During HIV-1 particle formation, the requisite plasma membrane curvature is thought to be solely driven by the retroviral Gag protein. Here, we reveal that the cellular I-BAR protein IRSp53 is required for the progression of HIV-1 membrane curvature to complete particle assembly. SiRNA-mediated knockdown of IRSp53 gene expression induces a decrease in viral particle production and a viral bud arrest at half completion. Single molecule localization microscopy at the cell plasma membrane shows a preferential localization of IRSp53 around HIV-1 Gag assembly sites. In addition, we observe the presence of IRSp53 in purified HIV-1 particles. Finally, HIV-1 Gag protein preferentially localizes to curved membranes induced by IRSp53 I-BAR domain on giant unilamellar vesicles. Overall, our data reveal a strong interplay between IRSp53 I-BAR and Gag at membranes during virus assembly. This highlights IRSp53 as a crucial host factor in HIV-1 membrane curvature and its requirement for full HIV-1 particle assembly.

MINimal fluorescence photon FLUXes (MINFLUX) nanoscopy, providing photon-efficient fluorophore localizations, has brought about three-dimensional resolution at nanometer scales. However, by using an intrinsic on–off switching process for single fluorophore separation, initial MINFLUX implementations have been limited to two color channels. Here we show that MINFLUX can be effectively combined with sequentially multiplexed DNA-based labeling (DNA-PAINT), expanding MINFLUX nanoscopy to multiple molecular targets. Our method is exemplified with three-color recordings of mitochondria in human cells.

The HIV-1 assembly process is a multi-complex mechanism that takes place at the host cell plasma membrane. It requires a spatio-temporal coordination of events to end up with a full mature and infectious virus. The molecular mechanisms of HIV-1 assembly have been extensively studied during the past decades, in order to dissect the respective roles of the structural and non-structural viral proteins of the viral RNA genome and of some host cell factors. Nevertheless, the time course of HIV-1 assembly was observed in living cells only a decade ago. The very recent revolution of optical microscopy, combining high speed and high spatial resolution, in addition to improved fluorescent tags for proteins, now permits study of HIV-1 assembly at the single molecule level within living cells. In this review, after a short description of these new approaches, we will discuss how HIV-1 assembly at the cell plasma membrane has been revisited using advanced super resolution microscopy techniques and how it can bridge the study of viral assembly from the single molecule to the entire host cell.