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  • 1. Alberione, Maria Pia
    et al.
    Moeller, Rebecca
    Kirui, Jared
    Ginkel, Corinne
    Doepke, Mandy
    Stroeh, Luisa J.
    Machtens, Jan-Philipp
    Pietschmann, Thomas
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Single-nucleotide variants in human CD81 influence hepatitis C virus infection of hepatoma cells2020In: Medical Microbiology and Immmunology, ISSN 0300-8584, E-ISSN 1432-1831, Vol. 209, no 4, p. 499-514Article in journal (Refereed)
    Abstract [en]

    An estimated number of 71 million people are living with chronic hepatitis C virus (HCV) infection worldwide and 400,000 annual deaths are related to the infection. HCV entry into the hepatocytes is complex and involves several host factors. The tetraspanin human CD81 (hCD81) is one of the four essential entry factors and is composed of one large extracellular loop, one small extracellular loop, four transmembrane domains, one intracellular loop and two intracellular tails. The large extracellular loop interacts with the E2 glycoprotein of HCV. Regions outside the large extracellular loop (backbone) of hCD81 have a critical role in post-binding entry steps and determine susceptibility of hepatocytes to HCV. Here, we investigated the effect of five non-synonymous single-nucleotide variants in the backbone of hCD81 on HCV susceptibility. We generated cell lines that stably express the hCD81 variants and infected the cells using HCV pseudoparticles and cell culture-derived HCV. Our results show that all the tested hCD81 variants support HCV pseudoparticle entry with similar efficiency as wild-type hCD81. In contrast, variants A54V, V211M and M220I are less supportive to cell culture-derived HCV infection. This altered susceptibility is HCV genotype dependent and specifically affected the cell entry step. Our findings identify three hCD81 genetic variants that are impaired in their function as HCV host factors for specific viral genotypes. This study provides additional evidence that genetic host variation contributes to inter-individual differences in HCV infection and outcome.

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  • 2. Banse, Pia
    et al.
    Moeller, Rebecca
    Bruening, Janina
    Lasswitz, Lisa
    Kahl, Sina
    Khan, Abdul G
    Marcotrigiano, Joseph
    Pietschmann, Thomas
    Gerold, Gisa
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, 30625 Hannover, Germany.
    CD81 receptor regions outside the large extracellular loop determine hepatitis C virus entry into hepatoma cells2018In: Viruses, E-ISSN 1999-4915, Vol. 10, no 4, article id 207Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) enters human hepatocytes using four essential entry factors, one of which is human CD81 (hCD81). The tetraspanin hCD81 contains a large extracellular loop (LEL), which interacts with the E2 glycoprotein of HCV. The role of the non-LEL regions of hCD81 (intracellular tails, four transmembrane domains, small extracellular loop and intracellular loop) is poorly understood. Here, we studied the contribution of these domains to HCV susceptibility of hepatoma cells by generating chimeras of related tetraspanins with the hCD81 LEL. Our results show that non-LEL regions in addition to the LEL determine susceptibility of cells to HCV. While closely related tetraspanins (X. tropicalis CD81 and D. rerio CD81) functionally complement hCD81 non-LEL regions, distantly related tetraspanins (C. elegans TSP9 amd D. melanogaster TSP96F) do not and tetraspanins with intermediate homology (hCD9) show an intermediate phenotype. Tetraspanin homology and susceptibility to HCV correlate positively. For some chimeras, infectivity correlates with surface expression. In contrast, the hCD9 chimera is fully surface expressed, binds HCV E2 glycoprotein but is impaired in HCV receptor function. We demonstrate that a cholesterol-coordinating glutamate residue in CD81, which hCD9 lacks, promotes HCV infection. This work highlights the hCD81 non-LEL regions as additional HCV susceptibility-determining factors.

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  • 3.
    Becker, Miriam
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Department of Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Conca, Dario Valter
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Dorma, Noemi
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Mistry, Nitesh
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Hahlin, Elin
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Frängsmyr, Lars
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bally, Marta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Department of Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Efficient clathrin-mediated entry of enteric adenoviruses in human duodenal cells2023In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 97, no 10Article in journal (Refereed)
    Abstract [en]

    Enteric adenovirus types F40 and 41 (EAdVs) are a leading cause of diarrhea and diarrhea-associated death in young children and have recently been proposed to cause acute hepatitis in children. EAdVs have a unique capsid architecture and exhibit — unlike other human adenoviruses — a relatively strict tropism for gastrointestinal tissues with, to date, understudied infection mechanism and unknown target cells. In this study, we turn to potentially limiting host factors by comparing EAdV entry in cell lines with respiratory and intestinal origin by cellular perturbation, virus particle tracking, and transmission electron microscopy. Our analyses highlight kinetic advantages for EAdVs in duodenal HuTu80 cell infection and reveal a larger fraction of mobile particles, faster virus uptake, and infectious particle entry in intestinal cells. Moreover, EAdVs display a dependence on clathrin- and dynamin-dependent pathways in intestinal cells. Detailed knowledge of virus entry routes and host factor requirements is essential to understanding pathogenesis and developing new countermeasures. Hence, this study provides novel insights into the entry mechanisms of a medically important virus with emerging tropism in a cell line originating from a relevant tissue. IMPORTANCE Enteric adenoviruses have historically been difficult to grow in cell culture, which has resulted in lack of knowledge of host factors and pathways required for infection of these medically relevant viruses. Previous studies in non-intestinal cell lines showed slow infection kinetics and generated comparatively low virus yields compared to other adenovirus types. We suggest duodenum-derived HuTu80 cells as a superior cell line for studies to complement efforts using complex intestinal tissue models. We show that viral host cell factors required for virus entry differ between cell lines from distinct origins and demonstrate the importance of clathrin-mediated endocytosis.

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  • 4. Brown, Richard J. P.
    et al.
    Tegtmeyer, Birthe
    Sheldon, Julie
    Khera, Tanvi
    Anggakusuma,
    Todt, Daniel
    Vieyres, Gabrielle
    Weller, Romy
    Joecks, Sebastian
    Zhang, Yudi
    Sake, Svenja
    Bankwitz, Dorothea
    Welsch, Kathrin
    Ginkel, Corinne
    Engelmann, Michael
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Department of Physiological Chemistry, University of Veterinary Medicine Hannover, Hannover, Germany.
    Steinmann, Eike
    Yuan, Qinggong
    Ott, Michael
    Vondran, Florian W. R.
    Krey, Thomas
    Ströh, Luisa J.
    Miskey, Csaba
    Ivics, Zoltán
    Herder, Vanessa
    Baumgärtner, Wolfgang
    Lauber, Chris
    Seifert, Michael
    Tarr, Alexander W.
    McClure, C. Patrick
    Randall, Glenn
    Baktash, Yasmine
    Ploss, Alexander
    Thi, Viet Loan Dao
    Michailidis, Eleftherios
    Saeed, Mohsan
    Verhoye, Lieven
    Meuleman, Philip
    Goedecke, Natascha
    Wirth, Dagmar
    Rice, Charles M.
    Pietschmann, Thomas
    Liver-expressed Cd302 and Cr1l limit hepatitis C virus cross-species transmission to mice2020In: Science Advances, E-ISSN 2375-2548, Vol. 6, no 45, article id eabd3233Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma cells impeded HCV uptake and cooperatively mediated transcriptional dysregulation of a noncanonical program of immunity genes. Murine hepatocyte expression of both factors was constitutive and not interferon inducible, while differences in liver expression and the ability to restrict HCV were observed between the murine orthologs and their human counterparts. Genetic ablation of endogenous Cd302 expression in human HCV entry factor transgenic mice increased hepatocyte permissiveness for an adapted HCV strain and dysregulated expression of metabolic process and host defense genes. These findings highlight human-mouse differences in liver-intrinsic antiviral immunity and facilitate the development of next-generation murine models for preclinical testing of HCV vaccine candidates.

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  • 5. Bruening, Janina
    et al.
    Lasswitz, Lisa
    Banse, Pia
    Kahl, Sina
    Marinach, Carine
    Vondran, Florian W.
    Kaderali, Lars
    Silvie, Olivier
    Pietschmann, Thomas
    Meissner, Felix
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Insitute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Hepatitis C virus enters liver cells using the CD81 receptor complex proteins calpain-5 and CBLB2018In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 14, no 7, article id e1007111Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) and the malaria parasite Plasmodium use the membrane protein CD81 to invade human liver cells. Here we mapped 33 host protein interactions of CD81 in primary human liver and hepatoma cells using high-resolution quantitative proteomics. In the CD81 protein network, we identified five proteins which are HCV entry factors or facilitators including epidermal growth factor receptor (EGFR). Notably, we discovered calpain-5 (CAPN5) and the ubiquitin ligase Casitas B-lineage lymphoma proto-oncogene B (CBLB) to form a complex with CD81 and support HCV entry. CAPN5 and CBLB were required for a post-binding and pre-replication step in the HCV life cycle. Knockout of CAPN5 and CBLB reduced susceptibility to all tested HCV genotypes, but not to other enveloped viruses such as vesicular stomatitis virus and human coronavirus. Furthermore, Plasmodium sporozoites relied on a distinct set of CD81 interaction partners for liver cell entry. Our findings reveal a comprehensive CD81 network in human liver cells and show that HCV and Plasmodium highjack selective CD81 interactions, including CAPN5 and CBLB for HCV, to invade cells.

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  • 6. Bruening, Janina
    et al.
    Weigel, Bettina
    Gerold, Gisa
    Institute for Experimental Virology, Centre for Experimental and Clinical Infection Research (TWINCORE), Hannover, Germany.
    The Role of Type III Interferons in Hepatitis C Virus Infection and Therapy2017In: Journal of Immunology Research, ISSN 2314-8861, E-ISSN 2314-7156, article id 7232361Article in journal (Refereed)
    Abstract [en]

    The human interferon (IFN) response is a key innate immune mechanism to fight virus infection. IFNs are host-encoded secreted proteins, which induce IFN-stimulated genes (ISGs) with antiviral properties. Among the three classes of IFNs, type III IFNs, also called IFN lambdas (IFNLs), are an essential component of the innate immune response to hepatitis C virus (HCV). In particular, human polymorphisms in IFNL gene loci correlate with hepatitis C disease progression and with treatment response. To date, the underlying mechanisms remain mostly elusive; however it seems clear that viral infection of the liver induces IFNL responses. As IFNL receptors show a more restricted tissue expression than receptors for other classes of IFNs, IFNL treatment has reduced side effects compared to the classical type I IFN treatment. In HCV therapy, however, IFNL will likely not play an important role as highly effective direct acting antivirals (DAA) exist. Here, we will review our current knowledge on IFNL gene expression, protein properties, signaling, ISG induction, and its implications on HCV infection and treatment. Finally, we will discuss the lessons learnt from the HCV and IFNL field for virus infections beyond hepatitis C.

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  • 7.
    Carriquí-Madroñal, Belén
    et al.
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany.
    Lasswitz, Lisa
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany.
    von Hahn, Thomas
    Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hannover, Germany; Department of Gastroenterology, Hepatology and Interventional Endoscopy, Asklepios Hospital Barmbek, Semmelweis University, Campus Hamburg, Hamburg, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, the Medical School Hannover/the Helmholtz Centre for Infection Research, Hanover, Germany.
    Genetic and pharmacological perturbation of hepatitis-C virus entry2023In: Current Opinion in Virology, ISSN 1879-6257, E-ISSN 1879-6265, Vol. 62, article id 101362Article, review/survey (Refereed)
    Abstract [en]

    Hepatitis-C virus (HCV) chronically infects 58 million individuals worldwide with variable disease outcome. While a subfraction of individuals exposed to the virus clear the infection, the majority develop chronic infection if untreated. Another subfraction of chronically ill proceeds to severe liver disease. The underlying causes of this interindividual variability include genetic polymorphisms in interferon genes. Here, we review available data on the influence of genetic or pharmacological perturbation of HCV host dependency factors on the clinically observed interindividual differences in disease outcome. We focus on host factors mediating virus entry into human liver cells. We assess available data on genetic variants of the major entry factors scavenger receptor class-B type I, CD81, claudin-1, and occludin as well as pharmacological perturbation of these entry factors. We review cell culture experimental and clinical cohort study data and conclude that entry factor perturbation may contribute to disease outcome of hepatitis C.

