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.