Soft X-ray cryo-microscopy/tomography of vitreous samples is becoming a valuable tool in structural cell biology. Within the 'water-window' wavelength region (2.34-4.37nm), it provides absorption contrast images with high signal to noise ratio and resolution of a few tens of nanometer. Soft X-rays with wavelengths close to the K-absorption edge of oxygen penetrate biological samples with thicknesses in the micrometer range. Here, we report on the application of a recently established extension of the transmission soft X-ray cryo-microscope (HZB TXM) at the beamline U41-XM of the BESSY II electron storage ring by an in-column epi-fluorescence and reflected light cryo-microscope. We demonstrate the new capability for correlative fluorescence and soft X-ray cryo-microscopy/tomography of this instrument along a typical life science experimental approach - the correlation of a fluorophore-tagged protein (pUL34-GFP of pseudorabies virus, PrV, the nuclear membrane-anchored component of the nuclear egress complex of the Herpesviridae which interacts with viral pUL31) in PrV pUL34-GFP/pUL31 coexpressing mammalian cells, with virus-induced vesicular structures in the nucleus, expanding the nucleoplasmic reticulum. Taken together, our results demonstrate new possibilities to study the role of specific proteins in substructures of adherent cells, especially of the nucleus in toto, accessible to electron microscopy in thinned samples only.

Correlative fluorescence and soft X-ray cryo-microscopy/tomography on flat sample holders is perfectly suited to study the uncompromised physiological status of adherent cells at its best possible preservation by imaging after fast cryo-immobilization. To understand the mechanism by which herpesviruses induce nucleoplasmic reticulum, i.e. invaginations of the nuclear envelope, during their egress from the host cell nucleus, morphologically similar structures found in laminopathies and after chemical induction were investigated as a potentially more easily accessible model system. For example, anti-retroviral protease inhibitors like Saquinavir also induce invaginations of the nuclear membranes. With the help of newly designed multimodal nanoparticles as alignment and correlation markers, and by optimizing fluorescence cryo-microscopy data acquisition, an elaborate three-dimensional network of nucleoplasmic reticulum was demonstrated in nuclei of Saquinavir-treated rabbit kidney cells expressing a fluorescently labeled inner nuclear membrane protein. In part of the protease inhibitor-treated samples, nuclei exhibited dramatic ultrastructural changes indicative of programmed cell death/apoptosis. This unexpected observation highlights another unique feature of soft X-ray microscopy, i.e. high absorption contrast information not relying on labeled cellular components, at a 3D resolution of approximately 40 nm (half-pitch) and through a sample thickness of several micrometers. These properties make it a valuable part of the cell biology imaging toolbox to visualize the cellular ultrastructure in its completeness.

Background: Somatosensation of the mammalian head is mainly mediated by the trigeminal nerve that provides innervation of diverse tissues like the face skin, the conjunctiva of the eyes, blood vessels and the mucouse membranes of the oral and nasal cavities. Trigeminal perception encompasses thermosensation, touch, and pain. Trigeminal chemosensation from the nasal epithelia mainly evokes stinging, burning, or pungent sensations. In vitro characterization of trigeminal primary sensory neurons derives largely from analysis of complete neuronal populations prepared from sensory ganglia. Thus, functional properties of primary trigeminal afferents depending on the area of innervation remain largely unclear. Results: We established a PrV based tracing technique to identify nasal and cutaneous trigeminal neurons in vitro. This approach allowed analysis and comparison of identified primary afferents by means of electrophysiological and imaging measurement techniques. Neurons were challenged with several agonists that were reported to exhibit specificity for known receptors, including TRP channels and purinergic receptors. In addition, TTX sensitivity of sodium currents and IB4 binding was investigated. Compared with cutaneous neurons, a larger fraction of nasal trigeminal neurons showed sensitivity for menthol and capsaicin. These findings pointed to TRPM8 and TRPV1 receptor protein expression largely in nasal neurons whereas for cutaneous neurons these receptors are present only in a smaller fraction. The majority of nasal neurons lacked P2X₃ receptor-mediated currents but showed P2X₂-mediated responses when stimulated with ATP. Interestingly, cutaneous neurons revealed largely TTX resistant sodium currents. A significantly higher fraction of nasal and cutaneous afferents showed IB4 binding when compared to randomly chosen trigeminal neurons. Conclusion: In conclusion, the usability of PrV mediated tracing of primary afferents was demonstrated. Using this technique it could be shown that compared with neurons innervating the skin nasal trigeminal neurons reveal pronounced chemosensitivity for TRPM8 and TRPV1 channel agonists and only partially meet properties typical for nociceptors. In contrast to P2X₃ receptors, TRPM8 and TRPV1 receptors seem to be of pronounced physiological relevance for intranasal trigeminal sensation.

Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD^–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD^–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD^–Pass in rabbit RK13-sgms1_KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD^–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked.

Envelope glycoprotein (g)B is conserved throughout the Herpesviridae and mediates fusion of the viral envelope with cellular membranes for infectious entry and spread. Like all viral envelope fusion proteins, gB is modified by asparagine (N)-linked glycosylation. Glycans can contribute to protein function, intracellular transport, trafficking, structure and immune evasion. gB of the alphaherpesvirus pseudorabies virus (PrV) contains six consensus sites for N-linked glycosylation, but their functional relevance is unknown. Here, we investigated the occupancy and functional relevance of N-glycosylation sites in PrV gB. To this end, all predicted N-glycosylation sites were inactivated either singly or in combination by the introduction of conservative mutations (N➔Q). The resulting proteins were tested for expression, fusion activity in cell–cell fusion assays and complementation of a gB-deficient PrV mutant. Our results indicate that all six sites are indeed modified. However, while glycosylation at most sites was dispensable for gB expression and fusogenicity, inactivation of N154 and N700 affected gB processing by furin cleavage and surface localization. Although all single mutants were functional in cell–cell fusion and viral entry, simultaneous inactivation of all six N-glycosylation sites severely impaired fusion activity and viral entry, suggesting a critical role of N-glycans for maintaining gB structure and function.

Newly assembled herpesvirus nucleocapsids traverse the intact nuclear envelope by a vesicle-mediated nucleo-cytoplasmic transport for final virion maturation in the cytoplasm. For this, they bud at the inner nuclear membrane resulting in primary enveloped particles in the perinuclear space (PNS) followed by fusion of the primary envelope with the outer nuclear membrane (ONM). While the conserved viral nuclear egress complex orchestrates the first steps, effectors of fusion of the primary virion envelope with the ONM are still mostly enigmatic but might include cellular proteins like SUN2 or ESCRT-III components. Here, we analyzed the influence of the only known AAA+ ATPases located in the endoplasmic reticulum and the PNS, the Torsins (Tor), on nuclear egress of the alphaherpesvirus pseudorabies virus. For this overexpression of wild type and mutant proteins as well as CRISPR/Cas9 genome editing was applied. Neither single overexpression nor gene knockout (KO) of TorA or TorB had a significant impact. However, TorA/B double KO cells showed decreased viral titers at early time points of infection and an accumulation of primary virions in the PNS pointing to a delay in capsid release during nuclear egress.