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, 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.

3D Ex vivo tissue platforms to investigate the early phases of influenza a virus- and SARS-CoV-2-induced respiratory diseases.

Pandemic outbreaks of viruses such as influenza virus or SARS-CoV-2 are associated with high morbidity and mortality and thus pose a massive threat to global health and economics. Physiologically relevant models are needed to study the viral life cycle, describe the pathophysiological consequences of viral infection, and explore possible drug targets and treatment options. While simple cell culture-based models do not reflect the tissue environment and systemic responses, animal models are linked with huge direct and indirect costs and ethical questions. Ex vivo platforms based on tissue explants have been introduced as suitable platforms to bridge the gap between cell culture and animal models. We established a murine lung tissue explant platform for two respiratory viruses, influenza A virus (IAV) and SARS-CoV-2. We observed efficient viral replication, associated with the release of inflammatory cytokines and the induction of an antiviral interferon response, comparable to ex vivo infection in human lung explants. Endolysosomal entry could be confirmed as a potential host target for pharmacological intervention, and the potential repurposing potentials of fluoxetine and interferons for host-directed therapy previously seen in vitro could be recapitulated in the ex vivo model.

The Evolution of Complex Muscle Cell In Vitro Models to Study Pathomechanisms and Drug Development of Neuromuscular Disease.

Many neuromuscular disease entities possess a significant disease burden and therapeutic options remain limited. Innovative human preclinical models may help to uncover relevant disease mechanisms and enhance the translation of therapeutic findings to strengthen neuromuscular disease precision medicine. By concentrating on idiopathic inflammatory muscle disorders, we summarize the recent evolution of the novel in vitro models to study disease mechanisms and therapeutic strategies. A particular focus is laid on the integration and simulation of multicellular interactions of muscle tissue in disease phenotypes in vitro. Finally, the requirements of a neuromuscular disease drug development workflow are discussed with a particular emphasis on cell sources, co-culture systems (including organoids), functionality, and throughput.

Generation of Human Lung Organoid Cultures from Healthy and Tumor Tissue to Study Infectious Diseases.

Respiratory viruses cause mild to severe diseases in humans every year, constituting a major public health problem. Characterizing the pathogenesis in physiologically relevant models is crucial for developing efficient vaccines and therapeutics. Here, we show that lung organoids derived from human primary or lung tumor tissue maintain the cellular composition and characteristics of the original tissue. Moreover, we show that these organoids sustain viral replication with particular infection foci formation, and they activate the expression of interferon-associated and proinflammatory genes responsible for mediating a robust innate immune response. All together, we show that three-dimensional (3D) lung organoids constitute a relevant platform to model diseases and enable the development of drug screenings. IMPORTANCE Three-dimensional (3D) human lung organoids reflect the native cell composition of the lung as well as its physiological properties. Human 3D lung organoids offer ideal conditions, such as timely availability in large quantities and high physiological relevance for reassessment and prediction of disease outbreaks of respiratory pathogens and pathogens that use the lung as a primary entry portal. Human lung organoids can be used in basic research and diagnostic settings as early warning cell culture systems and also serve as a relevant platform for modeling infectious diseases and drug development. They can be used to characterize pathogens and analyze the influence of infection on, for example, immunological parameters, such as the expression of interferon-associated and proinflammatory genes in the context of cancer. In our study, we found that cancer-derived lung organoids were more sensitive to influenza A virus infection than those derived from healthy tissue and demonstrated a decreased innate immune response.

Henipaviruses-A constant threat to livestock and humans.

In this review, we highlight the risk to livestock and humans from infections with henipaviruses, which belong to the virus family Paramyxoviridae. We provide a comprehensive overview of documented outbreaks of Nipah and Hendra virus infections affecting livestock and humans and assess the burden on the economy and health systems. In an increasingly globalized and interconnected world, attention must be paid to emerging viruses and infectious diseases, as transmission routes can be rapid and worldwide.

Pharmacologically induced endolysosomal cholesterol imbalance through clinically licensed drugs itraconazole and fluoxetine impairs Ebola virus infection in vitro.

Ebola virus disease (EVD) is a severe and frequently lethal disease caused by Ebola virus (EBOV). The latest occasional EVD outbreak (2013-2016) in Western African, which was accompanied by a high fatality rate, showed the great potential of epidemic and pandemic spread. Antiviral therapies against EBOV are very limited, strain-dependent (only antibody therapies are available) and mostly restricted to symptomatic treatment, illustrating the urgent need for novel antiviral strategies. Thus, we evaluated the effect of the clinically widely used antifungal itraconazole and the antidepressant fluoxetine for a repurposing against EBOV infection. While itraconazole, similar to U18666A, directly binds to and inhibits the endosomal membrane protein Niemann-Pick C1 (NPC1), fluoxetine, which belongs to the structurally unrelated group of weakly basic, amphiphile so-called "functional inhibitors of acid sphingomyelinase" (FIASMA) indirectly acts on the lysosome-residing acid sphingomyelinase via enzyme detachment leading to subsequent lysosomal degradation. Both, the drug-induced endolysosomal cholesterol accumulation and the altered endolysosomal pH, might interfere with the fusion of viral and endolysosomal membrane, preventing infection with EBOV. We further provide evidence that cholesterol imbalance is a conserved cross-species mechanism to hamper EBOV infection. Thus, exploring the endolysosomal host-pathogen interface as a suitable antiviral treatment may offer a general strategy to combat EBOV infection.

