Elucidating virus uptake and fusion by single virus tracing

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

Differential pH-dependent cellular uptake pathways among foamy viruses elucidated using dual-colored fluorescent particles

Background: It is thought that foamy viruses (FVs) enter host cells via endocytosis because all FV glycoproteins examined display pH-dependent fusion activities. Only the prototype FV (PFV) glycoprotein has also significant fusion activity at neutral pH, suggesting that its uptake mechanism may deviate from other FVs. To gain new insights into the uptake processes of FV in individual live host cells, we developed fluorescently labeled infectious FVs. Results: N-terminal tagging of the FV envelope leader peptide domain with a fluorescent protein resulted in efficient incorporation of the fluorescently labeled glycoprotein into secreted virions without interfering with their infectivity. Double-tagged viruses consisting of an eGFP-tagged PFV capsid (Gag-eGFP) and mCherry-tagged Env (Ch-Env) from either PFV or macaque simian FV (SFVmac) were observed during early stages of the infection pathway. PFV Env, but not SFVmac Env, containing particles induced strong syncytia formation on target cells. Both virus types showed trafficking of double-tagged virions towards the cell center. Upon fusion and subsequent capsid release into the cytosol, accumulation of naked capsid proteins was observed within four hours in the perinuclear region, presumably representing the centrosomes. Interestingly, virions harboring fusion-defective glycoproteins still promoted virus attachment and uptake, but failed to show syncytia formation and perinuclear capsid accumulation. Non-fused or non-fusogenic viruses are rapidly cleared from the cells by putative lysosomal degradation. Monitoring the fraction of viruses containing both Env and capsid signals as a function of time demonstrated that PFV virions fused within the first few minutes, whereas fusion of SFVmac virions was less pronounced and observed over the entire 90 minutes measured. Conclusions: The characterized double-labeled FVs described here provide new mechanistic insights into FV early entry steps, demonstrating that productive viral fusion occurs early after target cell attachment and uptake. The analysis highlights apparent differences in the uptake pathways of individual FV species. Furthermore, the infectious double-labeled FVs promise to provide important tools for future detailed analyses on individual FV fusion events in real time using advanced imaging techniques

Hyperon signatures in the PANDA experiment at FAIR

We present a detailed simulation study of the signatures from the sequential decays of the triple-strange pbar p -> Ω+Ω- -> K+ΛbarK- Λ -> K+pbarπ+K-pπ- process in the PANDA central tracking system with focus on hit patterns and precise time measurement. We present a systematic approach for studying physics channels at the detector level and develop input criteria for tracking algorithms and trigger lines. Finally, we study the beam momentum dependence on the reconstruction efficiency for the PANDA detector