  • 8.
    Carriquí-Madroñal, Belén
    et al.
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hanover, Germany.
    Sheldon, Julie
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany.
    Duven, Mara
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hanover, Germany.
    Stegmann, Cora
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hanover, Germany.
    Cirksena, Karsten
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hanover, Germany.
    Wyler, Emanuel
    Max Delbrück Center for Molecular Medicine in the Helmholtz Association (MDC), Berlin Institute for Medical Systems Biology (BIMSB), Berlin, Germany.
    Zapatero-Belinchón, Francisco J.
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hanover, Germany; Gladstone Institutes, CA, San Francisco, United States.
    Vondran, Florian W. R.
    Department of General, Visceral and Transplant Surgery, Regenerative Medicine and Experimental Surgery, Hannover Medical School, Hannover, Germany; German Center for Infection Research Partner Site Hannover-Braunschweig Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hanover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany.
    The matrix metalloproteinase ADAM10 supports hepatitis C virus entry and cell-to-cell spread via its sheddase activity2023In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 19, no 11, article id e1011759Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) exploits the four entry factors CD81, scavenger receptor class B type I (SR-BI, also known as SCARB1), occludin, and claudin-1 as well as the co-factor epidermal growth factor receptor (EGFR) to infect human hepatocytes. Here, we report that the disintegrin and matrix metalloproteinase 10 (ADAM10) associates with CD81, SR-BI, and EGFR and acts as HCV host factor. Pharmacological inhibition, siRNA-mediated silencing and genetic ablation of ADAM10 reduced HCV infection. ADAM10 was dispensable for HCV replication but supported HCV entry and cell-to-cell spread. Substrates of the ADAM10 sheddase including epidermal growth factor (EGF) and E-cadherin, which activate EGFR family members, rescued HCV infection of ADAM10 knockout cells. ADAM10 did not influence infection with other enveloped RNA viruses such as alphaviruses and a common cold coronavirus. Collectively, our study reveals a critical role for the sheddase ADAM10 as a HCV host factor, contributing to EGFR family member transactivation and as a consequence to HCV uptake.

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  • 9. de Diego, J L
    et al.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Zychlinsky, A
    Sensing, presenting, and regulating PAMPs.2007In: Ernst Schering Foundation symposium proceedings, no 3, p. 83-95Article in journal (Refereed)
    Abstract [en]

    Recognition of microbial infection and initiation of immune responses are controlled by multiple mechanisms. Toll-like receptors (TLRs) are key components of the innate immune system that detect microbial infection. TLR activation helps to eliminate the invading pathogens, coordinate systemic defenses, and initiate adaptive immune responses. Despite progress elucidating the TLR signaling aspects and the physiological relevance of TLRs in microbial infections, the molecular basis of microbial recognition by TLRs is still not fully understood. In this article we focus on the availability of microbial ligands to regulate presentation to TLRs and assist in our understanding of TLR-mediated microbial recognition.

  • 10.
    Dong, Thi Ngan
    et al.
    L3S Research Center, Leibniz University Hannover, Hannover, Germany.
    Brogden, Graham
    Institute for Biochemistry, University of Veterinary Medicine, Hannover, Germany; Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Institute for Biochemistry, University of Veterinary Medicine, Hannover, Germany; Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hannover, Germany.
    Khosla, Megha
    L3S Research Center, Leibniz University Hannover, Hannover, Germany.
    A multitask transfer learning framework for the prediction of virus-human protein–protein interactions2021In: BMC Bioinformatics, E-ISSN 1471-2105, Vol. 22, no 1, article id 572Article in journal (Refereed)
    Abstract [en]

    Background: Viral infections are causing significant morbidity and mortality worldwide. Understanding the interaction patterns between a particular virus and human proteins plays a crucial role in unveiling the underlying mechanism of viral infection and pathogenesis. This could further help in prevention and treatment of virus-related diseases. However, the task of predicting protein–protein interactions between a new virus and human cells is extremely challenging due to scarce data on virus-human interactions and fast mutation rates of most viruses.

    Results: We developed a multitask transfer learning approach that exploits the information of around 24 million protein sequences and the interaction patterns from the human interactome to counter the problem of small training datasets. Instead of using hand-crafted protein features, we utilize statistically rich protein representations learned by a deep language modeling approach from a massive source of protein sequences. Additionally, we employ an additional objective which aims to maximize the probability of observing human protein–protein interactions. This additional task objective acts as a regularizer and also allows to incorporate domain knowledge to inform the virus-human protein–protein interaction prediction model.

    Conclusions: Our approach achieved competitive results on 13 benchmark datasets and the case study for the SARS-CoV-2 virus receptor. Experimental results show that our proposed model works effectively for both virus-human and bacteria-human protein–protein interaction prediction tasks. We share our code for reproducibility and future research at https://git.l3s.uni-hannover.de/dong/multitask-transfer.

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  • 11. Fedeli, Chiara
    et al.
    Torriani, Giulia
    Galan-Navarro, Clara
    Moraz, Marie-Laurence
    Moreno, Hector
    Gerold, Gisa
    TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Kunz, Stefan
    Axl can serve as entry factor for lassa virus depending on the functional glycosylation of dystroglycan2018In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 92, no 5, article id e01613-17Article in journal (Refereed)
    Abstract [en]

    The highly pathogenic arenavirus Lassa virus (LASV) represents a serious public health problem in Africa. Although the principal LASV receptor, dystroglycan (DG), is ubiquitously expressed, virus binding critically depends on DG's posttranslational modification, which does not always correlate with tissue tropism. The broadly expressed phosphatidylserine receptor Axl was recently identified as an alternative LASV receptor candidate, but its role in LASV entry is unclear. Here, we investigate the exact role of Axl in LASV entry as a function of DG's posttranslational modification. We found that in the absence of functional DG, Axl can mediate LASV entry via apoptotic mimicry. Productive entry requires virus-induced receptor activation, involves macropinocytosis, and critically depends on LAMP-1. In endothelial cells that express low levels of glycosylated DG, both receptors can promote LASV entry. In sum, our study defines the roles of Axl in LASV entry and provides a rationale for targeting Axl in antiviral therapy.

  • 12.
    Gerold, Gisa
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Abu Ajaj, Khalid
    Bienert, Michael
    Laws, Hans-Jürgen
    Zychlinsky, Arturo
    de Diego, Juana L
    A Toll-like receptor 2-integrin beta3 complex senses bacterial lipopeptides via vitronectin.2008In: Nature Immunology, ISSN 1529-2908, E-ISSN 1529-2916, Vol. 9, no 7, p. 761-8Article in journal (Refereed)
    Abstract [en]

    Toll-like receptor 2 (TLR2) initiates inflammation in response to bacterial lipopeptide (BLP). However, the molecular mechanisms enabling the detection of BLP by TLR2 are unknown. Here we investigated the interaction of BLP with human serum proteins and identified vitronectin as a BLP-recognition molecule. Vitronectin and its receptor, integrin beta(3), were required for BLP-induced TLR2-mediated activation of human monocytes. Furthermore, monocytes from patients with Glanzmann thrombasthenia, which lack integrin beta(3), were completely unresponsive to BLP. In addition, integrin beta(3) formed a complex with TLR2 and this complex dissociated after BLP stimulation. Notably, vitronectin and integrin beta(3) coordinated responses to other TLR2 agonists such as lipoteichoic acid and zymosan. Our findings show that vitronectin and integrin beta(3) contribute to the initiation of TLR2 responses.

  • 13.
    Gerold, Gisa
    et al.
    Insitute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, A joint Venture Between the Medical School Hannover and the Helmholtz Centre for Infection Research, 30652 Hannover, Germany.
    Bruening, Janina
    Pietschmann, Thomas
    Decoding protein networks during virus entry by quantitative proteomics2016In: Virus Research, ISSN 0168-1702, E-ISSN 1872-7492, Vol. 218, p. 25-39Article in journal (Refereed)
    Abstract [en]

    Virus entry into host cells relies on interactions between viral and host structures including lipids, carbohydrates and proteins. Particularly, protein-protein interactions between viral surface proteins and host proteins as well as secondary host protein-protein interactions play a pivotal role in coordinating virus binding and uptake. These interactions are dynamic and frequently involve multiprotein complexes. In the past decade mass spectrometry based proteomics methods have reached sensitivities and high throughput compatibilities of genomics methods and now allow the reliable quantitation of proteins in complex samples from limited material. As proteomics provides essential information on the biologically active entity namely the protein, including its posttranslational modifications and its interactions with other proteins, it is an indispensable method in the virologist's toolbox. Here we review protein interactions during virus entry and compare classical biochemical methods to study entry with novel technically advanced quantitative proteomics techniques. We highlight the value of quantitative proteomics in mapping functional virus entry networks, discuss the benefits and limitations and illustrate how the methodology will help resolve unsettled questions in virus entry research in the future.

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  • 14.
    Gerold, Gisa
    et al.
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Bruening, Janina
    Weigel, Bettina
    Pietschmann, Thomas
    Protein Interactions during the Flavivirus and Hepacivirus Life Cycle2017In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 16, no 4, p. 75-91Article in journal (Refereed)
    Abstract [en]

    interaction proteomics and why we believe these challenges should be met.

  • 15. Gerold, Gisa
    et al.
    Meissner, Felix
    Bruening, Janina
    Welsch, Kathrin
    Perin, Paula M
    Baumert, Thomas F
    Vondran, Florian W
    Kaderali, Lars
    Marcotrigiano, Joseph
    Khan, Abdul G
    Mann, Matthias
    Rice, Charles M
    Pietschmann, Thomas
    Quantitative Proteomics Identifies Serum Response Factor Binding Protein 1 as a Host Factor for Hepatitis C Virus Entry2015In: Cell Reports, E-ISSN 2211-1247, Vol. 12, no 5, p. 864-878, article id S2211-1247(15)00689-0Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) enters human hepatocytes through a multistep mechanism involving, among other host proteins, the virus receptor CD81. How CD81 governs HCV entry is poorly characterized, and CD81 protein interactions after virus binding remain elusive. We have developed a quantitative proteomics protocol to identify HCV-triggered CD81 interactions and found 26 dynamic binding partners. At least six of these proteins promote HCV infection, as indicated by RNAi. We further characterized serum response factor binding protein 1 (SRFBP1), which is recruited to CD81 during HCV uptake and supports HCV infection in hepatoma cells and primary human hepatocytes. SRFBP1 facilitates host cell penetration by all seven HCV genotypes, but not of vesicular stomatitis virus and human coronavirus. Thus, SRFBP1 is an HCV-specific, pan-genotypic host entry factor. These results demonstrate the use of quantitative proteomics to elucidate pathogen entry and underscore the importance of host protein-protein interactions during HCV invasion.

  • 16.
    Gerold, Gisa
    et al.
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. TWINCORE, Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Moeller, Rebecca
    Pietschmann, Thomas
    Hepatitis C Virus Entry: Protein Interactions and Fusion Determinants Governing Productive Hepatocyte Invasion2020In: Cold Spring Harbor Perspectives in Medicine, E-ISSN 2157-1422, Vol. 10, no 2, article id a036830Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) entry is among the best-studied uptake processes for human pathogenic viruses. Uptake follows a spatially and temporally tightly controlled program. Numerous host factors including proteins, lipids, and glycans promote productive uptake of HCV particles into human liver cells. The virus initially attaches to surface proteoglycans, lipid receptors such as the scavenger receptor BI (SR-BI), and to the tetraspanin CD81. After lateral translocation of virions to tight junctions, claudin-1 (CLDN1) and occludin (OCLN) are essential for entry. Clathrin-mediated endocytosis engulfs HCV particles, which fuse with endosoma I membranes after pH drop. Uncoating of the viral RNA genome in the cytoplasm completes the entry process. Here we systematically review and classify HCV entry factors by their mechanistic role, relevance, and level of evidence. Finally, we report on more recent knowledge on determinants of membrane fusion and close with an outlook on future implications of HCV entry research.

  • 17.
    Gerold, Gisa
    et al.
    Center for Experimental and Clinical Infection Research Institute of Experimental Virology, TWINCORE Hannover Germany.
    Pietschmann, Thomas
    A circuit of paracrine signals between liver sinusoid endothelial cells and hepatocytes regulates hepatitis C virus replication2014In: Hepatology, ISSN 0270-9139, E-ISSN 1527-3350, Vol. 59, no 2, p. 363-5Article in journal (Refereed)
  • 18.
    Gerold, Gisa
    et al.
    Institute of Experimental Virology Twincore – Center for Experimental and Clinical Infectious Disease Research Hannover, Germany.
    Pietschmann, Thomas
    Hepatitis C virus NS5B polymerase primes innate immune signaling2013In: Hepatology, ISSN 0270-9139, E-ISSN 1527-3350, Vol. 57, no 3, p. 1275-1277Article in journal (Refereed)
    Abstract [en]

    Innate immunity controls pathogen replication and spread. Yet, certain pathogens, such as Hepatitis C Virus (HCV), escape immune elimination and establish persistent infections that promote chronic inflammation and related diseases. Whereas HCV regulatory proteins that attenuate antiviral responses are known, those that promote inflammation and liver injury remain to be identified. Here, we show that transient expression of HCV RNA-dependent RNA polymerase (RdRp), NS5B, in mouse liver and human hepatocytes results in production of small RNA species that activate innate immune signaling via TBK1-IRF3 and NF-kappa B and induce cytokine production, including type I interferons (IFN) and IL-6. NS5B-expression also results in liver damage.

