Publications by authors named "Frank Kirchhoff"

297 Publications

Systematic functional analysis of SARS-CoV-2 proteins uncovers viral innate immune antagonists and remaining vulnerabilities.

Cell Rep 2021 Apr 27:109126. Epub 2021 Apr 27.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany. Electronic address:

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) evades most innate immune responses but may still be vulnerable to some. Here, we systematically analyze the impact of SARS-CoV-2 proteins on interferon (IFN) responses and autophagy. We show that SARS-CoV-2 proteins synergize to counteract anti-viral immune responses. For example, Nsp14 targets the type I IFN receptor for lysosomal degradation, ORF3a prevents fusion of autophagosomes and lysosomes, and ORF7a interferes with autophagosome acidification. Most activities are evolutionarily conserved. However, SARS-CoV-2 Nsp15 antagonizes IFN signaling less efficiently than the orthologs of closely related RaTG13-CoV and SARS-CoV-1. Overall, SARS-CoV-2 proteins counteract autophagy and type I IFN more efficiently than type II or III IFN signaling, and infection experiments confirm potent inhibition by IFN-γ and -λ1. Our results define the repertoire and selected mechanisms of SARS-CoV-2 innate immune antagonists but also reveal vulnerability to type II and III IFN that may help to develop safe and effective anti-viral approaches.
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http://dx.doi.org/10.1016/j.celrep.2021.109126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8078906PMC
April 2021

Pen-2 negatively regulates the differentiation of oligodendrocyte precursor cells into astrocytes in the central nervous system.

J Neurosci 2021 May 7. Epub 2021 May 7.

State Key Laboratory of Pharmaceutical Biotechnology, MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center, Nanjing University, 12 Xuefu Avenue, Nanjing, Jiangsu Province, China, 210061.

Mutations on γ-secretase subunits are associated with neurological diseases. Whereas the role of γ-secretase in neurogenesis has been intensively studied, little is known about its role in astrogliogenesis. Recent evidence has demonstrated that astrocytes can be generated from oligodendrocyte (OL) precursor cells (OPCs). However, it is not well understood what mechanism may control OPCs to differentiate into astrocytes. To address the above questions, we generated two independent lines of OL lineage specific presenilin enhancer 2 () conditional knockout (cKO) mice. Both male and female mice were used. Here we demonstrate that conditional inactivation of Pen-2 mediated by or causes enhanced generation of astrocytes. Lineage-tracing experiments indicate that abnormally generated astrocytes are derived from Cre-expressing OPCs in the central nervous system (CNS) in cKO mice. Mechanistic analysis reveals that deletion of Pen-2 inhibits the Notch signaling to up-regulate signal transducer and activator of transcription 3 (Stat3), which triggers activation of GFAP to promote astrocyte differentiation. Together, these novel findings indicate that Pen-2 regulates the specification of astrocytes from OPCs through the Stat3 signaling.Astrocytes and oligodendrocyte (OLs) play critical roles in the brain. Recent evidence has demonstrated that astrocytes can be generated from OL precursor cells (OPCs). However, it remains poorly understood what mechanism governs the differentiation of OPCs into astrocytes. In this study, we took advantage of OL lineage cells specific presenilin enhancer 2 (Pen-2) conditional knockout (cKO) mice. We show that deletion of Pen-2 leads to dramatically enhanced astrocyte differentiation from OPCs in the central nervous system. Mechanistic analysis reveals that deletion of Pen-2 inhibits Hes1 and activates Stat3 to trigger GFAP activation which promotes astrocyte differentiation. Overall, this study identifies a novel function of Pen-2 in astrogliogenesis from OPCs.
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http://dx.doi.org/10.1523/JNEUROSCI.2455-19.2021DOI Listing
May 2021

Oligodendroglial GABAergic Signaling: More Than Inhibition!

Neurosci Bull 2021 Apr 29. Epub 2021 Apr 29.

Molecular Physiology, Center for Integrative Physiology and Molecular Medicine (CIPMM), University of Saarland, 66421, Homburg, Germany.

GABA is the main inhibitory neurotransmitter in the CNS acting at two distinct types of receptor: ligand-gated ionotropic GABA receptors and G protein-coupled metabotropic GABA receptors, thus mediating fast and slow inhibition of excitability at central synapses. GABAergic signal transmission has been intensively studied in neurons in contrast to oligodendrocytes and their precursors (OPCs), although the latter express both types of GABA receptor. Recent studies focusing on interneuron myelination and interneuron-OPC synapses have shed light on the importance of GABA signaling in the oligodendrocyte lineage. In this review, we start with a short summary on GABA itself and neuronal GABAergic signaling. Then, we elaborate on the physiological role of GABA receptors within the oligodendrocyte lineage and conclude with a description of these receptors as putative targets in treatments of CNS diseases.
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http://dx.doi.org/10.1007/s12264-021-00693-wDOI Listing
April 2021

Structural basis for GTP-induced dimerization and antiviral function of guanylate-binding proteins.

