Abstract
The ongoing threat of human immunodeficiency virus (HIV-1) requires continued, detailed investigations of its replication cycle, especially when combined with the most physiologically relevant, fully infectious model systems. Here, we demonstrate the application of the combination of stimulated emission depletion (STED) super-resolution microscopy with beam-scanning fluorescence correlation spectroscopy (sSTED-FCS) as a powerful tool for the interrogation of the molecular dynamics of HIV-1 virus assembly on the cell plasma membrane in the context of a fully infectious virus. In this process, HIV-1 envelope glycoprotein (Env) becomes incorporated into the assembling virus by interacting with the nascent Gag structural protein lattice. Molecular dynamics measurements at these distinct cell surface sites require a guiding strategy, for which we have used a two-colour implementation of sSTED-FCS to simultaneously target individual HIV-1 assembly sites via the aggregated Gag signal. We then compare the molecular mobility of Env proteins at the inside and outside of the virus assembly area. Env mobility was shown to be highly reduced at the assembly sites, highlighting the distinct trapping of Env as well as the usefulness of our methodological approach to study the molecular mobility of specifically targeted sites at the plasma membrane, even under high-biosafety conditions.
Highlights
Plasma membrane lipid and protein organisation modulates a wide range of biological processes [1]
fluorescence correlation spectroscopy (FCS) is characterised by high temporal resolution, in its classical form, it is unsuitable for observation of processes such as endocytosis, virus assembly and entry, as well as cell surface receptor clustering, as these occur at spatial scales below the resolution limit of conventional light microscopy
This limitation can be remedied by the combination of FCS recordings with stimulated emission depletion (STED) superresolution microscopy (STED-FCS), which allows for observations at sub-diffraction spatial scales [5]
Summary
Plasma membrane lipid and protein organisation modulates a wide range of biological processes [1]. To effectively study the membrane behaviour in the context of cellular processes such as endocytosis or virus entry assembly, it is important to set the FCS scanning line into the precise area of interest This can be achieved by fluorescently labelling another molecule which participates in the process and using it a guide for FCS data acquisition. Such a two-colour sSTED-FCS approach has recently been utilised to study the behaviour of fluorescent lipid analogues inside and outside of HIV-1 assembly sites, using the structural protein Gag to guide FCS observation points to the relevant areas [10]. The HIV-1 assembly site is a sub-diffraction-sized (≈140 nm) area of Gag protein multimerisation which leads to the release of the virus particle from the plasma membrane of the infected cell. This study further highlights the usefulness of the two-colour sSTED-FCS approach in the study of the heterogeneous behaviour of molecules at discrete plasma membrane sites
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