The native structure of the assembled matrix protein 1 of influenza A virus.

Influenza A virus causes millions of severe cases of disease during annual epidemics. The most abundant protein in influenza virions is matrix protein 1 (M1), which mediates virus assembly by forming an endoskeleton beneath the virus membrane1. The structure of full-length M1, and how it oligomerizes to mediate the assembly of virions, is unknown. Here we determine the complete structure of assembled M1 within intact virus particles, as well as the structure of M1 oligomers reconstituted in vitro. We find that the C-terminal domain of M1 is disordered in solution but can fold and bind in trans to the N-terminal domain of another M1 monomer, thus polymerizing M1 into linear strands that coat the interior surface of the membrane of the assembling virion. In the M1 polymer, five histidine residues-contributed by three different monomers of M1-form a cluster that can serve as the pH-sensitive disassembly switch after entry into a target cell. These structures therefore reveal mechanisms of influenza virus assembly and disassembly.

NSs amyloid formation is associated with the virulence of Rift Valley fever virus in mice.

Amyloid fibrils result from the aggregation of host cell-encoded proteins, many giving rise to specific human illnesses such as Alzheimer's disease. Here we show that the major virulence factor of Rift Valley fever virus, the protein NSs, forms filamentous structures in the brain of mice and affects mortality. NSs assembles into nuclear and cytosolic disulfide bond-dependent fibrillary aggregates in infected cells. NSs structural arrangements exhibit characteristics typical for amyloids, such as an ultrastructure of 12 nm-width fibrils, a strong detergent resistance, and interactions with the amyloid-binding dye Thioflavin-S. The assembly dynamics of viral amyloid-like fibrils can be visualized in real-time. They form spontaneously and grow in an amyloid fashion within 5 hours. Together, our results demonstrate that viruses can encode amyloid-like fibril-forming proteins and have strong implications for future research on amyloid aggregation and toxicity in general.

IFITM3 Clusters on Virus Containing Endosomes and Lysosomes Early in the Influenza A Infection of Human Airway Epithelial Cells.

Interferon-induced transmembrane proteins (IFITMs) have been shown to strongly affect influenza A virus (IAV) infectivity in tissue culture. Moreover, polymorphisms in IFITM3 have been associated with the severity of the disease in humans. IFITM3 appears to act early in the infection, but its mechanism of action and potential interactions with incoming IAV structures are not yet defined. Here, we visualized endogenous IFITM3 interactions with IAV in the human lung epithelial cell line A549 and in primary human airway epithelial cells employing stimulated emission depletion super-resolution microscopy. By applying an iterative approach for the cluster definition and computational cluster analysis, we found that IFITM3 reorganizes into clusters as IAV infection progresses. IFITM3 cluster formation started at 2-3 h post infection and increased over time to finally coat IAV-containing endosomal vesicles. This IAV-induced phenotype was due to the endosomal recruitment of IFITM3 rather than to an overall increase in the IFITM3 abundance. While the IAV-induced IFITM3 clustering and localization to endosomal vesicles was comparable in primary human airway epithelial cells and the human lung epithelial cell line A549, the endogenous IFITM3 signal was higher in primary cells. Moreover, we observed IFITM3 signals adjacent to IAV-containing recycling endosomes.

Combination of microdissection and single cell quantitative real-time PCR revealed intercellular mitochondrial DNA heterogeneities in fibroblasts of Kearns-Sayre syndrome patients.

Kearns-Sayre syndrome (KSS) is a multisystemic disorder marked by aerobic cell metabolism dysfunction. Fibroblasts derived from KSS patient skin biopsy exhibit heterogeneous occurrence of mitochondrial genomes as those circular DNA molecules partially carry the common deletion. In our approach, we aim to evaluate the intercellular alterations in respect to mitochondrial DNA integrity by laser capture microdissection and multiplex quantitative real-time PCR in single cells. The obtained results give new insights into the understanding of mitochondrial genetics, e.g. postulated sorting of damaged mitochondria, and heterogeneity of cells. Further, we discuss the relevance of intercellular heterogeneities for human mitochondrial disorders in general.

Clathrin-adaptor ratio and membrane tension regulate the flat-to-curved transition of the clathrin coat during endocytosis.

Although essential for many cellular processes, the sequence of structural and molecular events during clathrin-mediated endocytosis remains elusive. While it was long believed that clathrin-coated pits grow with a constant curvature, it was recently suggested that clathrin first assembles to form flat structures that then bend while maintaining a constant surface area. Here, we combine correlative electron and light microscopy and mathematical growth laws to study the ultrastructural rearrangements of the clathrin coat during endocytosis in BSC-1 mammalian cells. We confirm that clathrin coats initially grow flat and demonstrate that curvature begins when around 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 notion that BSC-1 mammalian cells dynamically regulate the flat-to-curved transition in clathrin-mediated endocytosis by both biochemical and mechanical factors.