Proc Natl Acad Sci U S A 2021 Apr;118(15)

School of Life Sciences, Tianjin University, Tianjin, 300072, China;

Guanylate-binding proteins (GBPs) form a family of dynamin-related large GTPases which mediate important innate immune functions. They were proposed to form oligomers upon GTP binding/hydrolysis, but the molecular mechanisms remain elusive. Here, we present crystal structures of C-terminally truncated human GBP5 (hGBP5), comprising the large GTPase (LG) and middle (MD) domains, in both its nucleotide-free monomeric and nucleotide-bound dimeric states, together with nucleotide-free full-length human GBP2. Upon GTP-loading, hGBP5 forms a closed face-to-face dimer. The MD of hGBP5 undergoes a drastic movement relative to its LG domain and forms extensive interactions with the LG domain and MD of the pairing molecule. Disrupting the MD interface (for hGBP5) or mutating the hinge region (for hGBP2/5) impairs their ability to inhibit HIV-1. Our results point to a GTP-induced dimerization mode that is likely conserved among all GBP members and provide insights into the molecular determinants of their antiviral function.
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http://dx.doi.org/10.1073/pnas.2022269118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8054025PMC
April 2021

HIV protease: late action to prevent immune detection.

Signal Transduct Target Ther 2021 Apr 16;6(1):157. Epub 2021 Apr 16.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

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http://dx.doi.org/10.1038/s41392-021-00588-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8052432PMC
April 2021

Intravital quantification reveals dynamic calcium concentration changes across B cell differentiation stages.

Elife 2021 Mar 22;10. Epub 2021 Mar 22.

Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin and Humboldt-Universität zu Berlin, Department of Rheumatology and Clinical Immunology, Charitéplatz 1, Berlin, Germany.

Calcium is a universal second messenger present in all eukaryotic cells. The mobilization and storage of Ca ions drives a number of signaling-related processes, stress-responses, or metabolic changes, all of which are relevant for the development of immune cells and their adaption to pathogens. Here, we introduce the Förster resonance energy transfer (FRET)-reporter mouse YellowCaB expressing the genetically encoded calcium indicator TN-XXL in B lymphocytes. Calcium-induced conformation change of TN-XXL results in FRET-donor quenching measurable by two-photon fluorescence lifetime imaging. For the first time, using our novel numerical analysis, we extract absolute cytoplasmic calcium concentrations in activated B cells during affinity maturation in vivo. We show that calcium in activated B cells is highly dynamic and that activation introduces a persistent calcium heterogeneity to the lineage. A characterization of absolute calcium concentrations present at any time within the cytosol is therefore of great value for the understanding of long-lived beneficial immune responses and detrimental autoimmunity.
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http://dx.doi.org/10.7554/eLife.56020DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8060033PMC
March 2021

Alpha-1 antitrypsin inhibits TMPRSS2 protease activity and SARS-CoV-2 infection.

Nat Commun 2021 03 19;12(1):1726. Epub 2021 Mar 19.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

SARS-CoV-2 is a respiratory pathogen and primarily infects the airway epithelium. As our knowledge about innate immune factors of the respiratory tract against SARS-CoV-2 is limited, we generated and screened a peptide/protein library derived from bronchoalveolar lavage for inhibitors of SARS-CoV-2 spike-driven entry. Analysis of antiviral fractions revealed the presence of α-antitrypsin (αAT), a highly abundant circulating serine protease inhibitor. Here, we report that αAT inhibits SARS-CoV-2 entry at physiological concentrations and suppresses viral replication in cell lines and primary cells including human airway epithelial cultures. We further demonstrate that αAT binds and inactivates the serine protease TMPRSS2, which enzymatically primes the SARS-CoV-2 spike protein for membrane fusion. Thus, the acute phase protein αAT is an inhibitor of TMPRSS2 and SARS-CoV-2 entry, and may play an important role in the innate immune defense against the novel coronavirus. Our findings suggest that repurposing of αAT-containing drugs has prospects for the therapy of COVID-19.
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http://dx.doi.org/10.1038/s41467-021-21972-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7979852PMC
March 2021

SIV-induced terminally differentiated adaptive NK cells in lymph nodes associated with enhanced MHC-E restricted activity.

Nat Commun 2021 02 24;12(1):1282. Epub 2021 Feb 24.

Institut Pasteur, Unité HIV, Inflammation et Persistance, Paris, France.

Natural killer (NK) cells play a critical understudied role during HIV infection in tissues. In a natural host of SIV, the African green monkey (AGM), NK cells mediate a strong control of SIVagm infection in secondary lymphoid tissues. We demonstrate that SIVagm infection induces the expansion of terminally differentiated NKG2a NK cells in secondary lymphoid organs displaying an adaptive transcriptional profile and increased MHC-E-restricted cytotoxicity in response to SIV Env peptides while expressing little IFN-γ. Such NK cell differentiation was lacking in SIVmac-infected macaques. Adaptive NK cells displayed no increased NKG2C expression. This study reveals a previously unknown profile of NK cell adaptation to a viral infection, thus accelerating strategies toward NK-cell directed therapies and viral control in tissues.
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http://dx.doi.org/10.1038/s41467-021-21402-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7904927PMC
February 2021

Astrocyte Intracellular Caand TrkB Signaling in the Hippocampus Could Be Involved in the Beneficial Behavioral Effects of Antidepressant Treatment.

Neurotox Res 2021 Jun 22;39(3):860-871. Epub 2021 Feb 22.

Department of Pharmacology, School of Medicine of Ribeirão Preto, University of São Paulo, Ribeirão Preto-SP, Brazil.