  • 19.
    Gerold, Gisa
    et al.
    TWINCORE – Institute of Experimental Virology, Centre for Experimental and Clinical Infection Research, Hannover , Germany.
    Pietschmann, Thomas
    The HCV life cycle: in vitro tissue culture systems and therapeutic targets2014In: Digestive Diseases, ISSN 0257-2753, E-ISSN 1421-9875, Vol. 32, no 5, p. 525-537Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) is a highly variable plus-strand RNA virus of the family Flaviviridae. Viral strains are grouped into six epidemiologically relevant genotypes that differ from each other by more than 30% at the nucleotide level. The variability of HCV allows immune evasion and facilitates persistence. It is also a substantial challenge for the development of specific antiviral therapies effective across all HCV genotypes and for prevention of drug resistance. Novel HCV cell culture models were instrumental for identification and profiling of therapeutic strategies. Concurrently, these models revealed numerous host factors critical for HCV propagation, some of which have emerged as targets for antiviral therapy. It is generally assumed that the use of host factors is conserved among HCV isolates and genotypes. Additionally, the barrier to viral resistance is thought to be high when interfering with host factors. Therefore, current drug development includes both targeting of viral factors but also of host factors essential for virus replication. In fact, some of these host-targeting agents, for instance inhibitors of cyclophilin A, have advanced to late stage clinical trials. Here, we highlight currently available cell culture systems for HCV, review the most prominent host-targeting strategies against hepatitis C and critically discuss opportunities and risks associated with host-targeting antiviral strategies.

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  • 20.
    Gerold, Gisa
    et al.
    Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA.
    Rice, Charles M
    Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, New York, USA.
    Locking out hepatitis C2011In: Nature Medicine, ISSN 1078-8956, E-ISSN 1546-170X, Vol. 17, no 5, p. 542-4Article in journal (Refereed)
  • 21.
    Gerold, Gisa
    et al.
    Center for the Study of Hepatitis C Laboratory of Virology and Infectious Diseases Rockefeller University, New York, NY.
    Rice, Charles M
    Ploss, Alexander
    Teaching new tricks to an old foe: murinizing hepatitis C virus2010In: Hepatology, ISSN 0270-9139, E-ISSN 1527-3350, Vol. 52, no 6, p. 2233-2236Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) naturally infects only humans and chimpanzees. The determinants responsible for this narrow species tropism are not well defined. Virus cell entry involves human scavenger receptor class B type I (SR‐BI), CD81, claudin‐1 and occludin. Among these, at least CD81 and occludin are utilized in a highly species‐specific fashion, thus contributing to the narrow host range of HCV. We adapted HCV to mouse CD81 and identified three envelope glycoprotein mutations which together enhance infection of cells with mouse or other rodent receptors approximately 100‐fold. These mutations enhanced interaction with human CD81 and increased exposure of the binding site for CD81 on the surface of virus particles. These changes were accompanied by augmented susceptibility of adapted HCV to neutralization by E2‐specific antibodies indicative of major conformational changes of virus‐resident E1/E2‐complexes. Neutralization with CD81, SR‐BI‐ and claudin‐1‐specific antibodies and knock down of occludin expression by siRNAs indicate that the adapted virus remains dependent on these host factors but apparently utilizes CD81, SR‐BI and occludin with increased efficiency. Importantly, adapted E1/E2 complexes mediate HCV cell entry into mouse cells in the absence of human entry factors. These results further our knowledge of HCV receptor interactions and indicate that three glycoprotein mutations are sufficient to overcome the species‐specific restriction of HCV cell entry into mouse cells. Moreover, these findings should contribute to the development of an immunocompetent small animal model fully permissive to HCV.

  • 22.
    Gerold, Gisa
    et al.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Zychlinsky, Arturo
    de Diego, Juana L
    What is the role of Toll-like receptors in bacterial infections?2007In: Seminars in Immunology, ISSN 1044-5323, E-ISSN 1096-3618, Vol. 19, no 1, p. 41-7Article in journal (Refereed)
    Abstract [en]

    Innate immunity relies on signalling by Toll-like receptors (TLRs) to alert the immune system of the presence of invading bacteria. TLR activation leads to the release of cytokines that allow for effective innate and adaptive immune responses. However, the contribution of different TLRs depends on the site of the infection and the pathogen. This review will describe the involvement of TLRs in the development of three different bacterial infections as well as our current understanding of the role of TLRs during microbial pathogenesis.

  • 23.
    Haid, Sibylle
    et al.
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Matthaei, Alina
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Winkler, Melina
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Sake, Svenja M.
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Gunesch, Antonia P.
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Milke, Vanessa
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Köhler, Natalie M.
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Rückert, Jessica
    Institute of Virology, Hannover Medical School, Hannover, Germany; German Center for Infection Research, Hannover-Braunschweig Site, Hannover, Germany.
    Vieyres, Gabrielle
    Junior Research Group “Cell Biology of RNA Viruses”, Leibniz Institute of Experimental Virology, Hamburg, Germany; Integrative Analysis of Pathogen-Induced Compartments, Leibniz ScienceCampus InterACt, Hamburg, Germany.
    Kühl, David
    Junior Research Group “Cell Biology of RNA Viruses”, Leibniz Institute of Experimental Virology, Hamburg, Germany.
    Nguyen, Tu-Trinh
    Calibr, a Division, The Scripps Research Institute, La Jolla, CA, United States.
    Göhl, Matthias
    German Center for Infection Research, Hannover-Braunschweig Site, Hannover, Germany; Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Lasswitz, Lisa
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany; Department of Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Zapatero-Belinchón, Francisco J.
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany; Department of Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Brogden, Graham
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany; Department of Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany; Department of Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany.
    Wiegmann, Bettina
    Department of Cardiothoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hannover, Germany; Lower Saxony Center for Biomedical Engineering, Implant Research and Development, Hannover Medical School, Hannover, Germany; BREATH (Biomedical Research in Endstage and Obstructive Lung Disease Hannover), German Center for Lung Research (DZL), Carl-Neuberg Str. 1, Hannover, Germany.
    Bilitewski, Ursula
    Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Brown, Richard J.P.
    Division of Veterinary Medicine, Paul Ehrlich Institute, Langen, Germany; Department of Molecular and Medical Virology, Ruhr University, Bochum, Germany.
    Brönstrup, Mark
    German Center for Infection Research, Hannover-Braunschweig Site, Hannover, Germany; Helmholtz Centre for Infection Research, Braunschweig, Germany.
    Schulz, Thomas F.
    Institute of Virology, Hannover Medical School, Hannover, Germany; German Center for Infection Research, Hannover-Braunschweig Site, Hannover, Germany; Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany.
    Pietschmann, Thomas
    Institute for Experimental Virology, Twincore - Centre for Experimental and Clinical Infection Research, Hannover, Germany; German Center for Infection Research, Hannover-Braunschweig Site, Hannover, Germany; Cluster of Excellence RESIST (EXC 2155), Hannover Medical School, Hannover, Germany.
    Repurposing screen identifies novel candidates for broad-spectrum coronavirus antivirals and druggable host targets2024In: Antimicrobial Agents and Chemotherapy, ISSN 0066-4804, E-ISSN 1098-6596, Vol. 68, no 3, article id e01210-23Article in journal (Refereed)
    Abstract [en]

    Libraries composed of licensed drugs represent a vast repertoire of molecules modulating physiological processes in humans, providing unique opportunities for the discovery of host-targeting antivirals. We screened the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) repurposing library with approximately 12,000 molecules for broad-spectrum coronavirus antivirals and discovered 134 compounds inhibiting an alphacoronavirus and mapping to 58 molecular target categories. Dominant targets included the 5-hydroxytryptamine receptor, the dopamine receptor, and cyclin-dependent kinases. Gene knock-out of the drugs’ host targets including cathepsin B and L (CTSB/L; VBY-825), the aryl hydrocarbon receptor (AHR; Phortress), the farnesyl-diphosphate farnesyltransferase 1 (FDFT1; P-3622), and the kelch-like ECH-associated protein 1 (KEAP1; Omaveloxolone), significantly modulated HCoV-229E infection, providing evidence that these compounds inhibited the virus through acting on their respective host targets. Counter-screening of all 134 primary compound candidates with SARS-CoV-2 and validation in primary cells identified Phortress, an AHR activating ligand, P-3622-targeting FDFT1, and Omaveloxolone, which activates the NFE2-like bZIP transcription factor 2 (NFE2L2) by liberating it from its endogenous inhibitor KEAP1, as antiviral candidates for both an Alpha- and a Betacoronavirus. This study provides an overview of HCoV-229E repurposing candidates and reveals novel potentially druggable viral host dependency factors hijacked by diverse coronaviruses.

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  • 24. Herrador, Antonio
    et al.
    Fedeli, Chiara
    Radulovic, Emilia
    Campbell, Kevin P.
    Moreno, Hector
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology. TWINCORE - Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Kunz, Stefan
    Dynamic Dystroglycan Complexes Mediate Cell Entry of Lassa Virus2019In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 10, no 2, article id e02869-18Article in journal (Refereed)
    Abstract [en]

    Recognition of functional receptors by viruses is a key determinant for their host range, tissue tropism, and disease potential. The highly pathogenic Lassa virus (LASV) currently represents one of the most important emerging pathogens. The major cellular receptor for LASV in human cells is the ubiquitously expressed and evolutionary highly conserved extracellular matrix receptor dystroglycan (DG). In the host, DG interacts with many cellular proteins in a tissue-specific manner. The resulting distinct supramolecular complexes likely represent the functional units for viral entry, and preexisting protein-protein interactions may critically influence DG's function in productive viral entry. Using an unbiased shotgun proteomic approach, we define the largely unknown molecular composition of DG complexes present in highly susceptible epithelial cells that represent important targets for LASV during viral transmission. We further show that the specific composition of cellular DG complexes can affect DG's function in receptor-mediated endocytosis of the virus. Under steady-state conditions, epithelial DG complexes underwent rapid turnover via an endocytic pathway that shared some characteristics with DG-mediated LASV entry. However, compared to steady-state uptake of DG, LASV entry via DG occurred faster and critically depended on additional signaling by receptor tyrosine kinases and the downstream effector p21-activating kinase. In sum, we show that the specific molecular composition of DG complexes in susceptible cells is a determinant for productive virus entry and that the pathogen can manipulate the existing DG-linked endocytic pathway. This highlights another level of complexity of virus-receptor interaction and provides possible cellular targets for therapeutic antiviral intervention.

    Importance: Recognition of cellular receptors allows emerging viruses to break species barriers and is an important determinant for their disease potential. Many virus receptors have complex tissue-specific interactomes, and preexisting protein-protein interactions may influence their function. Combining shotgun proteomics with a biochemical approach, we characterize the molecular composition of the functional receptor complexes used by the highly pathogenic Lassa virus (LASV) to invade susceptible human cells. We show that the specific composition of the receptor complexes affects productive entry of the virus, providing proof-of-concept. In uninfected cells, these functional receptor complexes undergo dynamic turnover involving an endocytic pathway that shares some characteristics with viral entry. However, steady-state receptor uptake and virus endocytosis critically differ in kinetics and underlying signaling, indicating that the pathogen can manipulate the receptor complex according to its needs. Our study highlights a remarkable complexity of LASV-receptor interaction and identifies possible targets for therapeutic antiviral intervention.

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  • 25. Kapoor, Amit
    et al.
    Simmonds, Peter
    Gerold, Gisa
    Center for the Study of Hepatitis C, Laboratory of Virology and Infectious Disease, The Rockefeller University, New York, NY 10065.
    Qaisar, Natasha
    Jain, Komal
    Henriquez, Jose A
    Firth, Cadhla
    Hirschberg, David L
    Rice, Charles M
    Shields, Shelly
    Lipkin, W Ian
    Characterization of a canine homolog of hepatitis C virus2011In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 108, no 28, p. 11608-11613Article in journal (Refereed)
    Abstract [en]

    An estimated 3% of the world's population is chronically infected with hepatitis C virus (HCV). Although HCV was discovered more than 20 y ago, its origin remains obscure largely because no closely related animal virus homolog has been identified; furthermore, efforts to understand HCV pathogenesis have been hampered by the absence of animal models other than chimpanzees for human disease. Here we report the identification in domestic dogs of a nonprimate hepacivirus. Comparative phylogenetic analysis of the canine hepacivirus (CHV) confirmed it to be the most genetically similar animal virus homolog of HCV. Bayesian Markov chains Monte Carlo and associated time to most recent common ancestor analyses suggest a mean recent divergence time of CHV and HCV clades within the past 500-1,000 y, well after the domestication of canines. The discovery of CHV may provide new insights into the origin and evolution of HCV and a tractable model system with which to probe the pathogenesis, prevention, and treatment of diseases caused by hepacivirus infection.

  • 26. Kirui, Jared
    et al.
    Abidine, Yara
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Lenman, Annasara
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Centre for Experimental and Clinical Infection Research, TWINCORE, Institute for Experimental Virology, a Joint Venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Islam, Md. Koushikul
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Infectious Diseases.
    Yong-Dae, Gwon
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Lasswitz, Lisa
    Evander, Magnus
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology.
    Bally, Marta
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Centre for Experimental and Clinical Infection Research, TWINCORE, Institute for Experimental Virology, a Joint Venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany; Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    The Phosphatidylserine Receptor TIM-1 Enhances Authentic Chikungunya Virus Cell Entry2021In: Cells, E-ISSN 2073-4409, Vol. 10, no 7, article id 1828Article in journal (Refereed)
    Abstract [en]

    Chikungunya virus (CHIKV) is a re-emerging, mosquito-transmitted, enveloped positive stranded RNA virus. Chikungunya fever is characterized by acute and chronic debilitating arthritis. Although multiple host factors have been shown to enhance CHIKV infection, the molecular mechanisms of cell entry and entry factors remain poorly understood. The phosphatidylserine-dependent receptors, T-cell immunoglobulin and mucin domain 1 (TIM-1) and Axl receptor tyrosine kinase (Axl), are transmembrane proteins that can serve as entry factors for enveloped viruses. Previous studies used pseudoviruses to delineate the role of TIM-1 and Axl in CHIKV entry. Conversely, here, we use the authentic CHIKV and cells ectopically expressing TIM-1 or Axl and demonstrate a role for TIM-1 in CHIKV infection. To further characterize TIM-1-dependent CHIKV infection, we generated cells expressing domain mutants of TIM-1. We show that point mutations in the phosphatidylserine binding site of TIM-1 lead to reduced cell binding, entry, and infection of CHIKV. Ectopic expression of TIM-1 renders immortalized keratinocytes permissive to CHIKV, whereas silencing of endogenously expressed TIM-1 in human hepatoma cells reduces CHIKV infection. Altogether, our findings indicate that, unlike Axl, TIM-1 readily promotes the productive entry of authentic CHIKV into target cells.

