Investigation Of Lentiviruses And Their Initial Contacts With Cells Using Real-Time 3d Tracking

Mature lentiviruses live fast and die young, thus, to study their infection pathway requires a microscope that can keep up. In this work, we track, at unprecedented timescales (10 µsec to minutes), single rapidly diffusing and fluorescent virus-like-particles (VLPs) in solution and on the cell membrane using a 3D Dynamic Photon Localization Tracking (3D-DyPLoT) microscope. Active feedback tracking by 3D-DyPLoT is achieved using a dynamically scanning laser pattern and converting photon arrival times into 3D positional information. In real-time, the diffusing particles’ position is continually updated and held within the centre of the continually scanning laser spot using a piezoelectric stage, relaying high spatiotemporal diffusion and intensity information. 3D-DyPLoT can adapt to different diffusive regimes enabling us to track VLPs on their journey to the cell surface and initial membrane-binding events taking advantage of the full potential of this system to characterize envelope glycoproteins (Env)-receptor interactions. The generation of lentiviral vectors (LVs) through replacement of cognate Envs with versatile, more promiscuous, classes of Envs has become a main gene-editing delivery system. Here we focus on one of the most commonly used lentiviral vectors, whereby the G protein of vesicular stomatitis virus (VSV-G) decorates the surface of the viral membrane. We find that the number of VSV-G Envs incorporated into integration-competent lentiviral vectors is low, on average there were 12 ± 2 Envs per VLP, but the vast majority of VLPs were skewed to a very low number, one or two, of incorporated Envs. With LVs being more commonly used in clinical applications, successful targeting of LVs to nominated cells is largely dependent on the interaction of Envs with endogenous membrane receptors. Ultimately, results here can help to unveil the initial stages of viral infection.