Structural Analysis of the Roles of Influenza A Virus Membrane-Associated Proteins in Assembly and Morphology.

UNLABELLED: The assembly of influenza A virus at the plasma membrane of infected cells leads to release of enveloped virions that are typically round in tissue culture-adapted strains but filamentous in strains isolated from patients. The viral proteins hemagglutinin (HA), neuraminidase (NA), matrix protein 1 (M1), and M2 ion channel all contribute to virus assembly. When expressed individually or in combination in cells, they can all, under certain conditions, mediate release of membrane-enveloped particles, but their relative roles in virus assembly, release, and morphology remain unclear. To investigate these roles, we produced membrane-enveloped particles by plasmid-derived expression of combinations of HA, NA, and M proteins (M1 and M2) or by infection with influenza A virus. We monitored particle release, particle morphology, and plasma membrane morphology by using biochemical methods, electron microscopy, electron tomography, and cryo-electron tomography. Our data suggest that HA, NA, or HANA (HA plus NA) expression leads to particle release through nonspecific induction of membrane curvature. In contrast, coexpression with the M proteins clusters the glycoproteins into filamentous membrane protrusions, which can be released as particles by formation of a constricted neck at the base. HA and NA are preferentially distributed to differently curved membranes within these particles. Both the budding intermediates and the released particles are morphologically similar to those produced during infection with influenza A virus. Together, our data provide new insights into influenza virus assembly and show that the M segment together with either of the glycoproteins is the minimal requirement to assemble and release membrane-enveloped particles that are truly virus-like. IMPORTANCE: Influenza A virus is a major respiratory pathogen. It assembles membrane-enveloped virus particles whose shapes vary from spherical to filamentous. Here we examine the roles of individual viral proteins in mediating virus assembly and determining virus shape. To do this, we used a range of electron microscopy techniques to obtain and compare two- and three-dimensional images of virus particles and virus-like particles during and after assembly. The virus-like particles were produced using different combinations of viral proteins. Among our results, we found that coexpression of one or both of the viral surface proteins (hemagglutinin and neuraminidase) with the viral membrane-associated proteins encoded by the M segment results in assembly and release of filamentous virus-like particles in a manner very similar to that of the budding and release of influenza virions. These data provide novel insights into the roles played by individual viral proteins in influenza A virus assembly.

Alteration of protein levels during influenza virus H1N1 infection in host cells: a proteomic survey of host and virus reveals differential dynamics.

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co-regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level.

Sequence-specific imaging of influenza A mRNA in living infected cells using fluorescent FIT-PNA.

Significant efforts have been devoted to the development of techniques allowing the investigation of viral mRNA progression during the replication cycle. We herein describe the use of sequence-specific FIT-PNA (Forced Intercalation Peptide Nucleic Acids) probes which contain a single intercalator serving as an artificial fluorescent nucleobase. FIT-PNA probes are not degraded by enzymes, neither by nucleases nor by proteases, and provide for both high sensitivity and high target specificity under physiological conditions inside the infected living host cell.

PNA FIT-probes for the dual color imaging of two viral mRNA targets in influenza H1N1 infected live cells.

Fluorogenic hybridization probes that allow RNA imaging provide information as to how the synthesis and transport of particular RNA molecules is orchestrated in living cells. In this study, we explored the peptide nucleic acid (PNA)-based FIT-probes in the simultaneous imaging of two different viral mRNA molecules expressed during the replication cycle of the H1N1 influenza A virus. PNA FIT-probes are non-nucleotidic, nonstructured probes and contain a single asymmetric cyanine dye which serves as a fluorescent base surrogate. The fluorochrome acts as a local intercalator probe and reports hybridization of target DNA/RNA by enhancement of fluorescence. Though multiplexed hybridization probes are expected to facilitate the analysis of RNA expression, there are no previous reports on the dual color imaging of two different viral mRNA targets. In this work, we developed a set of two differently colored PNA FIT-probes that allow the spectrally resolved imaging of mRNA coding for neuraminidase (NA) and matrix protein 1 (M1); proteins which execute distinct functions during the replication of the influenza A virus. The probes are characterized by a wide range of applicable hybridization temperatures. The same probe sequence enabled live-cell RNA imaging (at 37 °C) as well as real-time PCR measurements (at 60 °C annealing temperature). This facilitated a comprehensive analysis of RNA expression by quantitative (qPCR) and qualitative (imaging) means. Confocal laser scanning microscopy showed that the viral-RNA specific PNA FIT-probes neither stained noninfected cells nor cells infected by a control virus. The joint use of differently colored PNA FIT-probes in this feasibility study revealed significant differences in the expression pattern of influenza H1N1 mRNAs coding for NA or M1. These experiments provide evidence for the usefulness of PNA FIT-probes in investigations on the temporal and spatial progression of mRNA synthesis in living cells for two mRNA species.