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  • 27.
    Krüger, Nadine
    et al.
    Infection Biology Unit, German Primate Center, Göttingen, Germany.
    Rocha, Cheila
    Infection Biology Unit, German Primate Center, Göttingen, Germany.
    Runft, Sandra
    Department of Pathology, University of Veterinary Medicine, Foundation, Hannover, Germany.
    Krüger, Johannes
    Department of Pathology, University of Veterinary Medicine, Foundation, Hannover, Germany.
    Färber, Iris
    Department of Pathology, University of Veterinary Medicine, Foundation, Hannover, Germany.
    Armando, Federico
    Department of Pathology, University of Veterinary Medicine, Foundation, Hannover, Germany.
    Leitzen, Eva
    Department of Pathology, University of Veterinary Medicine, Foundation, Hannover, Germany.
    Brogden, Graham
    Department of Biochemistry, University of Veterinary Medicine, Foundation, Hannover, Germany; Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Foundation, Hannover, Germany; Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Department of Biochemistry, University of Veterinary Medicine, Foundation, Hannover, Germany; Research Center for Emerging Infections and Zoonoses, University of Veterinary Medicine, Foundation, Hannover, Germany; Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hannover, Germany.
    Pöhlmann, Stefan
    Infection Biology Unit, German Primate Center, Göttingen, Germany.
    Hoffmann, Markus
    Infection Biology Unit, German Primate Center, Göttingen, Germany.
    Baumgärtner, Wolfgang
    Department of Pathology, University of Veterinary Medicine, Foundation, Hannover, Germany.
    The upper respiratory tract of felids is highly susceptible to sars‐cov‐2 infection2021In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 22, no 19, article id 10636Article in journal (Refereed)
    Abstract [en]

    Natural or experimental infection of domestic cats and virus transmission from humans to captive predatory cats suggest that felids are highly susceptible to SARS‐CoV‐2 infection. However, it is unclear which cells and compartments of the respiratory tract are infected. To address this question, primary cell cultures derived from the nose, trachea, and lungs of cat and lion were inoculated with SARS‐CoV‐2. Strong viral replication was observed for nasal mucosa explants and tracheal air–liquid interface cultures, whereas replication in lung slices was less efficient. Infection was mainly restricted to epithelial cells and did not cause major pathological changes. Detection of high ACE2 levels in the nose and trachea but not lung further suggests that susceptibility of feline tissues to SARS‐CoV‐2 correlates with ACE2 expression. Collectively, this study demonstrates that SARS‐ CoV‐2 can efficiently replicate in the feline upper respiratory tract ex vivo and thus highlights the risk of SARS‐CoV‐2 spillover from humans to felids.

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  • 28. Lasswitz, Lisa
    et al.
    Chandra, Naresh
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Arnberg, Niklas
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Glycomics and Proteomics Approaches to Investigate Early Adenovirus-Host Cell Interactions2018In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 430, no 13, p. 1863-1882Article in journal (Refereed)
    Abstract [en]

    Adenoviruses as most viruses rely on glycan and protein interactions to attach to and enter susceptible host cells. The Adenoviridae family comprises more than 80 human types and they differ in their attachment factor and receptor usage, which likely contributes to the diverse tropism of the different types. In the past years, methods to systematically identify glycan and protein interactions have advanced. In particular sensitivity, speed and coverage of mass spectrometric analyses allow for high-throughput identification of glycans and peptides separated by liquid chromatography. Also, developments in glycan microarray technologies have led to targeted, high-throughput screening and identification of glycan-based receptors. The mapping of cell surface interactions of the diverse adenovirus types has implications for cell, tissue, and species tropism as well as drug development. Here we review known adenovirus interactions with glycan- and protein-based receptors, as well as glycomics and proteomics strategies to identify yet elusive virus receptors and attachment factors. We finally discuss challenges, bottlenecks, and future research directions in the field of non-enveloped virus entry into host cells.

  • 29.
    Lasswitz, Lisa
    et al.
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    Zapatero-Belinchón, Francisco J.
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    Moeller, Rebecca
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    Hülskötter, Kirsten
    Department of Pathology, University of Veterinary Medicine Hanover, Hanover, Germany.
    Laurent, Timothée
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Carlson, Lars-Anders
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Medical Biochemistry and Biophysics. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Goffinet, Christine
    Freie Universität Berlin and Humboldt-Universität zu Berlin, Institute of Virology, Charité-Universitätsmedizin Berlin, Berlin, Germany; Berlin Institute of Health at Charité-Universitätsmedizin Berlin, Berlin, Germany.
    Simmons, Graham
    Vitalant Research Institute, University of California, CA, San Francisco, United States; Department of Laboratory Medicine, University of California, CA, San Francisco, United States.
    Baumgärtner, Wolfgang
    Department of Pathology, University of Veterinary Medicine Hanover, Hanover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hanover, Germany; Institute for Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hanover, Hanover, Germany.
    The tetraspanin CD81 is a host factor for Chikungunya virus replication2022In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 13, no 3, article id e0073122Article in journal (Refereed)
    Abstract [en]

    Chikungunya virus (CHIKV) is an arthritogenic reemerging virus replicating in plasma membrane-derived compartments termed "spherules." Here, we identify the human transmembrane protein CD81 as host factor required for CHIKV replication. Ablation of CD81 results in decreased CHIKV permissiveness, while overexpression enhances infection. CD81 is dispensable for virus uptake but critically required for viral genome replication. Likewise, murine CD81 is crucial for CHIKV permissiveness and is expressed in target cells such as dermal fibroblasts, muscle and liver cells. Whereas related alphaviruses, including Ross River virus (RRV), Semliki Forest virus (SFV), Sindbis virus (SINV) and Venezuelan equine encephalitis virus (VEEV), also depend on CD81 for infection, RNA viruses from other families, such as coronaviruses, replicate independently of CD81. Strikingly, the replication-enhancing function of CD81 is linked to cholesterol binding. These results define a mechanism exploited by alphaviruses to hijack the membrane microdomain-modeling protein CD81 for virus replication through interaction with cholesterol.

    IMPORTANCE: In this study, we discover the tetraspanin CD81 as a host factor for the globally emerging chikungunya virus and related alphaviruses. We show that CD81 promotes replication of viral genomes in human and mouse cells, while virus entry into cells is independent of CD81. This provides novel insights into how alphaviruses hijack host proteins to complete their life cycle. Alphaviruses replicate at distinct sites of the plasma membrane, which are enriched in cholesterol. We found that the cholesterol-binding ability of CD81 is important for its function as an alphavirus host factor. This discovery thus broadens our understanding of the alphavirus replication process and the use of host factors to reprogram cells into virus replication factories.

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  • 30.
    Li, Dandan
    et al.
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Bühler, Melanie
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Runft, Sandra
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry, University of Veterinary Medicine Hannover, Foundation, Bünteweg 17, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Marek, Katarzyna
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Baumgärtner, Wolfgang
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Strowig, Till
    Department for Microbial Immune Regulation, Helmholtz Centre for Infection Research, Braunschweig, Germany; Hannover Medical School, Hannover, Germany.
    Gerhauser, Ingo
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    ASC- and caspase-1-deficient C57BL/6 mice do not develop demyelinating disease after infection with Theiler's murine encephalomyelitis virus2023In: Scientific Reports, E-ISSN 2045-2322, Vol. 13, no 1, article id 10960Article in journal (Refereed)
    Abstract [en]

    Theiler's murine encephalomyelitis virus (TMEV) induces an acute polioencephalomyelitis and a chronic demyelinating leukomyelitis in SJL mice. C57BL/6 (B6) mice generally do not develop TMEV-induced demyelinating disease (TMEV-IDD) due to virus elimination. However, TMEV can persist in specific immunodeficient B6 mice such as IFNβ-/- mice and induce a demyelinating process. The proinflammatory cytokines IL-1β and IL-18 are activated by the inflammasome pathway, which consists of a pattern recognition receptor molecule sensing microbial pathogens, the adaptor molecule Apoptosis-associated speck-like protein containing a CARD (ASC), and the executioner caspase-1. To analyze the contribution of the inflammasome pathway to the resistance of B6 mice to TMEV-IDD, ASC- and caspase-1-deficient mice and wild type littermates were infected with TMEV and investigated using histology, immunohistochemistry, RT-qPCR, and Western Blot. Despite the antiviral activity of the inflammasome pathway, ASC- and caspase-1-deficient mice eliminated the virus and did not develop TMEV-IDD. Moreover, a similar IFNβ and cytokine gene expression was found in the brain of immunodeficient mice and their wild type littermates. Most importantly, Western Blot showed cleavage of IL-1β and IL-18 in all investigated mice. Consequently, inflammasome-dependent activation of IL-1β and IL-18 does not play a major role in the resistance of B6 mice to TMEV-IDD.

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  • 31. Loll, Bernhard
    et al.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Virology.
    Slowik, Daria
    Voelter, Wolfgang
    Jung, Christiane
    Saenger, Wolfram
    Irrgang, Klaus-Dieter
    Thermostability and Ca2+ binding properties of wild type and heterologously expressed PsbO protein from cyanobacterial photosystem II.2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 12, p. 4691-8Article in journal (Refereed)
    Abstract [en]

    Oxygenic photosynthesis takes place in the thylakoid membrane of cyanobacteria, algae, and higher plants. Initially light is absorbed by an oligomeric pigment-protein complex designated as photosystem II (PSII), which catalyzes light-induced water cleavage under release of molecular oxygen for the biosphere on our planet. The membrane-extrinsic manganese stabilizing protein (PsbO) is associated on the lumenal side of the thylakoids close to the redox-active (Mn)(4)Ca cluster at the catalytically active site of PSII. Recombinant PsbO from the thermophilic cyanobacterium Thermosynechococcus elongatus was expressed in Escherichia coli and spectroscopically characterized. The secondary structure of recombinant PsbO (recPsbO) was analyzed in the absence and presence of Ca(2+) using Fourier transform infrared spectroscopy (FTIR) and circular dichroism spectropolarimetry (CD). No significant structural changes could be observed when the PSII subunit was titrated with Ca(2+) in vitro. These findings are compared with data for spinach PsbO. Our results are discussed in the light of the recent 3D-structural analysis of the oxygen-evolving PSII and structural/thermodynamic differences between the two homologous proteins from thermophilic cyanobacteria and plants.

  • 32. Lump, Edina
    et al.
    Castellano, Laura M
    Meier, Christoph
    Seeliger, Janine
    Erwin, Nelli
    Sperlich, Benjamin
    Stürzel, Christina M
    Usmani, Shariq
    Hammond, Rebecca M
    von Einem, Jens
    Gerold, Gisa
    Kreppel, Florian
    Bravo-Rodriguez, Kenny
    Pietschmann, Thomas
    Holmes, Veronica M
    Palesch, David
    Zirafi, Onofrio
    Weissman, Drew
    Sowislok, Andrea
    Wettig, Burkhard
    Heid, Christian
    Kirchhoff, Frank
    Weil, Tanja
    Klärner, Frank-Gerrit
    Schrader, Thomas
    Bitan, Gal
    Sanchez-Garcia, Elsa
    Winter, Roland
    Shorter, James
    Münch, Jan
    A molecular tweezer antagonizes seminal amyloids and HIV infection2015In: eLIFE, E-ISSN 2050-084X, Vol. 4, article id e05397Article in journal (Refereed)
    Abstract [en]

    Semen is the main vector for HIV transmission and contains amyloid fibrils that enhance viral infection. Available microbicides that target viral components have proven largely ineffective in preventing sexual virus transmission. In this study, we establish that CLR01, a 'molecular tweezer' specific for lysine and arginine residues, inhibits the formation of infectivity-enhancing seminal amyloids and remodels preformed fibrils. Moreover, CLR01 abrogates semen-mediated enhancement of viral infection by preventing the formation of virion-amyloid complexes and by directly disrupting the membrane integrity of HIV and other enveloped viruses. We establish that CLR01 acts by binding to the target lysine and arginine residues rather than by a non-specific, colloidal mechanism. CLR01 counteracts both host factors that may be important for HIV transmission and the pathogen itself. These combined anti-amyloid and antiviral activities make CLR01 a promising topical microbicide for blocking infection by HIV and other sexually transmitted viruses.