Although monoaminergic-based antidepressant drugs are largely used to treat major depressive disorder (MDD), their mechanisms are still incompletely understood. Intracellular Ca (iCa) and Calmodulin 1(CaM-1) homeostasis have been proposed to participate in the therapeutic effects of these compounds. We investigated whether intra-hippocampal inhibition of CaM-1 would modulate the behavioral responses to chronic treatment with imipramine (IMI) or 7-nitroindazole (7-NI), a selective inhibitor of the neuronal nitric oxide synthase 1 (NOS1) enzyme that shows antidepressant-like effects. We also investigated the interactions of IMI and CaM-1 on transient astrocyte iCa evoked by glutamate stimuli. Intra-hippocampal microinjection of the lentiviral delivered (LV) short hairpin iRNA-driven against the CaM-1 mRNA (LV-shRNA-CaM-1) or the CaM-1 inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalene sulphonamide (W-7) blocked the antidepressant-like effect of chronic treatment with IMI or 7-NI. The shRNA also inhibited the mRNA expression of the tropomyosin receptor kinase B (TrkB) in the microinjection region. The iCa in ex vivo hippocampus slices stained with fluorescent Caindicator Oregon Green 488 BAPTA-1 revealed that IMI increased the intensity and duration of iCa oscillation and reduced the number of events evoked by glutamate stimuli, evaluated by using CCD imaging and the % ΔF/Fo parameters. The pre-treatment with W-7 fully antagonized this effect. The present results indicate that the behavioral benefits of chronic antidepressant treatment might be associated with astrocyte intracellular Cadynamics and TrkB mRNA expression in the hippocampus.
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http://dx.doi.org/10.1007/s12640-021-00334-0DOI Listing
June 2021

SERINC5 Can Enhance Proinflammatory Cytokine Production by Primary Human Myeloid Cells in Response to Challenge with HIV-1 Particles.

J Virol 2021 04 12;95(9). Epub 2021 Apr 12.

Department of Infectious Diseases, Integrative Virology, University Hospital Heidelberg, Heidelberg, Germany

HIV-1 has to overcome physical barriers posed by host cell restriction factors (RFs) for efficient replication. Some RFs, including Trim5α and tetherin, trigger antiviral signaling in addition to directly impairing HIV replication. SERINC5 (S5) is an RF that is incorporated into HIV-1 particles to potently impair their infectivity and is efficiently antagonized by the viral pathogenesis factor Nef. Since effects of S5 on HIV-1 infectivity were mostly studied in reporter cell lines, we analyzed the effects of S5 during infection of primary HIV-1 target cells. In activated CD4 T lymphocytes, virion incorporation of S5 only moderately impaired virion infectivity and was not associated with altered innate immune recognition. In contrast, in monocyte-derived macrophages, S5 virion incorporation potentiated the production of proinflammatory cytokines with very potent but donor-dependent effects on virion infectivity. Nef counteracted effects of S5 on both cytokine production and virion infectivity. Similar S5-induced cytokine production was observed in immature monocyte-derived dendritic cells. Notably, S5-mediated enhancement of cytokine production was not linked to the efficacy of productive infection and could be overcome by using vesicular stomatitis virus glycoprotein (VSV-G) but not infectivity restriction-insensitive HIV-1 Env for cell entry. Moreover, inhibiting entry of S5-negative HIV-1 ΔNef particles increased proinflammatory cytokine production comparably to virion incorporation of S5. Together, these results describe the sensitization of noninfectious HIV-1 particles to proinflammatory cytokine production by myeloid target cells as an additional and Nef-sensitive activity of S5. Moreover, the study reveals important cell-type and donor-dependent differences in the sensitivity of HIV target cells for antiviral effects of S5. SERINC5 (S5) is a host cell restriction factor (RF) that impairs the infectivity of HIV-1 particles in target cell lines. To assess the potential physiological relevance of this restriction, we assessed the effects of S5 on HIV-1 infection of relevant primary human target cells. We found that effects of S5 on infection of CD4 T lymphocytes were negligible. In myeloid target cells, however, virion incorporation of S5 potently suppressed infectivity and promoted innate immune recognition of HIV-1 particles characterized by proinflammatory cytokine production. Both effects were not observed in cells of all donors analyzed, were exerted independently of one another, and were counteracted by the HIV-1 pathogenesis factor Nef. These results identify the sensitization of HIV-1 particles for innate immune recognition by myeloid target cells as a novel activity of S5 and emphasize the need to study RF function in the context of primary target cells and taking donor variabilities into account.
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http://dx.doi.org/10.1128/JVI.02372-20DOI Listing
April 2021

SARS-CoV-2 infects and replicates in cells of the human endocrine and exocrine pancreas.

Nat Metab 2021 02 3;3(2):149-165. Epub 2021 Feb 3.

Institute of Diabetes and Regeneration Research, Helmholtz Zentrum München, Neuherberg, Germany.

Infection-related diabetes can arise as a result of virus-associated β-cell destruction. Clinical data suggest that the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the coronavirus disease 2019 (COVID-19), impairs glucose homoeostasis, but experimental evidence that SARS-CoV-2 can infect pancreatic tissue has been lacking. In the present study, we show that SARS-CoV-2 infects cells of the human exocrine and endocrine pancreas ex vivo and in vivo. We demonstrate that human β-cells express viral entry proteins, and SARS-CoV-2 infects and replicates in cultured human islets. Infection is associated with morphological, transcriptional and functional changes, including reduced numbers of insulin-secretory granules in β-cells and impaired glucose-stimulated insulin secretion. In COVID-19 full-body postmortem examinations, we detected SARS-CoV-2 nucleocapsid protein in pancreatic exocrine cells, and in cells that stain positive for the β-cell marker NKX6.1 and are in close proximity to the islets of Langerhans in all four patients investigated. Our data identify the human pancreas as a target of SARS-CoV-2 infection and suggest that β-cell infection could contribute to the metabolic dysregulation observed in patients with COVID-19.
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http://dx.doi.org/10.1038/s42255-021-00347-1DOI Listing
February 2021

Evolutionary conflicts and adverse effects of antiviral factors.

