Biology subjects

Kraeusslich, H.-G.

Publications and source records attributed to Kraeusslich, H.-G..

7 recordsLinked to original sources

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↗

bioRxiv2024

Passage of the HIV capsid cracks the nuclear pore

Upon infection, human immunodeficiency virus (HIV-1) releases its cone-shaped capsid into the cytoplasm of infected T-cells and macrophages. As its largest known cargo, the capsid enters the nuclear pore complex (NPC), driven by interactions with numerous FG-repeat nucleoporins (FG-Nups). Whether NPCs structurally adapt to capsid passage and whether capsids are modified during passage remains unknown, however. Here, we combined super-resolution and correlative microscopy with cryo electron tomography and molecular simulations to study nuclear entry of HIV-1 capsids in primary human macrophages. We found that cytosolically bound cyclophilin A is stripped off capsids entering the NPC, and the capsid hexagonal lattice remains largely intact inside and beyond the central channel. Strikingly, the NPC scaffold rings frequently crack during capsid passage, consistent with computer simulations indicating the need for NPC widening. The unique cone shape of the HIV-1 capsid facilitates its entry into NPCs and helps to crack their rings.

biophysics↗

bioRxiv2024

Nuclear pores safeguard the integrity of the nuclear envelope

Nuclear pore complexes (NPCs) constitute giant channels within the nuclear envelope that mediate nucleocytoplasmic exchange. NPC diameter is thought to be regulated by nuclear envelope tension, but how such diameter changes are physiologically linked to cell differentiation, where mechanical properties of nuclei are remodeled and nuclear mechanosensing occurs, remains unstudied. Here we used cryo-electron tomography to show that NPCs dilate during differentiation of mouse embryonic stem cells into neural progenitors. In Nup133-deficient cells, which are known to display impaired neural differentiation, NPCs however fail to dilate. By analyzing the architectures of individual NPCs with template matching, we revealed that the Nup133-deficient NPCs are structurally heterogeneous and frequently disintegrate, resulting in the formation of large nuclear envelope openings. We propose that the elasticity of the NPC scaffold mechanically safeguards the nuclear envelope. Our studies provide a molecular explanation for how genetic perturbation of scaffolding components of macromolecular complexes causes tissue-specific phenotypes.

cell biology↗

bioRxiv2023

The phenuivirus Toscana virus makes an atypical use of vacuolar acidity to enter host cells

Toscana virus is a major cause of arboviral disease in humans in the Mediterranean basin during summer. However, early virus-host cell interactions and entry mechanisms remain poorly characterized. Investigating iPSC-derived human neurons and cell lines, we found that virus binding to the cell surface was specific but inefficient, and 50% of bound virions were endocytosed within 10 min. Virions entered Rab5a+ early endosomes and, subsequently, Rab7a+ and LAMP-1+ late endosomal compartments. Penetration required intact late endosomes and occurred within 30 min following internalization. Virus entry relied on vacuolar acidification, with an optimal pH for viral membrane fusion at pH 5.5. The pH threshold increased to 5.8 with longer pre-exposure of virions to the slightly acidic pH in early endosomes. Strikingly, the particles remained infectious after entering late endosomes with a pH below the fusion threshold. Overall, our study establishes Toscana virus as a late-penetrating virus and reveals an atypical use of vacuolar acidity by this virus to enter host cells.

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↗

bioRxiv2020

Critical role of type III interferon in controlling SARS-CoV-2 infection, replication and spread in primary human intestinal epithelial cells

SARS-CoV-2 is an unprecedented worldwide health problem that requires concerted and global approaches to better understand the virus in order to develop novel therapeutic approaches to stop the COVID-19 pandemic and to better prepare against potential future emergence of novel pandemic viruses. Although SARS-CoV-2 primarily targets cells of the lung epithelium causing respiratory infection and pathologies, there is growing evidence that the intestinal epithelium is also infected. However, the importance of the enteric phase of SARS-CoV-2 for virus-induced pathologies, spreading and prognosis remains unknown. Here, using both colon-derived cell lines and primary non-transformed colon organoids, we engage in the first comprehensive analysis of SARS-CoV-2 lifecycle in human intestinal epithelial cells. Our results demonstrate that human intestinal epithelial cells fully support SARS-CoV-2 infection, replication and production of infectious de-novo virus particles. Importantly, we identified intestinal epithelial cells as the best culture model to propagate SARS-CoV-2. We found that viral infection elicited an extremely robust intrinsic immune response where, interestingly, type III interferon mediated response was significantly more efficient at controlling SARS-CoV-2 replication and spread compared to type I interferon. Taken together, our data demonstrate that human intestinal epithelial cells are a productive site of SARS-CoV-2 replication and suggest that the enteric phase of SARS-CoV-2 may participate in the pathologies observed in COVID-19 patients by contributing in increasing patient viremia and by fueling an exacerbated cytokine response.

microbiology↗

bioRxiv2017

Flat-to-curved transition during clathrin-mediated endocytosis correlates with a change in clathrin-adaptor ratio and is regulated by membrane tension

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form a flat structure and then bends while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical modelling to quantify the sequence of ultrastructural rearrangements of the clathrin coat during endocytosis in mammalian cells. We confirm that clathrin-coated structures can initially grow flat and that lattice curvature does not show a direct correlation with clathrin coat assembly. We demonstrate that curvature begins when 70% of the final clathrin content is acquired. We find that this transition is marked by a change in the clathrin to clathrin-adaptor protein AP2 ratio and that membrane tension suppresses this transition. Our results support the model that mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

cell biology↗