  • 33.
    Löw, Karin
    et al.
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland.
    Möller, Rebecca
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Stegmann, Cora
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Becker, Miriam
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Rehburg, Laura
    Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Obernolte, Helena
    Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Hannover, Germany; Fraunhofer International Consortium for Anti-Infective Research (iCAIR), Hannover, Germany; Fraunhofer Cluster of Excellence Immune-Mediated Diseases, (CIMD), Hannover, Germany; Member of the German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease (BREATH) Research Network, Hannover, Germany; Institute of Immunology, Hannover Medical School, Hannover, Germany.
    Schaudien, Dirk
    Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Hannover, Germany; Fraunhofer International Consortium for Anti-Infective Research (iCAIR), Hannover, Germany; Fraunhofer Cluster of Excellence Immune-Mediated Diseases, (CIMD), Hannover, Germany; Member of the German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease (BREATH) Research Network, Hannover, Germany; Institute of Immunology, Hannover Medical School, Hannover, Germany.
    Oestereich, Lisa
    Department of Virology, Bernhard-Nocht Institute for Tropical Medicine, Hamburg, Germany; German Center for Infectious Research (DZIF), Partner Site Hamburg-Lübeck-Borstel-Riems, Hamburg, Germany.
    Braun, Armin
    Fraunhofer Institute for Toxicology and Experimental Medicine (ITEM), Hannover, Germany; Fraunhofer International Consortium for Anti-Infective Research (iCAIR), Hannover, Germany; Fraunhofer Cluster of Excellence Immune-Mediated Diseases, (CIMD), Hannover, Germany; Member of the German Center for Lung Research (DZL), Biomedical Research in Endstage and Obstructive Lung Disease (BREATH) Research Network, Hannover, Germany; Institute of Immunology, Hannover Medical School, Hannover, Germany.
    Kunz, Stefan
    Institute of Microbiology, University Hospital Center and University of Lausanne, Lausanne, Switzerland.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Luminescent reporter cells enable the identification of broad-spectrum antivirals against emerging viruses2023In: Journal of Medical Virology, ISSN 0146-6615, E-ISSN 1096-9071, Vol. 95, no 11, article id e29211Article in journal (Refereed)
    Abstract [en]

    The emerging viruses SARS-CoV-2 and arenaviruses cause severe respiratory and hemorrhagic diseases, respectively. The production of infectious particles of both viruses and virus spread in tissues requires cleavage of surface glycoproteins (GPs) by host proprotein convertases (PCs). SARS-CoV-2 and arenaviruses rely on GP cleavage by PCs furin and subtilisin kexin isozyme-1/site-1 protease (SKI-1/S1P), respectively. We report improved luciferase-based reporter cell lines, named luminescent inducible proprotein convertase reporter cells that we employ to monitor PC activity in its authentic subcellular compartment. Using these sensor lines we screened a small compound library in high-throughput manner. We identified 23 FDA-approved small molecules, among them monensin which displayed broad activity against furin and SKI-1/S1P. Monensin inhibited arenaviruses and SARS-CoV-2 in a dose-dependent manner. We observed a strong reduction in infectious particle release upon monensin treatment with little effect on released genome copies. This was reflected by inhibition of SARS-CoV-2 spike processing suggesting the release of immature particles. In a proof of concept experiment using human precision cut lung slices, monensin potently inhibited SARS-CoV-2 infection, evidenced by reduced infectious particle release. We propose that our PC sensor pipeline is a suitable tool to identify broad-spectrum antivirals with therapeutic potential to combat current and future emerging viruses.

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  • 34.
    Marek, Katarzyna
    et al.
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; Center for Systems Neuroscience, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Armando, Federico
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Asawapattanakul, Thanaporn
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; Center for Systems Neuroscience, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Nippold, Vanessa Maria
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Plattet, Philippe
    Division of Experimental Clinical Research, Vetsuisse University Bern, Bern, Switzerland.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Baumgärtner, Wolfgang
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; Center for Systems Neuroscience, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Puff, Christina
    Department of Pathology, University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Functional Granulocyte–Macrophage Colony-Stimulating Factor (GM-CSF) Delivered by Canine Histiocytic Sarcoma Cells Persistently Infected with Engineered Attenuated Canine Distemper Virus2023In: Pathogens, E-ISSN 2076-0817, Vol. 12, no 7, article id 877Article in journal (Refereed)
    Abstract [en]

    The immune response plays a key role in the treatment of malignant tumors. One important molecule promoting humoral and cellular immunity is granulocyte–macrophage colony-stimulating factor (GM-CSF). Numerous successful trials have led to the approval of this immune-stimulating molecule for cancer therapy. However, besides immune stimulation, GM-CSF may also accelerate tumor cell proliferation, rendering this molecule a double-edged sword in cancer treatment. Therefore, detailed knowledge about the in vitro function of GM-CSF produced by infected tumor cells is urgently needed prior to investigations in an in vivo model. The aim of the present study was to functionally characterize a persistent infection of canine histiocytic sarcoma cells (DH82 cells) with the canine distemper virus strain Onderstepoort genetically engineered to express canine GM-CSF (CDV-Ondneon-GM-CSF). The investigations aimed (1) to prove the overall functionality of the virally induced production of GM-CSF and (2) to determine the effect of GM-CSF on the proliferation and motility of canine HS cells. Infected cells consistently produced high amounts of active, pH-stable GM-CSF, as demonstrated by increased proliferation of HeLa cells. By contrast, DH82 cells lacked increased proliferation and motility. The significantly increased secretion of GM-CSF by persistently CDV-Ondneon-GM-CSF-infected DH82 cells, the pH stability of this protein, and the lack of detrimental effects on DH82 cells renders this virus strain an interesting candidate for future studies aiming to enhance the oncolytic properties of CDV for the treatment of canine histiocytic sarcomas.

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  • 35.
    Marek, Katarzyna
    et al.
    Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany; Center for Systems Neuroscience, University of Veterinary Medicine Hannover, Hannover, Germany.
    Armando, Federico
    Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.
    Nippold, Vanessa Maria
    Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.
    Rohn, Karl
    Institute for Biometry, Epidemiology and Information Processing, University of Veterinary Medicine Hannover, Hannover, Germany.
    Plattet, Philippe
    Division of Experimental Clinical Research, Vetsuisse University Bern, Bern, Switzerland.
    Brogden, Graham
    Department of Biochemistry, University of Veterinary Medicine Hannover, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany; Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a Joint Venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry, University of Veterinary Medicine Hannover, Hannover, Germany; Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Hannover, Germany.
    Baumgärtner, Wolfgang
    Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany; Center for Systems Neuroscience, University of Veterinary Medicine Hannover, Hannover, Germany.
    Puff, Christina
    Department of Pathology, University of Veterinary Medicine Hannover, Hannover, Germany.
    Persistent Infection of a Canine Histiocytic Sarcoma Cell Line with Attenuated Canine Distemper Virus Expressing Vasostatin or Granulocyte-Macrophage Colony-Stimulating Factor2022In: International Journal of Molecular Sciences, ISSN 1661-6596, E-ISSN 1422-0067, Vol. 23, no 11, article id 6156Article in journal (Refereed)
    Abstract [en]

    Canine histiocytic sarcoma (HS) represents a neoplasia with poor prognosis. Due to the high metastatic rate of HS, there is urgency to improve treatment options and to prevent tumor metastases. Canine distemper virus (CDV) is a single-stranded negative-sense RNA (ssRNA (-)) virus with potentially oncolytic properties. Moreover, vasostatin and granulocyte-macrophage colony-stimulating factor (GM-CSF) are attractive molecules in cancer therapy research because of their anti-angiogenetic properties and potential modulation of the tumor microenvironment. In the present study, an in vitro characterization of two genetically engineered viruses based on the CDV strain Onderstepoort (CDV-Ond), CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF was performed. Canine histiocytic sarcoma cells (DH82 cells) were persistently infected with CDV-Ond, CDV-Ondneon, CDV-Ondneon-vasostatin and CDV-Ondneon-GM-CSF and characterized on a molecular and protein level regarding their vasostatin and GM-CSF production. Interestingly, DH82 cells persistently infected with CDV-Ondneon-vasostatin showed a significantly increased number of vasostatin mRNA transcripts. Similarly, DH82 cells persistently infected with CDV-Ondneon-GM-CSF displayed an increased number of GM-CSF mRNA transcripts mirrored on the protein level as confirmed by immunofluorescence and Western blot. In summary, modified CDV-Ond strains expressed GM-CSF and vasostatin, rendering them promising candidates for the improvement of oncolytic virotherapies, which should be further detailed in future in vivo studies.

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  • 36.
    Matthaei, Alina
    et al.
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany.
    Joecks, Sebastian
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany.
    Frauenstein, Annika
    RG Experimental Systems Immunology, Max-Planck Institute for Biochemistry, Bavaria, Planegg, Germany.
    Bruening, Janina
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany.
    Bankwitz, Dorothea
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany.
    Friesland, Martina
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany; Department of Biochemistry & Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Lower Saxony, Hannover, Germany.
    Vieyres, Gabrielle
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany; Junior Research Group "Cell Biology of RNA Viruses, Leibniz Institute of Experimental Virology, Hamburg, Germany.
    Kaderali, Lars
    Institute of Bioinformatics, Greifswald, University Medicine Greifswald, Germany.
    Meissner, Felix
    RG Experimental Systems Immunology, Max-Planck Institute for Biochemistry, Bavaria, Planegg, Germany; Systems Immunology and Proteomics, Institute of Innate Immunity, Medical Faculty, University of Bonn, Bonn, Germany.
    Pietschmann, Thomas
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Lower Saxony, Hannover, Germany.
    Landscape of protein-protein interactions during hepatitis C virus assembly and release2024In: Microbiology Spectrum, E-ISSN 2165-0497, Vol. 12, no 2, article id e0256222Article in journal (Refereed)
    Abstract [en]

    Assembly of infectious hepatitis C virus (HCV) particles requires multiple cellular proteins including for instance apolipoprotein E (ApoE). To describe these protein-protein interactions, we performed an affinity purification mass spectrometry screen of HCV-infected cells. We used functional viral constructs with epitope-tagged envelope protein 2 (E2), protein (p) 7, or nonstructural protein 4B (NS4B) as well as cells expressing a tagged variant of ApoE. We also evaluated assembly stage-dependent remodeling of protein complexes by using viral mutants carrying point mutations abrogating particle production at distinct steps of the HCV particle production cascade. Five ApoE binding proteins, 12 p7 binders, 7 primary E2 interactors, and 24 proteins interacting with NS4B were detected. Cell-derived PREB, STT3B, and SPCS2 as well as viral NS2 interacted with both p7 and E2. Only GTF3C3 interacted with E2 and NS4B, highlighting that HCV assembly and replication complexes exhibit largely distinct interactomes. An HCV core protein mutation, preventing core protein decoration of lipid droplets, profoundly altered the E2 interactome. In cells replicating this mutant, E2 interactions with HSPA5, STT3A/B, RAD23A/B, and ZNF860 were significantly enhanced, suggesting that E2 protein interactions partly depend on core protein functions. Bioinformatic and functional studies including STRING network analyses, RNA interference, and ectopic expression support a role of Rad23A and Rad23B in facilitating HCV infectious virus production. Both Rad23A and Rad23B are involved in the endoplasmic reticulum (ER)-associated protein degradation (ERAD). Collectively, our results provide a map of host proteins interacting with HCV assembly proteins, and they give evidence for the involvement of ER protein folding machineries and the ERAD pathway in the late stages of the HCV replication cycle.IMPORTANCEHepatitis C virus (HCV) establishes chronic infections in the majority of exposed individuals. This capacity likely depends on viral immune evasion strategies. One feature likely contributing to persistence is the formation of so-called lipo-viro particles. These peculiar virions consist of viral structural proteins and cellular lipids and lipoproteins, the latter of which aid in viral attachment and cell entry and likely antibody escape. To learn about how lipo-viro particles are coined, here, we provide a comprehensive overview of protein-protein interactions in virus-producing cells. We identify numerous novel and specific HCV E2, p7, and cellular apolipoprotein E-interacting proteins. Pathway analyses of these interactors show that proteins participating in processes such as endoplasmic reticulum (ER) protein folding, ER-associated protein degradation, and glycosylation are heavily engaged in virus production. Moreover, we find that the proteome of HCV replication sites is distinct from the assembly proteome, suggesting that transport process likely shuttles viral RNA to assembly sites.

