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978-3-8439-0633-3, Reihe Physikalische Chemie
Dorothee Carolin Schupp
Elucidating virus uptake and fusion by single virus tracing
210 Seiten, Dissertation Ludwig-Maximilians-Universität München (2012), Softcover, A5
Viruses are known to cause many diseases, from the common cold and cold sores to more serious diseases such as the Ebola virus disease and AIDS. Viruses have evolved different strategies to enter and infect cells. In order to infect a cell, viruses have to overcome the cell membrane barrier to deliver their genome to the site of replication. Enveloped viruses can either fuse directly at the plasma membrane or with an endosomal membrane after endocytic uptake. In this work, I studied the early steps in virus entry of herpes simplex virus 1 (HSV-1) and foamy virus (FV) by means of fluorescence microscopy. The virus particles contain two different labels, one located at the envelope and the other at the capsid so that fusion can be detected upon separation of the two colors in space.
The virus preparations were optimized for a high dual-color virus yield and live-cell imaging experiments were performed with spinning-disk confocal microscopy in 3D to gain insights into the entry kinetics. In order to determine the time-scale when virus fusion occurs, the percentage of virions containing both envelope and capsid signals was evaluated over time.
Virus particles that are taken up by endocytosis face an increasing proton concentration within maturing endosomes. However, the emission of some fluorescent proteins is known to be pH-dependent and the use of pH-sensitive fluorescent proteins, such as GFP, can result in critical artifacts in live-cell imaging. Therefore, experimental approaches are presented to circumvent this issue. To obtain dynamic information on virus fusion, single virus tracing experiments were performed with high time resolution to investigate individual fusion events in real-time. In the case of foamy virus, sixteen fusion events, visualized by color separation, were observed. Thereof, four fusion events were observed at the plasma membrane and twelve fused with an endosomal membrane after endocytic uptake. Moreover, an intermediate stage during the fusion process of foamy viruses was identified that lasted over minutes. This stage was characterized by an increase in the distance between the fluorescent envelope and capsid signals before the final color separation event. Hence, it was possible for the first time to visualize single fusion events of foamy virus in real-time and characterize the corresponding dynamics. The results provide new insights into the entry pathway and fusion process of this unconventional retrovirus.