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Elucidating Virus Uptake and Fusion by Single Virus Tracing

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Elucidating Virus Uptake and Fusion by Single Virus Tracing Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Elucidating virus uptake and fusion by single virus tracing Dorothee Carolin Schupp aus Ravensburg, Deutschland 2012 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. Novem- ber 2011 von Herrn Professor Don C. Lamb, PhD, betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München, den 07. Juni 2012 (Dorothee Carolin Schupp) Dissertation eingereicht am 11. Juni 2012 1. Gutachter: Prof. Don C. Lamb, PhD 2. Gutachter: Prof. Dr. Christoph Bräuchle Mündliche Prüfung am 16. Juli 2012 für Benno Summary 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. Most viruses have evolved with their host over thousands of years and are experts in cell entry. On the one hand, this can complicate the development of efficient drugs against virus infections. On the other hand, one can make use of the virus entry skills to cure diseases using them as shuttles to target a therapeutic agent to particular cells in the body. Prior to their application as therapeutic agents, it is crucial to understand how cells are infected by these viruses. 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. Upon fusion the viral capsid is released into the cytoplasm. The aim of this work is to investigate 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 first dual-color HSV-1 preparations contained only very few dual-color particles. Therefore, a screen for the best preparation conditions was performed. The amount of capsid particles colocalizing with an additional envelope signal was increased from about 40 % to around 70 %. In addition, the presence of envelope-only particles was significantly reduced although still about 50 % envelope-only particles were present in the optimized virus preparations. Live-cell imaging experiments in Vero cells revealed a decrease in the colocalization percentage over time, indicating fusion. However, pH-dependent quenching of the flu- orescence intensity in acidic cellular compartments could also lead to this observation. Evaluation of the fluorescence intensity of the pH-sensitive GFP-tag, located at the in- I Summary side of the virus envelope, revealed significant quenching at acidic pH values. In order to account for pH-dependent quenching, colocalization experiments were per- formed in fixed and permeabilized Vero and HeLa cells. Permeabilization ensures neutral pH throughout all cellular compartments. In the case of Vero cells, no clear decay in the colocalization percentage was observed during 3.5 hours post attachment. In contrast, HeLa cells showed a small decay over time. The use of fixed cells is intrinsically accompanied by a lack of kinetics on a single-cell level over time. Consequently, colocalization analysis of dual-color viruses was performed in live HeLa cells with an additional marker of endosomal compartments. This 3-color experiment revealed that fusion takes place to a small extend in HeLa cells, consistent with the experiment in fixed HeLa cells. Moreover, the uptake of individual virus parti- cles was investigated in real-time, but no fusion event was detected. Thus, the use of GFP can result in critical artifacts in live-cell imaging, but tools are presented to circumvent this issue. Furthermore, one can take advantage of the pH- sensitivity to distinguish extra- and intracellular virus particles. The entry of two types of dual-color foamy virus particles was investigated, namely of prototype foamy virus (PFV) and macaque simian foamy virus (SFVmac). The virus particles contained a GFP-tag at the capsid and an mCherry-tag at the envelope. First, the percentage of dual-color virus particles was characterized for each virus preparation. In all virus preparations, more than 90 % of detected capsid particles colocalized with an mCherry-envelope signal. The amount of envelope particles that contained an additional GFP-capsid signal ranged between 30 to 60 %. FV glycoproteins possess highest fusion activity at acidic pH values which suggests a pH-dependent endocytic uptake pathway. However, PFV in contrast to the other FV species, already shows significant fusion activity at neutral pH. Hence, it may take an additional pH-independent entry route. In the case of endocytosis, the virions encounter a low pH environment on their journey towards the nucleus in late endosomes. Under these pH conditions, the fluorescence intensity of the dual-color particles was still de- tectable and a slight reduction was only observed for the GFP-tag. The pH-dependent GFP-tag is located at the capsid and protected by the surrounding lipid bilayer envelope. Live-cell imaging experiments were performed with spinning-disk confocal microscopy in 3D to gain insights into virus uptake and fusion. 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 in live HeLa cells for PFV and SFVmac particles. In the case of PFV, the obtained colocalization percentage already decreased significantly within the first few minutes post attachment whereas for SFVmac, the decay was less II Summary pronounced. This experiment demonstrates that there are differences between the up- take of PFV and SFVmac in HeLa cells. PFV already fuses within the first minutes. In contrast, fusion of SFVmac occurs at later times and less fusion events were observed during the measurement time of 90 minutes. The highest probability to detect FV fusion is expected during the first 30 minutes post attachment. Subsequently, single virus tracing experiments were performed with high time resolution to investigate individual fusion events in real-time. Sixteen fusion events, visualized by color separation, were observed. In the case of PFV, four fusion events were observed at the plasma membrane and nine fused with an endosomal membrane after endocytic uptake. For SFVmac, only three intracellular fusion events were observed in total, con- sistent with the previous colocalization experiment. 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 mCherry-envelope and GFP-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. III Contents 1 Introduction 1 2 Fluorescence microscopy 5 2.1 Principlesoffluorescence. 6 2.2 Fluorescent microscope techniques . 8 2.2.1 Wide-field microscopy . 9 2.2.2 Confocal microscopy . 10 2.3 Fluorescentproteins ............................. 12 3 Introduction to viruses 17 3.1 Herpesvirales ................................. 18 3.1.1 Herpessimplexvirus1. 21 3.2 Retroviridae.................................. 24 3.2.1 Foamyvirus.............................. 26 4 Virus entry and replication 31 4.1 Endocyticmodesofentry .......................... 31 4.1.1 Theendosomalsystem ........................ 33 4.2 Virusmembranefusion............................ 33 4.2.1 Virustrafficking ........................... 37 4.3 Herpessimplexvirus1 ............................ 39 4.3.1 Attachmentandentry . 39 4.3.2 Assemblyandegress ......................... 49 4.4 Foamyvirus.................................. 52 4.4.1 Attachmentandentry . 52 4.4.2 Assemblyandegress ......................... 54 5 Materials and methods 57 V Contents 5.1 Samplepreparation.............................. 57 5.1.1 Herpes simplex virus 1 particles (HSV-1) . 57 5.1.2 Foamy virus particles (FV) . 59 5.1.3 Cellculture .............................. 60 5.1.4 Live-cell imaging conditions . 61 5.1.5 Fixation and permeabilization . 62 5.1.6 Quenching of virus particles at low pH . 64 5.2 Experimentalsetupindetails . 64 5.2.1 Wide-field microscopy . 66 5.2.2 Spinning-disk confocal microscopy . 68 5.3 Dataanalysis ................................. 70 5.3.1 Imagecalibration........................... 71 5.3.2 Colocalization analysis of virus-only samples in 2D . 71 5.3.3 Colocalization analysis of virus-incubated cells in 3D ....... 72 5.3.4 Singleparticletracking. 74 5.3.5 Dynamic colocalization analysis of single virus trajectories . 75 6 Study of herpes simplex virus entry 79 6.1 Optimizationofviruspreparation . 80 6.1.1 First screening round of virus optimization . 82 6.1.2 Second screening round of virus optimization . 90 6.1.3 Colocalization analysis of purified dual-color virus preparations . 94 6.2 Colocalization analysis in live cells . ..... 97 6.3 pH-dependency of fluorescent proteins in the viral context ........ 100 6.4 Colocalization analysis in fixed cells . 102 6.5 Studying influence of pH dependency in live cells
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