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  • 37.
    Mhlekude, Baxolele
    et al.
    University of Cape Town, Department of Surgery, Groote Schuur Hospital, Observatory, South Africa; TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, Institute of Experimental Virology, Hannover; Charité - Universitätsmedizin Berlin, Institute of Virology, Charité Campus Mitte; Berlin Institute of Health, Berlin, Germany.
    Lenman, Annasara
    TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, Institute of Experimental Virology, Hannover.
    Sidoyi, Phikolomzi
    Faculty of Health Sciences, School of Medicine, Walter Sisulu University, Mthatha, South Africa.
    Joseph, Jim
    Department of Human Biology, Walter Sisulu University, Mthatha, South Africa.
    Kruppa, Jochen
    Charité - Universitätsmedizin Berlin, Institut für Biometrie und Klinische Epidemiologie, Charité Campus Mitte, Berlin, Germany.
    Businge, Charles Bitamazire
    Department of Obstetrics and Gynaecology, Walter Sisulu University/Nelson Mandela Academic Hospital.
    Mdaka, Mana Lungisa
    Department of Obstetrics and Gynaecology, Walter Sisulu University/Nelson Mandela Academic Hospital.
    Konietschke, Frank
    Berlin Institute of Health, Berlin, Germany; Charité - Universitätsmedizin Berlin, Institut für Biometrie und Klinische Epidemiologie, Charité Campus Mitte, Berlin, Germany.
    Pich, Andreas
    Hannover Medical School, Institute of Toxicology, Core Facility Proteomics, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, Institute of Experimental Virology, Hannover; Department of Biochemistry, University of Veterinary Medicine Hannover, Hanover, Germany.
    Goffinet, Christine
    TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Hannover Medical School and the Helmholtz Centre for Infection Research, Institute of Experimental Virology, Hannover; Charité - Universitätsmedizin Berlin, Institute of Virology, Charité Campus Mitte; Berlin Institute of Health, Berlin, Germany.
    Mall, Anwar Suleman
    University of Cape Town, Department of Surgery, Groote Schuur Hospital, Observatory, South Africa.
    The barrier functions of crude cervical mucus plugs against HIV-1 infection in the context of cell-free and cell-to-cell transmission2021In: AIDS, ISSN 0269-9370, E-ISSN 1473-5571, Vol. 35, no 13, p. 2105-2117Article in journal (Refereed)
    Abstract [en]

    OBJECTIVE: The cervical mucus plugs are enriched with proteins of known immunological functions. We aimed to characterize the anti-HIV-1 activity of the cervical mucus plugs against a panel of different HIV-1 strains in the contexts of cell-free and cell-associated virus.

    DESIGN: A cohort of consenting HIV-1-negative and HIV-1-positive pregnant women in labour was recruited from Mthatha General Hospital in the Eastern Cape province of South Africa, from whom the cervical mucus plugs were collected in 6 M guanidinium chloride with protease inhibitors and transported to our laboratories at -80 °C.

    METHODS: Samples were centrifuged to remove insoluble material and dialysed before freeze--drying and subjecting them to the cell viability assays. The antiviral activities of the samples were studied using luminometric reporter assays and flow cytometry. Time-of-addition and BlaM-Vpr virus-cell fusion assays were used to pin-point the antiviral mechanisms of the cervical mucus plugs, before proteomic profiling using liquid chromatography-tandem mass spectrometry.

    RESULTS: The proteinaceous fraction of the cervical mucus plugs exhibited anti-HIV-1 activity with inter-individual variations and some degree of specificity among different HIV-1 strains. Cell-associated HIV-1 was less susceptible to inhibition by the potent samples whenever compared with the cell-free HIV-1. The samples with high antiviral potency exhibited a distinct proteomic profile when compared with the less potent samples.

    CONCLUSION: The crude cervical mucus plugs exhibit anti-HIV-1 activity, which is defined by a specific proteomic profile.

  • 38.
    Mhlekude, Baxolele
    et al.
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany; Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany; Virology and Innate Immunity Research Group, Helmholtz Centre for Infection Research, Braunschweig, Germany; Institute of Genetics, Technische Universität Braunschweig, Braunschweig, Germany.
    Postmus, Dylan
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany; Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany.
    Stenzel, Saskia
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany; Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany.
    Weiner, January
    Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany.
    Jansen, Jenny
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany; Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany.
    Zapatero-Belinchón, Francisco J.
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry, University of Veterinary Medicine Hannover, Hannover, Germany; Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, The Hannover Medical School, The Helmholtz Centre for Infection Research, Hannover, Germany.
    Olmer, Ruth
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH—Center for Translational Regenerative Medicine, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover Medical School, Hannover, Germany.
    Richter, Anja
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Heinze, Julian
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Heinemann, Nicolas
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Mühlemann, Barbara
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Schroeder, Simon
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Jones, Terry C.
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany; Centre for Pathogen Evolution, Department of Zoology, University of Cambridge, Cambridge, United Kingdom.
    Müller, Marcel A.
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Drosten, Christian
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Pich, Andreas
    Institute of Toxicology, Hannover Medical School, Core Facility Proteomics, Hannover, Germany.
    Thiel, Volker
    Institute of Virology and Immunology (IVI), University of Bern, Bern, Switzerland; Department of Infectious Diseases and Pathobiology, Vetsuisse Faculty, University of Bern, Bern, Switzerland.
    Martin, Ulrich
    Leibniz Research Laboratories for Biotechnology and Artificial Organs (LEBAO), Department of Cardiothoracic, Transplantation and Vascular Surgery, REBIRTH—Center for Translational Regenerative Medicine, Biomedical Research in Endstage and Obstructive Lung Disease Hannover (BREATH), German Center for Lung Research (DZL), Hannover Medical School, Hannover, Germany.
    Niemeyer, Daniela
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Department of Biochemistry, University of Veterinary Medicine Hannover, Hannover, Germany; Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, The Hannover Medical School, The Helmholtz Centre for Infection Research, Hannover, Germany.
    Beule, Dieter
    Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany.
    Goffinet, Christine
    Institute of Virology, Campus Charité Mitte, Charité—Universitätsmedizin Berlin, Freie Universität Berlin, Humboldt-Universität zu Berlin, Berlin, Germany; Berlin Institute of Health at Charité, Universitätsmedizin Berlin, Berlin, Germany; Department of Tropical Disease Biology, Liverpool School of Tropical Medicine, Liverpool, United Kingdom.
    Pharmacological inhibition of bromodomain and extra-terminal proteins induces an NRF-2-mediated antiviral state that is subverted by SARS-CoV-2 infection2023In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 19, no 9, article id e1011657Article in journal (Refereed)
    Abstract [en]

    Inhibitors of bromodomain and extra-terminal proteins (iBETs), including JQ-1, have been suggested as potential prophylactics against SARS-CoV-2 infection. However, molecular mechanisms underlying JQ-1-mediated antiviral activity and its susceptibility to viral subversion remain incompletely understood. Pretreatment of cells with iBETs inhibited infection by SARS-CoV-2 variants and SARS-CoV, but not MERS-CoV. The antiviral activity manifested itself by reduced reporter expression of recombinant viruses, and reduced viral RNA quantities and infectious titers in the culture supernatant. While we confirmed JQ-1-mediated downregulation of expression of angiotensin-converting enzyme 2 (ACE2) and interferon-stimulated genes (ISGs), multi-omics analysis addressing the chromatin accessibility, transcriptome and proteome uncovered induction of an antiviral nuclear factor erythroid 2-related factor 2 (NRF-2)-mediated cytoprotective response as an additional mechanism through which JQ-1 inhibits SARS-CoV-2 replication. Pharmacological inhibition of NRF-2, and knockdown of NRF-2 and its target genes reduced JQ-1-mediated inhibition of SARS-CoV-2 replication. Serial passaging of SARS-CoV-2 in the presence of JQ-1 resulted in predominance of ORF6-deficient variant, which exhibited resistance to JQ-1 and increased sensitivity to exogenously administered type I interferon (IFN-I), suggesting a minimised need for SARS-CoV-2 ORF6-mediated repression of IFN signalling in the presence of JQ-1. Importantly, JQ-1 exhibited a transient antiviral activity when administered prophylactically in human airway bronchial epithelial cells (hBAECs), which was gradually subverted by SARS-CoV-2, and no antiviral activity when administered therapeutically following an established infection. We propose that JQ-1 exerts pleiotropic effects that collectively induce an antiviral state in the host, which is ultimately nullified by SARS-CoV-2 infection, raising questions about the clinical suitability of the iBETs in the context of COVID-19.

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  • 39. Moreno, Hector
    et al.
    Moeller, Rebecca
    Fedeli, Chiara
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. TWINCORE–Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Kunz, Stefan
    Comparison of the Innate Immune Responses to Pathogenic and Nonpathogenic Clade B New World Arenaviruses2019In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 93, no 19, article id e00148-19Article in journal (Refereed)
    Abstract [en]

    The New World (NW) arenaviruses are a diverse group of zoonotic viruses, including several causative agents of severe hemorrhagic fevers in humans. All known human-pathogenic NW arenaviruses belong to Glade B, where they group into sublineages with phylogenetically closely related nonpathogenic viruses, e.g., the highly pathogenic Junin (JUNV) and Machupo viruses with the nonpathogenic Tacaribe virus (TCRV). Considering the close genetic relationship of nonpathogenic and pathogenic NW arenaviruses, the identification of molecular determinants of virulence is of great importance. The host cell's innate antiviral defense represents a major barrier for zoonotic infection. Here, we performed a side-by-side comparison of the innate immune responses against JUNV and TCRV in human cells. Despite similar levels of viral replication, infection with TCRV consistently induced a stronger type I interferon (IFN-I) response than JUNV infection did. Transcriptome profiling revealed upregulation of a largely overlapping set of interferon-stimulated genes in cells infected with TCRV and JUNV. Both viruses were relatively insensitive to IFN-I treatment of human cells and induced similar levels of apoptosis in the presence or absence of an IFN-I response. However, in comparison to JUNV, TCRV induced stronger activation of the innate sensor double-strand RNA-dependent protein kinase R (PKR), resulting in phosphorylation of eukaryotic translation initiation factor eIF2 alpha. Confocal microscopy studies revealed similar subcellular colocalizations of the JUNV and TCRV viral replication-transcription complexes with PKR. However, deletion of PKR by CRISPR/Cas9 hardly affected JUNV but promoted TCRV multiplication, providing the first evidence for differential innate recognition and control of pathogenic and nonpathogenic NW arenaviruses by PKR.

    IMPORTANCE New World (NW) arenaviruses are a diverse family of emerging zoonotic viruses that merit significant attention as important public health problems. The close genetic relationship of nonpathogenic NW arenaviruses with their highly pathogenic cousins suggests that few mutations may be sufficient to enhance virulence. The identification of molecular determinants of virulence of NW arenaviruses is therefore of great importance. Here we undertook a side-by-side comparison of the innate immune responses against the highly pathogenic Junin virus (JUNV) and the related nonpathogenic Tacaribe virus (TCRV) in human cells. We consistently found that TCRV induces a stronger type I interferon (IFN-I) response than JUNV. Transcriptome profiling revealed an overlapping pattern of IFN-induced gene expression and similar low sensitivities to IFN-I treatment. However, the double-stranded RNA (dsRNA)-dependent protein kinase R (PKR) contributed to the control of TCRV, but not JUNV, providing the first evidence for differential innate recognition and control of JUNV and TCRV.

  • 40. Moreno, Hector
    et al.
    Rastrojo, Alberto
    Pryce, Rhys
    Fedeli, Chiara
    Zimmer, Gert
    Bowden, Thomas A.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). TWINCORE -Center for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany; Department of Biochemistry, University of Veterinary Medicine Hannover, Hannover Germany.
    Kunz, Stefan
    A novel circulating tamiami mammarenavirus shows potential for zoonotic spillover2020In: PLoS Neglected Tropical Diseases, ISSN 1935-2727, E-ISSN 1935-2735, Vol. 14, no 12, article id e0009004Article in journal (Refereed)
    Abstract [en]

    A detailed understanding of the mechanisms underlying the capacity of a virus to break the species barrier is crucial for pathogen surveillance and control. New World (NW) mammarenaviruses constitute a diverse group of rodent-borne pathogens that includes several causative agents of severe viral hemorrhagic fever in humans. The ability of the NW mammarenaviral attachment glycoprotein (GP) to utilize human transferrin receptor 1 (hTfR1) as a primary entry receptor plays a key role in dictating zoonotic potential. The recent isolation of Tacaribe and lymphocytic choriominingitis mammarenaviruses from host-seeking ticks provided evidence for the presence of mammarenaviruses in arthropods, which are established vectors for numerous other viral pathogens. Here, using next generation sequencing to search for other mammarenaviruses in ticks, we identified a novel replication-competent strain of the NW mammarenavirus Tamiami (TAMV-FL), which we found capable of utilizing hTfR1 to enter mammalian cells. During isolation through serial passaging in mammalian immunocompetent cells, the quasispecies of TAMV-FL acquired and enriched mutations leading to the amino acid changes N151K and D156N, within GP. Cell entry studies revealed that both substitutions, N151K and D156N, increased dependence of the virus on hTfR1 and binding to heparan sulfate proteoglycans. Moreover, we show that the substituted residues likely map to the sterically constrained trimeric axis of GP, and facilitate viral fusion at a lower pH, resulting in viral egress from later endosomal compartments. In summary, we identify and characterize a naturally occurring TAMV strain (TAMV-FL) within ticks that is able to utilize hTfR1. The TAMV-FL significantly diverged from previous TAMV isolates, demonstrating that TAMV quasispecies exhibit striking genetic plasticity that may facilitate zoonotic spillover and rapid adaptation to new hosts.

