusion behavior, we measured the mobility of a number of mutant Gag molecules where different interaction sites have been altered. To investigate the fusion of viral particles, we have combined the advantages of single-particle-tracking with image correlation spectroscopy. Individual viruses are tracked in three-dimensions and the signal from the different channels in the volume surrounding the particle are cross-correlated. Using the TRacking Image Correlation analysis (TRIC), we detected multiple fusion events of Foamy Virus. The analysis revealed a novel intermediate step during the fusion process where the envelope and capsid are still connected although they are separated by several hundred nanometers.