Elife 2021 Jan 15;10. Epub 2021 Jan 15.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

Human cells are equipped with a plethora of antiviral proteins protecting them against invading viral pathogens. In contrast to apoptotic or pyroptotic cell death, which serves as ultima ratio to combat viral infections, these cell-intrinsic restriction factors may prevent or at least slow down viral spread while allowing the host cell to survive. Nevertheless, their antiviral activity may also have detrimental effects on the host. While the molecular mechanisms underlying the antiviral activity of restriction factors are frequently well investigated, potential undesired effects of their antiviral functions on the host cell are hardly explored. With a focus on antiretroviral proteins, we summarize in this review how individual restriction factors may exert adverse effects as trade-off for efficient defense against attacking pathogens.
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http://dx.doi.org/10.7554/eLife.65243DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7811402PMC
January 2021

Natural cystatin C fragments inhibit GPR15-mediated HIV and SIV infection without interfering with GPR15L signaling.

Proc Natl Acad Sci U S A 2021 01;118(3)

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany;

GPR15 is a G protein-coupled receptor (GPCR) proposed to play a role in mucosal immunity that also serves as a major entry cofactor for HIV-2 and simian immunodeficiency virus (SIV). To discover novel endogenous GPR15 ligands, we screened a hemofiltrate (HF)-derived peptide library for inhibitors of GPR15-mediated SIV infection. Our approach identified a C-terminal fragment of cystatin C (CysC95-146) that specifically inhibits GPR15-dependent HIV-1, HIV-2, and SIV infection. In contrast, GPR15L, the chemokine ligand of GPR15, failed to inhibit virus infection. We found that cystatin C fragments preventing GPR15-mediated viral entry do not interfere with GPR15L signaling and are generated by proteases activated at sites of inflammation. The antiretroviral activity of CysC95-146 was confirmed in primary CD4 T cells and is conserved in simian hosts of SIV infection. Thus, we identified a potent endogenous inhibitor of GPR15-mediated HIV and SIV infection that does not interfere with the physiological function of this GPCR.
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http://dx.doi.org/10.1073/pnas.2023776118DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7826402PMC
January 2021

IFI16 knockdown in primary HIV-1 target cells.

STAR Protoc 2021 Mar 19;2(1):100236. Epub 2020 Dec 19.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany.

IFI16 is an important player of the host intrinsic immune response. Among others, it has been reported to sense intermediate products of HIV-1 reverse transcription in the cytosol and to sequester the transcription factor Sp1 in the nucleus to attenuate viral gene expression. Here, we present three different methods to reduce IFI16 protein expression levels in HIV-1 primary target cells. These techniques can be adapted for the investigation of other cellular factors in primary macrophages and CD4 T lymphocytes. For complete details on the use and execution of this protocol, please refer to Hotter et al. (2019) and Bosso et al. (2020).
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http://dx.doi.org/10.1016/j.xpro.2020.100236DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7753938PMC
March 2021

Emerging Role of PYHIN Proteins as Antiviral Restriction Factors.

Viruses 2020 12 18;12(12). Epub 2020 Dec 18.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany.

Innate immune sensors and restriction factors are cellular proteins that synergize to build an effective first line of defense against viral infections. Innate sensors are usually constitutively expressed and capable of detecting pathogen-associated molecular patterns (PAMPs) via specific pattern recognition receptors (PRRs) to stimulate the immune response. Restriction factors are frequently upregulated by interferons (IFNs) and may inhibit viral pathogens at essentially any stage of their replication cycle. Members of the Pyrin and hematopoietic interferon-inducible nuclear (HIN) domain (PYHIN) family have initially been recognized as important sensors of foreign nucleic acids and activators of the inflammasome and the IFN response. Accumulating evidence shows, however, that at least three of the four members of the human PYHIN family restrict viral pathogens independently of viral sensing and innate immune activation. In this review, we provide an overview on the role of human PYHIN proteins in the innate antiviral immune defense and on viral countermeasures.
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http://dx.doi.org/10.3390/v12121464DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7767131PMC
December 2020

The Paradox of Astroglial Ca Signals at the Interface of Excitation and Inhibition.

Front Cell Neurosci 2020 26;14:609947. Epub 2020 Nov 26.

Department of Molecular Physiology, Center for Integrative Physiology and Molecular Medicine, University of Saarland, Homburg, Germany.

Astroglial networks constitute a non-neuronal communication system in the brain and are acknowledged modulators of synaptic plasticity. A sophisticated set of transmitter receptors in combination with distinct secretion mechanisms enables astrocytes to sense and modulate synaptic transmission. This integrative function evolved around intracellular Ca signals, by and large considered as the main indicator of astrocyte activity. Regular brain physiology meticulously relies on the constant reciprocity of excitation and inhibition (E/I). Astrocytes are metabolically, physically, and functionally associated to the E/I convergence. Metabolically, astrocytes provide glutamine, the precursor of both major neurotransmitters governing E/I in the central nervous system (CNS): glutamate and γ-aminobutyric acid (GABA). Perisynaptic astroglial processes are structurally and functionally associated with the respective circuits throughout the CNS. Astonishingly, in astrocytes, glutamatergic as well as GABAergic inputs elicit similar rises in intracellular Ca that in turn can trigger the release of glutamate and GABA as well. Paradoxically, as gliotransmitters, these two molecules can thus strengthen, weaken or even reverse the input signal. Therefore, the net impact on neuronal network function is often convoluted and cannot be simply predicted by the nature of the stimulus itself. In this review, we highlight the ambiguity of astrocytes on discriminating and affecting synaptic activity in physiological and pathological state. Indeed, aberrant astroglial Ca signaling is a key aspect of pathological conditions exhibiting compromised network excitability, such as epilepsy. Here, we gather recent evidence on the complexity of astroglial Ca signals in health and disease, challenging the traditional, neuro-centric concept of segregating E/I, in favor of a non-binary, mutually dependent perspective on glutamatergic and GABAergic transmission.
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http://dx.doi.org/10.3389/fncel.2020.609947DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726216PMC
November 2020