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  • 41.
    Nicolay, Wiebke
    et al.
    TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Moeller, Rebecca
    TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany; Center for Emerging Infections and Zoonoses (RIZ), Institute of Biochemistry & Research, University of Veterinary Medicine Hannover, Hannover, Germany.
    Kahl, Sina
    TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Vondran, Florian W. R.
    Department of General, Visceral and Transplant Surgery, Hannover Medical School, Hannover, Germany; German Centre for Infection Research (DZIF), Braunschweig, Germany.
    Pietschmann, Thomas
    TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany.
    Kunz, Stefan
    Institute of Microbiology, Lausanne University Hospital, Lausanne, Switzerland.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. TWINCORE—Centre for Experimental and Clinical Infection Research, Institute for Experimental Virology, Hannover, Germany; Center for Emerging Infections and Zoonoses (RIZ), Institute of Biochemistry & Research, University of Veterinary Medicine Hannover, Hannover, Germany.
    Characterization of rna sensing pathways in hepatoma cell lines and primary human hepatocytes2021In: Cells, E-ISSN 2073-4409, Vol. 10, no 11, article id 3019Article in journal (Refereed)
    Abstract [en]

    The liver is targeted by several human pathogenic RNA viruses for viral replication and dissemination; despite this, the extent of innate immune sensing of RNA viruses by human hepatocytes is insufficiently understood to date. In particular, for highly human tropic viruses such as hepatitis C virus, cell culture models are needed to study immune sensing. However, several human hepatoma cell lines have impaired RNA sensing pathways and fail to mimic innate immune responses in the human liver. Here we compare the RNA sensing properties of six human hepatoma cell lines, namely Huh-6, Huh-7, HepG2, HepG2-HFL, Hep3B, and HepaRG, with primary human hepatocytes. We show that primary liver cells sense RNA through retinoic acid-inducible gene I (RIG-I) like receptor (RLR) and Toll-like receptor 3 (TLR3) pathways. Of the tested cell lines, Hep3B cells most closely mimicked the RLR and TLR3 mediated sensing in primary hepatocytes. This was shown by the expression of RLRs and TLR3 as well as the expression and release of bioactive interferon in primary hepatocytes and Hep3B cells. Our work shows that Hep3B cells partially mimic RNA sensing in primary hepatocytes and thus can serve as in vitro model to study innate immunity to RNA viruses in hepatocytes.

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  • 42. Palor, Machaela
    et al.
    Stejskal, Lenka
    Mandal, Piya
    Lenman, Annasara
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover and the Helmholtz Centre for Infection Research, Hannover, Germany.
    Alberione, Maria Pia
    Kirui, Jared
    Moeller, Rebecca
    Ebner, Stefan
    Meissner, Felix
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Shepherd, Adrian J.
    Grove, Joe
    Cholesterol sensing by CD81 is important for hepatitis C virus entry2020In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 295, no 50, p. 16931-16948Article in journal (Refereed)
    Abstract [en]

    CD81 plays a role in a variety of physiological and pathological processes. Recent structural analysis of CD81 indicates that it contains an intramembrane cholesterol-binding pocket and that interaction with cholesterol may regulate a conformational switch in the extracellular domain of CD81. Therefore, CD81 possesses a potential cholesterol sensing mechanism; however, its relevance for protein function is thus far unknown. In this study we investigate CD81 cholesterol sensing in the context of its activity as a receptor for hepatitis C virus. Structure-led mutagenesis of the cholesterol-binding pocket reduced CD81-cholesterol association, but had disparate effects on HCV, both reducing and enhancing CD81 receptor activity. We reasoned that this could be explained by alterations in the consequences of cholesterol binding. To investigate this further we performed molecular dynamic simulations of CD81 with and without cholesterol; this identified an allosteric mechanism by which cholesterol binding regulates the conformation of CD81. To test this, we designed further mutations to force CD81 into either the open (cholesterol unbound) or closed (cholesterol bound) conformation. The open mutant of CD81 exhibited reduced receptor activity whereas the closed mutant was enhanced. These data are consistent with cholesterol switching CD81 between a receptor active and inactive state. CD81 interactome analysis also suggests that conformational switching may modulate the assembly of CD81-partner networks. This work furthers our understanding of the molecular mechanism of CD81 cholesterol sensing, how this relates to HCV entry and CD81's function as a molecular scaffold; these insights are relevant to CD81's varied roles in health and disease.

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  • 43.
    Passos, Vania
    et al.
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Henkel, Lisa M.
    Department of Neurology, Hannover Medical School, Hannover, Germany.
    Wang, Jiayi
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Zapatero-Belinchón, Francisco J.
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, Hannover, Germany.
    Möller, Rebecca
    University of Veterinary Medicine Hannover, Foundation, Hannover, Germany.
    Sun, Guorong
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Waltl, Inken
    Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany.
    Schneider, Talia
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Wachs, Amelie
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Ritter, Birgit
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Kropp, Kai A.
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Zhu, Shuyong
    Hannover Medical School, Institute of Virology, Hannover, Germany.
    Deleidi, Michela
    Center of Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Tübingen, Germany; German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany.
    Kalinke, Ulrich
    Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, Hannover, Germany; Institute for Experimental Infection Research, TWINCORE, Centre for Experimental and Clinical Infection Research, A Joint Venture between the Helmholtz Centre for Infection Research and the Hannover Medical School, Hannover, Germany.
    Schulz, Thomas F.
    Hannover Medical School, Institute of Virology, Hannover, Germany; Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, Hannover, Germany.
    Höglinger, Günter
    Department of Neurology, Hannover Medical School, Hannover, Germany; Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology. Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). University of Veterinary Medicine Hannover, Foundation, Hannover, Germany; Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, Hannover, Germany.
    Wegner, Florian
    Department of Neurology, Hannover Medical School, Hannover, Germany.
    Viejo-Borbolla, Abel
    Hannover Medical School, Institute of Virology, Hannover, Germany; Cluster of Excellence-Resolving Infection Susceptibility (RESIST), Hannover Medical School, Hannover, Germany.
    Innate immune response to SARS-CoV-2 infection contributes to neuronal damage in human iPSC-derived peripheral neurons2024In: Journal of Medical Virology, ISSN 0146-6615, E-ISSN 1096-9071, Vol. 96, no 2, article id e29455Article in journal (Refereed)
    Abstract [en]

    Severe acute respiratory coronavirus 2 (SARS-CoV-2) causes neurological disease in the peripheral and central nervous system (PNS and CNS, respectively) of some patients. It is not clear whether SARS-CoV-2 infection or the subsequent immune response are the key factors that cause neurological disease. Here, we addressed this question by infecting human induced pluripotent stem cell-derived CNS and PNS neurons with SARS-CoV-2. SARS-CoV-2 infected a low number of CNS neurons and did not elicit a robust innate immune response. On the contrary, SARS-CoV-2 infected a higher number of PNS neurons. This resulted in expression of interferon (IFN) λ1, several IFN-stimulated genes and proinflammatory cytokines. The PNS neurons also displayed alterations characteristic of neuronal damage, as increased levels of sterile alpha and Toll/interleukin receptor motif-containing protein 1, amyloid precursor protein and α-synuclein, and lower levels of cytoskeletal proteins. Interestingly, blockade of the Janus kinase and signal transducer and activator of transcription pathway by Ruxolitinib did not increase SARS-CoV-2 infection, but reduced neuronal damage, suggesting that an exacerbated neuronal innate immune response contributes to pathogenesis in the PNS. Our results provide a basis to study coronavirus disease 2019 (COVID-19) related neuronal pathology and to test future preventive or therapeutic strategies.

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  • 44.
    Ricke-Hoch, Melanie
    et al.
    Department of Cardiology and Angiology, Hannover Medical School, Hanover, Germany.
    Stelling, Elisabeth
    Department of Cardiology and Angiology, Hannover Medical School, Hanover, Germany.
    Lasswitz, Lisa
    Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hanover, Germany.
    Gunesch, Antonia P.
    Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hanover, Germany; German Center for Infection Research, Hanover-Braunschweig Site, Braunschweig, Germany; Department of Gastroenterology, Hepatology and Endocrinology, Hannover Medical School, Hanover, Germany.
    Kasten, Martina
    Department of Cardiology and Angiology, Hannover Medical School, Hanover, Germany.
    Zapatero-Belinchón, Francisco J.
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hanover, Germany.
    Brogden, Graham
    Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hanover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hanover, Germany; Department of Biochemistry, University of Veterinary Medicine Hannover, Hanover, Germany.
    Pietschmann, Thomas
    Institute of Experimental Virology, TWINCORE, Center for Experimental and Clinical Infection Research Hannover, Hanover, Germany; German Center for Infection Research, Hanover-Braunschweig Site, Braunschweig, Germany.
    Montiel, Virginie
    Pole of Pharmacology and Therapeutics, Institut de Recherche Expérimentale et Clinique, Cliniques Universitaires Saint-Luc, Université catholique de Louvain (UCLouvain), Brussels, Belgium.
    Balligand, Jean-Luc
    Pole of Pharmacology and Therapeutics, Institut de Recherche Expérimentale et Clinique, Cliniques Universitaires Saint-Luc, Université catholique de Louvain (UCLouvain), Brussels, Belgium.
    Facciotti, Federica
    Department of Experimental Oncology, European Institute of Oncology IRCCS, Milan, Italy.
    Hirsch, Emilio
    Department of Molecular Biotechnology and Health Sciences, Molecular Biotechnology Center, University of Torino, Torino, Italy.
    Gausepohl, Thomas
    Department of Cardiology and Angiology, Hannover Medical School, Hanover, Germany.
    Elbahesh, Husni
    Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine in Hannover (TiHo), Hannover, Germany.
    Rimmelzwaan, Guus F.
    Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine in Hannover (TiHo), Hannover, Germany.
    Höfer, Anne
    Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), German Center for Lung Research, Hanover, Germany; Institute for Pathology, Hannover Medical School, Hanover, Germany.
    Kühnel, Mark P.
    Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), German Center for Lung Research, Hanover, Germany; Institute for Pathology, Hannover Medical School, Hanover, Germany.
    Jonigk, Danny
    Biomedical Research in Endstage and Obstructive Lung Disease (BREATH), German Center for Lung Research, Hanover, Germany; Institute for Pathology, Hannover Medical School, Hanover, Germany.
    Eigendorf, Julian
    Institute of Sports Medicine, Hannover Medical School, Hanover, Germany.
    Tegtbur, Uwe
    Institute of Sports Medicine, Hannover Medical School, Hanover, Germany.
    Mink, Lena
    Institute of Sports Medicine, Hannover Medical School, Hanover, Germany.
    Scherr, Michaela
    Department of Hematology, Hemostasis, Oncology and Stem Cell Transplantation, Hannover Medical School, Hanover, Germany.
    Illig, Thomas
    Hannover Unified Biobank (HUB), Hannover Medical School, Hanover, Germany.
    Schambach, Axel
    Institute of Experimental Hematology, Hannover Medical School, Hanover, Germany; Division of Hematology and Oncology, Boston Children’s Hospital, Harvard Medical School, MA, Boston, United States.
    Pfeffer, Tobias J.
    Department of Cardiology and Angiology, Hannover Medical School, Hanover, Germany.
    Hilfiker, Andres
    Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hanover, Germany.
    Haverich, Axel
    Department of Cardiac, Thoracic, Transplantation and Vascular Surgery, Hannover Medical School, Hanover, Germany.
    Hilfiker-Kleiner, Denise
    Department of Cardiology and Angiology, Hannover Medical School, Hanover, Germany; Department of Cardiovascular Complications of Oncologic Therapies, Medical Faculty of the Philipps University Marburg, Marburg, Germany.
    Impaired immune response mediated by prostaglandin E2 promotes severe COVID-19 disease2021In: PLOS ONE, E-ISSN 1932-6203, Vol. 16, no 8, article id e0255335Article in journal (Refereed)
    Abstract [en]

    The SARS-CoV-2 coronavirus has led to a pandemic with millions of people affected. The present study finds that risk-factors for severe COVID-19 disease courses, i.e. male sex, older age and sedentary life style are associated with higher prostaglandin E2 (PGE2) serum levels in blood samples from unaffected subjects. In COVID-19 patients, PGE2 blood levels are markedly elevated and correlate positively with disease severity. SARS-CoV-2 induces PGE2 generation and secretion in infected lung epithelial cells by upregulating cyclo-oxygenase (COX)-2 and reducing the PG-degrading enzyme 15-hydroxyprostaglan-din-dehydrogenase. Also living human precision cut lung slices (PCLS) infected with SARS-CoV-2 display upregulated COX-2. Regular exercise in aged individuals lowers PGE2 serum levels, which leads to increased Paired-Box-Protein-Pax-5 (PAX5) expression, a master regulator of B-cell survival, proliferation and differentiation also towards long lived memory B-cells, in human pre-B-cell lines. Moreover, PGE2 levels in serum of COVID-19 patients lowers the expression of PAX5 in human pre-B-cell lines. The PGE2 inhibitor Taxifolin reduces SARS-CoV-2-induced PGE2 production. In conclusion, SARS-CoV-2, male sex, old age, and sedentary life style increase PGE2 levels, which may reduce the early anti-viral defense as well as the development of immunity promoting severe disease courses and multiple infections. Regular exercise and Taxifolin treatment may reduce these risks and prevent severe disease courses.

