Time-resolved single virus tracking and spectral imaging to understand HIV-1 entry and fusion.

Single Virus Tracking (SVT) is a key technique to understand how individual viral particles evolve during the infection cycle. In the case of the human immunodeficiency virus (HIV-1), this technology, which can be employed using a simple and affordable wide-field microscope, has proven to be very useful in the first steps of infection, such as the kinetics of the fusion reaction or the point of fusion within live cells. Here, we describe how SVT in combination with other spectral imaging approaches is a powerful technique to illuminate crucial mechanistic aspects of the HIV-1 fusion reaction. We also stress the role of our laboratory in elucidating a few mechanistic aspects of retroviral fusion employing SVT such as: i) the role of dynamin, ii) how metabolism modulates membrane composition and cholesterol and its impact in fusion, iii) the importance of envelope glycoprotein (Env) intra and intermolecular dynamics for neutralization or iv) the time-resolved fusion stoichiometry in three characteristic steps for the HIV-1 prefusion step. These observations constitute a good testimony of the complexity of retroviral fusion and show the strength of SVT when applied to live cells and combined with quantitative spectral approaches. Finally, we propose several crucial remaining questions around HIV-1 fusion and how the combined use of these technologies, always in live cells, will be able to shed light into the intricacies of arguably the most important step of HIV-1 infection cycle. This article is protected by copyright. All rights reserved.