Peptide and peptide-based inhibitors of SARS-CoV-2 entry.

Adv Drug Deliv Rev 2020 12 13;167:47-65. Epub 2020 Nov 13.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany. Electronic address:

To date, no effective vaccines or therapies are available against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative pandemic agent of the coronavirus disease 2019 (COVID-19). Due to their safety, efficacy and specificity, peptide inhibitors hold great promise for the treatment of newly emerging viral pathogens. Based on the known structures of viral proteins and their cellular targets, antiviral peptides can be rationally designed and optimized. The resulting peptides may be highly specific for their respective targets and particular viral pathogens or exert broad antiviral activity. Here, we summarize the current status of peptides inhibiting SARS-CoV-2 entry and outline the strategies used to design peptides targeting the ACE2 receptor or the viral spike protein and its activating proteases furin, transmembrane serine protease 2 (TMPRSS2), or cathepsin L. In addition, we present approaches used against related viruses such as SARS-CoV-1 that might be implemented for inhibition of SARS-CoV-2 infection.
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http://dx.doi.org/10.1016/j.addr.2020.11.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665879PMC
December 2020

Serotonin receptor 4 regulates hippocampal astrocyte morphology and function.

Glia 2021 Apr 6;69(4):872-889. Epub 2020 Nov 6.

Cellular Neurophysiology, Hannover Medical School, Hannover, Germany.

Astrocytes are an important component of the multipartite synapse and crucial for proper neuronal network function. Although small GTPases of the Rho family are powerful regulators of cellular morphology, the signaling modules of Rho-mediated pathways in astrocytes remain enigmatic. Here we demonstrated that the serotonin receptor 4 (5-HT R) is expressed in hippocampal astrocytes, both in vitro and in vivo. Through fluorescence microscopy, we established that 5-HT R activation triggered RhoA activity via Gα -mediated signaling, which boosted filamentous actin assembly, leading to morphological changes in hippocampal astrocytes. We investigated the effects of these 5-HT R-mediated changes in mixed cultures and in acute slices, in which 5-HT R was expressed exclusively in astrocytes. In both systems, 5-HT R-RhoA signaling changed glutamatergic synaptic transmission: It increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) in mixed cultures and reduced the paired-pulse-ratio (PPR) of field excitatory postsynaptic potentials (fEPSPs) in acute slices. Overall, our present findings demonstrate that astrocytic 5-HT R-Gα -RhoA signaling is a previously unrecognized molecular pathway involved in the functional regulation of excitatory synaptic circuits.
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http://dx.doi.org/10.1002/glia.23933DOI Listing
April 2021

SARS-CoV-2 Is Restricted by Zinc Finger Antiviral Protein despite Preadaptation to the Low-CpG Environment in Humans.

mBio 2020 10 16;11(5). Epub 2020 Oct 16.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany

Recent evidence shows that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is sensitive to interferons (IFNs). However, the most effective types of IFNs and the underlying antiviral effectors remain to be defined. Here, we show that zinc finger antiviral protein (ZAP), which preferentially targets CpG dinucleotides in viral RNA sequences, restricts SARS-CoV-2. We further demonstrate that ZAP and its cofactors KHNYN and TRIM25 are expressed in human lung cells. Type I, II, and III IFNs all strongly inhibited SARS-CoV-2 and further induced ZAP expression. Comprehensive sequence analyses revealed that SARS-CoV-2 and its closest relatives from horseshoe bats showed the strongest CpG suppression among all known human and bat coronaviruses, respectively. Nevertheless, endogenous ZAP expression restricted SARS-CoV-2 replication in human lung cells, particularly upon treatment with IFN-α or IFN-γ. Both the long and the short isoforms of human ZAP reduced SARS-CoV-2 RNA expression levels, but the former did so with greater efficiency. Finally, we show that the ability to restrict SARS-CoV-2 is conserved in ZAP orthologues of the reservoir bat and potential intermediate pangolin hosts of human coronaviruses. Altogether, our results show that ZAP is an important effector of the innate response against SARS-CoV-2, although this pandemic pathogen emerged from zoonosis of a coronavirus that was preadapted to the low-CpG environment in humans. Although interferons inhibit SARS-CoV-2 and have been evaluated for treatment of coronavirus disease 2019 (COVID-19), the most effective types and antiviral effectors remain to be defined. Here, we show that IFN-γ is particularly potent in restricting SARS-CoV-2 and in inducing expression of the antiviral factor ZAP in human lung cells. Knockdown experiments revealed that endogenous ZAP significantly restricts SARS-CoV-2. We further show that CpG dinucleotides which are specifically targeted by ZAP are strongly suppressed in the SARS-CoV-2 genome and that the two closest horseshoe bat relatives of SARS-CoV-2 show the lowest genomic CpG content of all coronavirus sequences available from this reservoir host. Nonetheless, both the short and long isoforms of human ZAP reduced SARS-CoV-2 RNA levels, and this activity was conserved in horseshoe bat and pangolin ZAP orthologues. Our findings indicating that type II interferon is particularly efficient against SARS-CoV-2 and that ZAP restricts this pandemic viral pathogen might promote the development of effective immune therapies against COVID-19.
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http://dx.doi.org/10.1128/mBio.01930-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7569149PMC
October 2020

Undermining breastfeeding will not alleviate the COVID-19 pandemic - Authors' reply.

