Biology subjects

Anders-Oesswein, M.

Publications and source records attributed to Anders-Oesswein, M..

3 recordsLinked to original sources

bioRxiv2026

Developing a fluorescent derivative of GagPol as a tool for live-cell imaging of HIV-1 assembly

The HIV-1 assembly process is driven by the structural polyprotein Gag, which forms small cytosolic oligomers that traffic to the plasma membrane. While Gag alone is sufficient to drive particle formation, the incorporation of GagPol, a polyprotein comprising Gag and viral enzymes, is essential for productive infection. Maintenance of the proper Gag:GagPol ratio is crucial for virion infectivity. Yet, the mechanisms regulating their cytosolic interactions remain incompletely understood, in part because most studies rely on ensemble biochemical assays that hide cell-to-cell heterogeneity and lack spatial and temporal resolution. To overcome these limitations and investigate the dynamics of GagPol at the molecule level, we systematically tested different approaches to fluorescently label it. We successfully produced two functional fluorescent GagPol variants: one single-labeled GagPol, and a second double-labeled variant with a fluorescent protein added within Gag, that allows concurrent visualization of Gag and GagPol. These labeled versions, in combination with the use of raster image (cross-) correlation spectroscopy, enabled the quantification of Gag and GagPol relative concentrations and intermolecular interactions at the single-cell level. Overall, these variants set the stage for in-depth investigations of GagPol during HIV assembly providing insights into cytoplasmic trafficking, particle assembly, and the kinetics of these processes. ImportanceThe participation of Gag and GagPol in HIV-1 assembly is central to the generation of infectious virions. While the structural role of Gag and the enzymatic functions of GagPol are well described, little is known regarding the mechanisms underlying their cytosolic interactions, spatial organization, and relative incorporation into assembling particles. Here, we successfully developed two stable fluorescently labeled variants of GagPol; a single labeled variant with a label in the Pol domain and double-labeled construct in which the Gag domain is also tagged. Using these constructs, we can quantitatively investigate Gag and GagPol interactions on the single-cell level. Hence, they provide a framework for a comprehensive study of the oligomerization properties and trafficking of these polyproteins at different stages of HIV-1 assembly, including membrane recruitment and incorporation into nascent virions.

biophysics↗

bioRxiv2025

Lenacapavir-induced capsid damage exposes HIV-1 genomes from nuclear speckles.

Following cell entry, HIV-1 capsids enter the nucleus by passage through nuclear pores and reach nuclear speckles, where uncoating of the reverse-transcribed genome and integration occur. Here, we characterized the ultrastructure of HIV-1 subviral complexes in nuclei of primary monocyte-derived macrophages and cell lines using live-cell imaging, super-resolution microscopy and correlative light and electron tomography in the absence and presence of capsid-targeting inhibitors Lenacapavir and PF74. Capsid-like structures containing viral DNA as well as broken capsids clustered in nuclear speckles and were displaced from speckles by drug treatment. This was accompanied by alteration of the nuclear capsid structure, with electron-dense protrusions emanating from the narrow end of capsid cones and exposure of complete, or almost complete, genomic HIV-1 DNA. Our data indicate that synthesis of genomic dsDNA can be completed inside the closed HIV-1 capsid, and speckle-associated factors appear to be required to promote genome uncoating. This may ensure that genome uncoating occurs at optimal sites for integration into transcriptionally active chromatin. The results also shed further light on the mechanism of action of Lenacapavir.

microbiology↗

bioRxiv2021

Direct capsid labeling of infectious HIV-1 by genetic code expansion allows detection of largely complete nuclear capsids and suggests nuclear entry of HIV-1 complexes via common routes

The cone-shaped mature HIV-1 capsid is the main orchestrator of early viral replication. After cytosolic entry, it transports the viral replication complex along microtubules towards the nucleus. While it was initially believed that the reverse transcribed genome is released from the capsid in the cytosol, recent observations indicate that a high amount of capsid protein (CA) remains associated with subviral complexes during import through the nuclear pore complex (NPC). Observation of post-entry events via microscopic detection of HIV-1 CA is challenging, since epitope shielding limits immunodetection and the genetic fragility of CA hampers direct labeling approaches. Here, we present a minimally invasive strategy based on genetic code expansion and click chemistry that allows for site-directed fluorescent labeling of HIV-1 CA, while retaining virus morphology and infectivity. Thereby, we could directly visualize virions and subviral complexes using advanced microscopy, including nanoscopy and correlative imaging. Quantification of signal intensities of subviral complexes revealed an amount of CA associated with nuclear complexes in HeLa-derived cells and primary T cells consistent with a complete capsid and showed that treatment with the small molecule inhibitor PF74 did not result in capsid dissociation from nuclear complexes. Cone-shaped objects detected in the nucleus by electron tomography were clearly identified as capsid-derived structures by correlative microscopy. High-resolution imaging revealed dose-dependent clustering of nuclear capsids, suggesting that incoming particles may follow common entry routes.

microbiology↗