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  • 45. Scull, Margaret A
    et al.
    Shi, Chao
    de Jong, Ype P
    Gerold, Gisa
    Ries, Moritz
    von Schaewen, Markus
    Donovan, Bridget M
    Labitt, Rachael N
    Horwitz, Joshua A
    Gaska, Jenna M
    Hrebikova, Gabriela
    Xiao, Jing W
    Flatley, Brenna
    Fung, Canny
    Chiriboga, Luis
    Walker, Christopher M
    Evans, David T
    Rice, Charles M
    Ploss, Alexander
    Hepatitis C virus infects rhesus macaque hepatocytes and simianized mice.2015In: Hepatology, ISSN 0270-9139, E-ISSN 1527-3350, Vol. 62, no 1, p. 57-67Article in journal (Refereed)
    Abstract [en]

    UNLABELLED: At least 170 million people are chronically infected with hepatitis C virus (HCV). Owing to the narrow host range of HCV and restricted use of chimpanzees, there is currently no suitable animal model for HCV pathogenesis studies or the development of a HCV vaccine. To identify cellular determinants of interspecies transmission and establish a novel immunocompetent model system, we examined the ability of HCV to infect hepatocytes from a small nonhuman primate, the rhesus macaque (Macaca mulatta). We show that the rhesus orthologs of critical HCV entry factors support viral glycoprotein-dependent virion uptake. Primary hepatocytes from rhesus macaques are also permissive for HCV-RNA replication and particle production, which is enhanced when antiviral signaling is suppressed. We demonstrate that this may be owing to the diminished capacity of HCV to antagonize mitochondrial antiviral-signaling protein-dependent innate cellular defenses. To test the ability of HCV to establish persistent replication in vivo, we engrafted primary rhesus macaque hepatocytes into immunocompromised xenorecipients. Inoculation of resulting simian liver chimeric mice with either HCV genotype 1a or 2a resulted in HCV serum viremia for up to 10 weeks.

    CONCLUSION: Together, these data indicate that rhesus macaques may be a viable model for HCV and implicate host immunity as a potential species-specific barrier to HCV infection. We conclude that suppression of host immunity or further viral adaptation may allow robust HCV infection in rhesus macaques and creation of a new animal model for studies of HCV pathogenesis, lentivirus coinfection, and vaccine development.

  • 46. Uckeley, Zina M.
    et al.
    Moeller, Rebecca
    Kuhn, Lars I.
    Nilsson, Emma
    Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS).
    Robens, Claudia
    Lasswitz, Lisa
    Lindquist, Richard
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Lenman, Annasara
    Passos, Vania
    Voss, Yannik
    Sommerauer, Christian
    Kampmann, Martin
    Goffinet, Christine
    Meissner, Felix
    Överby, Anna K.
    Umeå University, Faculty of Medicine, Molecular Infection Medicine Sweden (MIMS). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology.
    Lozach, Pierre-Yves
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM).
    Quantitative Proteomics of Uukuniemi Virus-host Cell Interactions Reveals GBF1 as Proviral Host Factor for Phleboviruses2019In: Molecular & Cellular Proteomics, ISSN 1535-9476, E-ISSN 1535-9484, Vol. 18, no 12, p. 2401-2417Article in journal (Refereed)
    Abstract [en]

    Novel tick-borne phleboviruses in the Phenuiviridae family, which are highly pathogenic in humans and all closely related to Uukuniemi virus (UUKV), have recently emerged on different continents. How phleboviruses assemble, bud, and exit cells remains largely elusive. Here, we performed high-resolution, label-free mass spectrometry analysis of UUKV immunoprecipitated from cell lysates and identified 39 cellular partners interacting with the viral envelope glycoproteins. The importance of these host factors for UUKV infection was validated by silencing each host factor by RNA interference. This revealed Golgi-specific brefeldin A-resistance guanine nucleotide exchange factor 1 (GBF1), a guanine nucleotide exchange factor resident in the Golgi, as a critical host factor required for the UUKV life cycle. An inhibitor of GBF1, Golgicide A, confirmed the role of the cellular factor in UUKV infection. We could pinpoint the GBF1 requirement to UUKV replication and particle assembly. When the investigation was extended to viruses from various positive and negative RNA viral families, we found that not only phleboviruses rely on GBF1 for infection, but also Flavi-, Corona-, Rhabdo-, and Togaviridae. In contrast, silencing or blocking GBF1 did not abrogate infection by the human adenovirus serotype 5 and immunodeficiency retrovirus type 1, the replication of both requires nuclear steps. Together our results indicate that UUKV relies on GBF1 for viral replication, assembly and egress. This study also highlights the proviral activity of GBF1 in the infection by a broad range of important zoonotic RNA viruses. Ticks are important vectors of infectious emerging diseases and tick-borne phleboviruses represent a growing threat to humans globally. We employed here a high-resolution, label-free mass spectrometry and RNA interference screen approach to reveal the host cell protein GBF1 as a proviral factor, not only for tick-borne phleboviruses, but also for many other important zoonotic RNA viruses. This study lays the basis for the development of innovative antiviral strategies against a broad range of human pathogenic viruses.

  • 47.
    Victoria, Catherine
    et al.
    Institute of Organic Chemistry, Leibniz University Hannover, Schneiderberg 1B, Hannover, Germany.
    Schulz, Göran
    Institute of Organic Chemistry, Leibniz University Hannover, Schneiderberg 1B, Hannover, Germany.
    Klöhn, Mara
    Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany.
    Weber, Saskia
    Federal Research Institute in Animal Health (FLI), Südufer 10, Insel Riems, Greifswald, Germany.
    Holicki, Cora M.
    Federal Research Institute in Animal Health (FLI), Südufer 10, Insel Riems, Greifswald, Germany.
    Brüggemann, Yannick
    Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany.
    Becker, Miriam
    Institute for Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Bünteweg 2, Hannover, Germany.
    Gerold, Gisa
    Umeå University, Faculty of Medicine, Wallenberg Centre for Molecular Medicine at Umeå University (WCMM). Umeå University, Faculty of Medicine, Department of Clinical Microbiology, Section of Virology. Institute for Biochemistry, Research Center for Emerging Infections and Zoonoses (RIZ), University of Veterinary Medicine Hannover, Bünteweg 2, Hannover, Germany.
    Eiden, Martin
    Federal Research Institute in Animal Health (FLI), Südufer 10, Insel Riems, Greifswald, Germany.
    Groschup, Martin H.
    Federal Research Institute in Animal Health (FLI), Südufer 10, Insel Riems, Greifswald, Germany.
    Steinmann, Eike
    Department of Molecular and Medical Virology, Ruhr-University Bochum, Bochum, Germany.
    Kirschning, Andreas
    Institute of Organic Chemistry, Leibniz University Hannover, Schneiderberg 1B, Hannover, Germany.
    Halogenated rocaglate derivatives: pan-antiviral agents against hepatitis E virus and emerging viruses2024In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 67, no 1, p. 289-321Article in journal (Refereed)
    Abstract [en]

    The synthesis of a library of halogenated rocaglate derivatives belonging to the flavagline class of natural products, of which silvestrol is the most prominent example, is reported. Their antiviral activity and cytotoxicity profile against a wide range of pathogenic viruses, including hepatitis E, Chikungunya, Rift Valley Fever virus and SARS-CoV-2, were determined. The incorporation of halogen substituents at positions 4′, 6 and 8 was shown to have a significant effect on the antiviral activity of rocaglates, some of which even showed enhanced activity compared to CR-31-B and silvestrol.

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  • 48. Vieyres, Gabrielle
    et al.
    Welsch, Kathrin
    Gerold, Gisa
    Institute of Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research; a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Hannover, Germany.
    Gentzsch, Juliane
    Kahl, Sina
    Vondran, Florian W R
    Kaderali, Lars
    Pietschmann, Thomas
    ABHD5/CGI-58, the Chanarin-Dorfman Syndrome Protein, Mobilises Lipid Stores for Hepatitis C Virus Production2016In: PLoS Pathogens, ISSN 1553-7366, E-ISSN 1553-7374, Vol. 12, no 4, article id e1005568Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) particles closely mimic human very-low-density lipoproteins (VLDL) to evade humoral immunity and to facilitate cell entry. However, the principles that govern HCV association with VLDL components are poorly defined. Using an siRNA screen, we identified ABHD5 (α/β hydrolase domain containing protein 5, also known as CGI-58) as a new host factor promoting both virus assembly and release. ABHD5 associated with lipid droplets and triggered their hydrolysis. Importantly, ABHD5 Chanarin-Dorfman syndrome mutants responsible for a rare lipid storage disorder in humans were mislocalised, and unable to consume lipid droplets or support HCV production. Additional ABHD5 mutagenesis revealed a novel tribasic motif that does not influence subcellular localization but determines both ABHD5 lipolytic and proviral properties. These results indicate that HCV taps into the lipid droplet triglyceride reservoir usurping ABHD5 lipase cofactor function. They also suggest that the resulting lipid flux, normally devoted to VLDL synthesis, also participates in the assembly and release of the HCV lipo-viro-particle. Altogether, our study provides the first association between the Chanarin-Dorfman syndrome protein and an infectious disease and sheds light on the hepatic manifestations of this rare genetic disorder as well as on HCV morphogenesis.

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  • 49. Vogt, Alexander
    et al.
    Scull, Margaret A
    Friling, Tamar
    Horwitz, Joshua A
    Donovan, Bridget M
    Dorner, Marcus
    Gerold, Gisa
    Center for the Study of Hepatitis C, The Rockefeller University, New York, NY 10065, USA.
    Labitt, Rachael N
    Rice, Charles M
    Ploss, Alexander
    Recapitulation of the hepatitis C virus life-cycle in engineered murine cell lines2013In: Virology, ISSN 0042-6822, E-ISSN 1096-0341, Vol. 444, no 1-2, p. 1-11Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) remains a major medical problem. In-depth study of HCV pathogenesis and immune responses is hampered by the lack of suitable small animal models. The narrow host range of HCV remains incompletely understood. We demonstrate that the entire HCV life-cycle can be recapitulated in mouse cells. We show that antiviral signaling interferes with HCV RNA replication in mouse cells. We were able to infect mouse cells expressing human CD81 and occludin (OCLN)-the minimal set of entry factor factors required for HCV uptake into mouse cells. Infected mouse cells sustain HCV RNA replication in the presence of miR122 and release infectious particles when mouse apoE is supplied. Our data demonstrate that the barriers of HCV interspecies transmission can be overcome by engineering a suitable cellular environment and provide a blue-print towards constructing a small animal model for HCV infection.

  • 50. von Schaewen, Markus
    et al.
    Dorner, Marcus
    Hueging, Kathrin
    Foquet, Lander
    Gerges, Sherif
    Hrebikova, Gabriela
    Heller, Brigitte
    Bitzegeio, Julia
    Doerrbecker, Juliane
    Horwitz, Joshua A
    Gerold, Gisa
    Institute for Experimental Virology, TWINCORE, Centre for Experimental and Clinical Infection Research, Hannover, Germany.
    Suerbaum, Sebastian
    Rice, Charles M
    Meuleman, Philip
    Pietschmann, Thomas
    Ploss, Alexander
    Expanding the Host Range of Hepatitis C Virus through Viral Adaptation2016In: mBio, ISSN 2161-2129, E-ISSN 2150-7511, Vol. 7, no 6, article id e01915-16Article in journal (Refereed)
    Abstract [en]

    Hepatitis C virus (HCV) species tropism is incompletely understood. We have previously shown that at the level of entry, human CD81 and occludin (OCLN) comprise the minimal set of human factors needed for viral uptake into murine cells. As an alternative approach to genetic humanization, species barriers can be overcome by adapting HCV to use the murine orthologues of these entry factors. We previously generated a murine tropic HCV (mtHCV or Jc1/mCD81) strain harboring three mutations within the viral envelope proteins that allowed productive entry into mouse cell lines. In this study, we aimed to characterize the ability of mtHCV to enter and infect mouse hepatocytes in vivo and in vitro Using a highly sensitive, Cre-activatable reporter, we demonstrate that mtHCV can enter mouse hepatocytes in vivo in the absence of any human cofactors. Viral entry still relied on expression of mouse CD81 and SCARB1 and was more efficient when mouse CD81 and OCLN were overexpressed. HCV entry could be significantly reduced in the presence of anti-HCV E2 specific antibodies, suggesting that uptake of mtHCV is dependent on viral glycoproteins. Despite mtHCV's ability to enter murine hepatocytes in vivo, we did not observe persistent infection, even in animals with severely blunted type I and III interferon signaling and impaired adaptive immune responses. Altogether, these results establish proof of concept that the barriers limiting HCV species tropism can be overcome by viral adaptation. However, additional viral adaptations will likely be needed to increase the robustness of a murine model system for hepatitis C.

    IMPORTANCE: At least 150 million individuals are chronically infected with HCV and are at risk of developing serious liver disease. Despite the advent of effective antiviral therapy, the frequency of chronic carriers has only marginally decreased. A major roadblock in developing a vaccine that would prevent transmission is the scarcity of animal models that are susceptible to HCV infection. It is poorly understood why HCV infects only humans and chimpanzees. To develop an animal model for hepatitis C, previous efforts focused on modifying the host environment of mice, for example, to render them more susceptible to HCV infection. Here, we attempted a complementary approach in which a laboratory-derived HCV variant was tested for its ability to infect mice. We demonstrate that this engineered HCV strain can enter mouse liver cells but does not replicate efficiently. Thus, additional adaptations are likely needed to construct a robust animal model for HCV.

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