Lancet 2020 10;396(10257):1065-1066

Institute of Molecular Virology, Ulm University Medical Centre, Ulm 89081, Germany. Electronic address:

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http://dx.doi.org/10.1016/S0140-6736(20)32066-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7544464PMC
October 2020

HIV-1 infection activates endogenous retroviral promoters regulating antiviral gene expression.

Nucleic Acids Res 2020 11;48(19):10890-10908

Institute of Molecular Virology, Ulm University Medical Center, Ulm 89081, Germany.

Although endogenous retroviruses (ERVs) are known to harbor cis-regulatory elements, their role in modulating cellular immune responses remains poorly understood. Using an RNA-seq approach, we show that several members of the ERV9 lineage, particularly LTR12C elements, are activated upon HIV-1 infection of primary CD4+ T cells. Intriguingly, HIV-1-induced ERVs harboring transcription start sites are primarily found in the vicinity of immunity genes. For example, HIV-1 infection activates LTR12C elements upstream of the interferon-inducible genes GBP2 and GBP5 that encode for broad-spectrum antiviral factors. Reporter assays demonstrated that these LTR12C elements drive gene expression in primary CD4+ T cells. In line with this, HIV-1 infection triggered the expression of a unique GBP2 transcript variant by activating a cryptic transcription start site within LTR12C. Furthermore, stimulation with HIV-1-induced cytokines increased GBP2 and GBP5 expression in human cells, but not in macaque cells that naturally lack the GBP5 gene and the LTR12C element upstream of GBP2. Finally, our findings suggest that GBP2 and GBP5 have already been active against ancient viral pathogens as they suppress the maturation of the extinct retrovirus HERV-K (HML-2). In summary, our findings uncover how human cells can exploit remnants of once-infectious retroviruses to regulate antiviral gene expression.
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http://dx.doi.org/10.1093/nar/gkaa832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7641743PMC
November 2020

Real-Time Killing Assays to Assess the Potency of a New Anti-Simian Immunodeficiency Virus Chimeric Antigen Receptor T Cell.

AIDS Res Hum Retroviruses 2020 12 5;36(12):998-1009. Epub 2020 Nov 5.

Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA.

The success of chimeric antigen receptor (CAR) T cell therapies for treating leukemia has resulted in a booming interest for the technology. Expression of a CAR in T cells allows redirection of their natural cytolytic activity toward cells presenting a specific designated surface antigen. Although CAR T cell therapies have thus far shown promising results mostly in B cell malignancy trials, interest in their potential to treat other diseases is on the rise, including using CAR T cells to control human immunodeficiency virus infection. The assessment of CAR T cell potency toward specific targets is a critical preclinical step. In this study, we describe novel assays that monitor the cytotoxicity of candidate CAR T cells toward simian immunodeficiency virus (SIV) infected CD4 T cells. The assays involve live cell imaging using a fluorescence microscopy system that records in real time the disappearance or appearance of targets infected with SIV carrying a fluorescent protein gene. The assays are highly reproducible, and their rapid turn around and reduced cost present a significant advance regarding the efficient preclinical evaluation of CAR T cell constructs and are broadly applicable to potential human diseases that could benefit from CAR T cell therapy.
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http://dx.doi.org/10.1089/AID.2020.0163DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7703091PMC
December 2020

Nuclear PYHIN proteins target the host transcription factor Sp1 thereby restricting HIV-1 in human macrophages and CD4+ T cells.

PLoS Pathog 2020 08 6;16(8):e1008752. Epub 2020 Aug 6.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

Members of the family of pyrin and HIN domain containing (PYHIN) proteins play an emerging role in innate immunity. While absent in melanoma 2 (AIM2) acts a cytosolic sensor of non-self DNA and plays a key role in inflammasome assembly, the γ-interferon-inducible protein 16 (IFI16) restricts retroviral gene expression by sequestering the transcription factor Sp1. Here, we show that the remaining two human PYHIN proteins, i.e. myeloid cell nuclear differentiation antigen (MNDA) and pyrin and HIN domain family member 1 (PYHIN1 or IFIX) share this antiretroviral function of IFI16. On average, knock-down of each of these three nuclear PYHIN proteins increased infectious HIV-1 yield from human macrophages by more than an order of magnitude. Similarly, knock-down of IFI16 strongly increased virus transcription and production in primary CD4+ T cells. The N-terminal pyrin domain (PYD) plus linker region containing a nuclear localization signal (NLS) were generally required and sufficient for Sp1 sequestration and anti-HIV-1 activity of IFI16, MNDA and PYHIN1. Replacement of the linker region of AIM2 by the NLS-containing linker of IFI16 resulted in a predominantly nuclear localization and conferred direct antiviral activity to AIM2 while attenuating its ability to form inflammasomes. The reverse change caused nuclear-to-cytoplasmic relocalization of IFI16 and impaired its antiretroviral activity but did not result in inflammasome assembly. We further show that the Zn-finger domain of Sp1 is critical for the interaction with IFI16 supporting that pyrin domains compete with DNA for Sp1 binding. Finally, we found that human PYHIN proteins also inhibit Hepatitis B virus and simian vacuolating virus 40 as well as the LINE-1 retrotransposon. Altogether, our data show that IFI16, PYHIN1 and MNDA restrict HIV-1 and other viral pathogens by interfering with Sp1-dependent gene expression and support an important role of nuclear PYHIN proteins in innate antiviral immunity.
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http://dx.doi.org/10.1371/journal.ppat.1008752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7433898PMC
August 2020

An improved method for high-throughput quantification of autophagy in mammalian cells.

