Articles by Uta Haselmann in JoVE
Correlative Light Electron Microscopy (CLEM) for Tracking and Imaging Viral Protein Associated Structures in Cryo-immobilized Cells Rachel Santarella-Mellwig1, Uta Haselmann2, Nicole L. Schieber1, Paul Walther3, Yannick Schwab1, Claude Antony1, Ralf Bartenschlager2,4, Inés Romero-Brey2 1European Molecular Biology Laboratory, 2Department of Infectious Diseases, Molecular Virology, Heidelberg University, 3Central Facility for Electron Microscopy, Ulm University, 4Heidelberg Partner Site, German Center for Infection Research A correlative light electron microscopy (CLEM) method is applied to image virus-induced intracellular structures via electron microscopy (EM) in cells that are previously selected by light microscopy (LM). LM and EM are combined as a hybrid imaging approach to achieve an integrated view of virus-host interactions.
Other articles by Uta Haselmann on PubMed
Ultrastructural Characterization of Zika Virus Replication Factories Cell Reports. Feb, 2017 | Pubmed ID: 28249158 A global concern has emerged with the pandemic spread of Zika virus (ZIKV) infections that can cause severe neurological symptoms in adults and newborns. ZIKV is a positive-strand RNA virus replicating in virus-induced membranous replication factories (RFs). Here we used various imaging techniques to investigate the ultrastructural details of ZIKV RFs and their relationship with host cell organelles. Analyses of human hepatic cells and neural progenitor cells infected with ZIKV revealed endoplasmic reticulum (ER) membrane invaginations containing pore-like openings toward the cytosol, reminiscent to RFs in Dengue virus-infected cells. Both the MR766 African strain and the H/PF/2013 Asian strain, the latter linked to neurological diseases, induce RFs of similar architecture. Importantly, ZIKV infection causes a drastic reorganization of microtubules and intermediate filaments forming cage-like structures surrounding the viral RF. Consistently, ZIKV replication is suppressed by cytoskeleton-targeting drugs. Thus, ZIKV RFs are tightly linked to rearrangements of the host cell cytoskeleton.
Genome Packaging of Reovirus is Mediated by the Scaffolding Property of the Microtubule Network Cellular Microbiology. 12, 2017 | Pubmed ID: 28672089 Reovirus replication occurs in the cytoplasm of the host cell, in virally induced mini-organelles called virus factories. On the basis of the serotype of the virus, the virus factories can manifest as filamentous (type 1 Lang strain) or globular structures (type 3 Dearing strain). The filamentous factories morphology is dependent on the microtubule cytoskeleton; however, the exact function of the microtubule network in virus replication remains unknown. Using a combination of fluorescent microscopy, electron microscopy, and tomography of high-pressure frozen and freeze-substituted cells, we determined the ultrastructural organisation of reovirus factories. Cells infected with the reovirus microtubule-dependent strain display paracrystalline arrays of progeny virions resulting from their tiered organisation around microtubule filaments. On the contrary, in cells infected with the microtubule-independent strain, progeny virions lacked organisation. Conversely to the microtubule-dependent strain, around half of the viral particles present in these viral factories did not contain genomes (genome-less particles). Complementarily, interference with the microtubule filaments in cells infected with the microtubule-dependent strain resulted in a significant increase of genome-less particle number. This decrease of genome packaging efficiency could be rescued by rerouting viral factories on the actin cytoskeleton. These findings demonstrate that the scaffolding properties of the microtubule, and not biochemical nature of tubulin, are critical determinants for reovirus efficient genome packaging. This work establishes, for the first time, a functional correlation between ultrastructural organisation of reovirus factories with genome packaging efficiency and provides novel information on how viruses coordinate assembly of progeny particles.
Structural Studies of Self-Assembled Subviral Particles: Combining Cell-Free Expression with 110 kHz MAS NMR Spectroscopy Angewandte Chemie (International Ed. in English). Apr, 2018 | Pubmed ID: 29457857 Viral membrane proteins are prime targets in combatting infection. Still, the determination of their structure remains a challenge, both with respect to sample preparation and the need for structural methods allowing for analysis in a native-like lipid environment. Cell-free protein synthesis and solid-state NMR spectroscopy are promising approaches in this context, the former with respect to its great potential in the native expression of complex proteins, and the latter for the analysis of membrane proteins in lipids. Herein, we show that milligram amounts of the small envelope protein of the duck hepatitis B virus (DHBV) can be produced by cell-free expression, and that the protein self-assembles into subviral particles. Proton-detected 2D NMR spectra recorded at a magic-angle-spinning frequency of 110 kHz on