Sci Rep 2020 07 22;10(1):12241. Epub 2020 Jul 22.

Institute of Molecular Virology, Ulm University Medical Center, 89081, Ulm, Germany.

Autophagy is a cellular homeostatic pathway with functions ranging from cytoplasmic protein turnover to immune defense. Therapeutic modulation of autophagy has been demonstrated to positively impact the outcome of autophagy-dysregulated diseases such as cancer or microbial infections. However, currently available agents lack specificity, and new candidates for drug development or potential cellular targets need to be identified. Here, we present an improved method to robustly detect changes in autophagy in a high-throughput manner on a single cell level, allowing effective screening. This method quantifies eGFP-LC3B positive vesicles to accurately monitor autophagy. We have significantly streamlined the protocol and optimized it for rapid quantification of large numbers of cells in little time, while retaining accuracy and sensitivity. Z scores up to 0.91 without a loss of sensitivity demonstrate the robustness and aptness of this approach. Three exemplary applications outline the value of our protocols and cell lines: (I) Examining autophagy modulating compounds on four different cell types. (II) Monitoring of autophagy upon infection with e.g. measles or influenza A virus. (III) CRISPR/Cas9 screening for autophagy modulating factors in T cells. In summary, we offer ready-to-use protocols to generate sensitive autophagy reporter cells and quantify autophagy in high-throughput assays.
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http://dx.doi.org/10.1038/s41598-020-68607-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7376206PMC
July 2020

Vpu modulates DNA repair to suppress innate sensing and hyper-integration of HIV-1.

Nat Microbiol 2020 10 20;5(10):1247-1261. Epub 2020 Jul 20.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany.

To avoid innate sensing and immune control, human immunodeficiency virus type 1 (HIV-1) has to prevent the accumulation of viral complementary DNA species. Here, we show that the late HIV-1 accessory protein Vpu hijacks DNA repair mechanisms to promote degradation of nuclear viral cDNA in cells that are already productively infected. Vpu achieves this by interacting with RanBP2-RanGAP1*SUMO1-Ubc9 SUMO E3-ligase complexes at the nuclear pore to reprogramme promyelocytic leukaemia protein nuclear bodies and reduce SUMOylation of Bloom syndrome protein, unleashing end degradation of viral cDNA. Concomitantly, Vpu inhibits RAD52-mediated homologous repair of viral cDNA, preventing the generation of dead-end circular forms of single copies of the long terminal repeat and permitting sustained nucleolytic attack. Our results identify Vpu as a key modulator of the DNA repair machinery. We show that Bloom syndrome protein eliminates nuclear HIV-1 cDNA and thereby suppresses immune sensing and proviral hyper-integration. Therapeutic targeting of DNA repair may facilitate the induction of antiviral immunity and suppress proviral integration replenishing latent HIV reservoirs.
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http://dx.doi.org/10.1038/s41564-020-0753-6DOI Listing
October 2020

Structural basis for translational shutdown and immune evasion by the Nsp1 protein of SARS-CoV-2.

Science 2020 09 17;369(6508):1249-1255. Epub 2020 Jul 17.

Gene Center Munich, Department of Biochemistry, University of Munich, Munich, Germany.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the current coronavirus disease 2019 (COVID-19) pandemic. A major virulence factor of SARS-CoVs is the nonstructural protein 1 (Nsp1), which suppresses host gene expression by ribosome association. Here, we show that Nsp1 from SARS-CoV-2 binds to the 40 ribosomal subunit, resulting in shutdown of messenger RNA (mRNA) translation both in vitro and in cells. Structural analysis by cryo-electron microscopy of in vitro-reconstituted Nsp1-40 and various native Nsp1-40 and -80 complexes revealed that the Nsp1 C terminus binds to and obstructs the mRNA entry tunnel. Thereby, Nsp1 effectively blocks retinoic acid-inducible gene I-dependent innate immune responses that would otherwise facilitate clearance of the infection. Thus, the structural characterization of the inhibitory mechanism of Nsp1 may aid structure-based drug design against SARS-CoV-2.
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http://dx.doi.org/10.1126/science.abc8665DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7402621PMC
September 2020

Convergent Evolution of HLA-C Downmodulation in HIV-1 and HIV-2.

mBio 2020 07 14;11(4). Epub 2020 Jul 14.

Institute of Molecular Virology, Ulm University Medical Center, Ulm, Germany

HLA-C-mediated antigen presentation induces the killing of human immunodeficiency virus (HIV)-infected CD4 T cells by cytotoxic T lymphocytes (CTLs). To evade killing, many HIV-1 group M strains decrease HLA-C surface levels using their accessory protein Vpu. However, some HIV-1 group M isolates lack this activity, possibly to prevent the activation of natural killer (NK) cells. Analyzing diverse primate lentiviruses, we found that Vpu-mediated HLA-C downregulation is not limited to pandemic group M but is also found in HIV-1 groups O and P as well as several simian immunodeficiency viruses (SIVs). We show that Vpu targets HLA-C primarily at the protein level, independently of its ability to suppress NF-κB-driven gene expression, and that in some viral lineages, HLA-C downregulation may come at the cost of efficient counteraction of the restriction factor tetherin. Remarkably, HIV-2, which does not carry a gene, uses its accessory protein Vif to decrease HLA-C surface expression. This Vif activity requires intact binding sites for the Cullin5/Elongin ubiquitin ligase complex but is separable from its ability to counteract APOBEC3G. Similar to HIV-1 Vpu, the degree of HIV-2 Vif-mediated HLA-C downregulation varies considerably among different virus isolates. In agreement with opposing selection pressures , we show that the reduction of HLA-C surface levels by HIV-2 Vif is accompanied by increased NK cell-mediated killing. In summary, our results highlight the complex role of HLA-C in lentiviral infections and demonstrate that HIV-1 and HIV-2 have evolved at least two independent mechanisms to decrease HLA-C levels on infected cells. Genome-wide association studies suggest that HLA-C expression is a major determinant of viral load set points and CD4 T cell counts in HIV-infected individuals. On the one hand, efficient HLA-C expression enables the killing of infected cells by cytotoxic T lymphocytes (CTLs). On the other hand, HLA-C sends inhibitory signals to natural killer (NK) cells and enhances the infectivity of newly produced HIV particles. HIV-1 group M viruses modulate HLA-C expression using the accessory protein Vpu, possibly to balance CTL- and NK cell-mediated immune responses. Here, we show that the second human immunodeficiency virus, HIV-2, can use its accessory protein Vif to evade HLA-C-mediated restriction. Furthermore, our mutational analyses provide insights into the underlying molecular mechanisms. In summary, our results reveal how the two human AIDS viruses modulate HLA-C, a key component of the antiviral immune response.
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http://dx.doi.org/10.1128/mBio.00782-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7360927PMC
July 2020

The HIV-1 Env gp120 Inner Domain Shapes the Phe43 Cavity and the CD4 Binding Site.

mBio 2020 05 26;11(3). Epub 2020 May 26.

Centre de Recherche du CHUM, Montreal, Quebec, Canada

The HIV-1 envelope glycoproteins (Env) undergo conformational changes upon interaction of the gp120 exterior glycoprotein with the CD4 receptor. The gp120 inner domain topological layers facilitate the transition of Env to the CD4-bound conformation. CD4 engages gp120 by introducing its phenylalanine 43 (Phe43) in a cavity ("the Phe43 cavity") located at the interface between the inner and outer gp120 domains. Small CD4-mimetic compounds (CD4mc) can bind within the Phe43 cavity and trigger conformational changes similar to those induced by CD4. Crystal structures of CD4mc in complex with a modified CRF01_AE gp120 core revealed the importance of these gp120 inner domain layers in stabilizing the Phe43 cavity and shaping the CD4 binding site. Our studies reveal a complex interplay between the gp120 inner domain and the Phe43 cavity and generate useful information for the development of more-potent CD4mc. The Phe43 cavity of HIV-1 envelope glycoproteins (Env) is an attractive druggable target. New promising compounds, including small CD4 mimetics (CD4mc), were shown to insert deeply into this cavity. Here, we identify a new network of residues that helps to shape this highly conserved CD4 binding pocket and characterize the structural determinants responsible for Env sensitivity to small CD4 mimetics.
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http://dx.doi.org/10.1128/mBio.00280-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251204PMC
May 2020

Detection of SARS-CoV-2 in human breastmilk.

Lancet 2020 06 21;395(10239):1757-1758. Epub 2020 May 21.

Institute of Molecular Virology, Ulm University Medical Center, Ulm 89081, Germany. Electronic address:

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http://dx.doi.org/10.1016/S0140-6736(20)31181-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7241971PMC
June 2020

Cellular Factors Targeting HIV-1 Transcription and Viral RNA Transcripts.

Viruses 2020 04 29;12(5). Epub 2020 Apr 29.

Institute of Molecular Virology, Ulm University Medical Center, 89081 Ulm, Germany.

Restriction factors are structurally and functionally diverse cellular proteins that constitute a first line of defense against viral pathogens. Exceptions exist, but typically these proteins are upregulated by interferons (IFNs), target viral components, and are rapidly evolving due to the continuous virus-host arms race. Restriction factors may target HIV replication at essentially each step of the retroviral replication cycle, and the suppression of viral transcription and the degradation of viral RNA transcripts are emerging as major innate immune defense mechanisms. Recent data show that some antiviral factors, such as the tripartite motif-containing protein 22 (TRIM22) and the g-IFN-inducible protein 16 (IFI16), do not target HIV-1 itself but limit the availability of the cellular transcription factor specificity protein 1 (Sp1), which is critical for effective viral gene expression. In addition, several RNA-interacting cellular factors including RNAse L, the NEDD4-binding protein 1 (N4BP1), and the zinc finger antiviral protein (ZAP) have been identified as important immune effectors against HIV-1 that may be involved in the maintenance of the latent viral reservoirs, representing the major obstacle against viral elimination and cure. Here, we review recent findings on specific cellular antiviral factors targeting HIV-1 transcription or viral RNA transcripts and discuss their potential role in viral latency.
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http://dx.doi.org/10.3390/v12050495DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7290996PMC
April 2020