• Labyrinthopeptins exert broad-spectrum antiviral activity through lipid-binding-mediated virolysis.

      Prochnow, Hans; Rox, Katharina; Birudukota, N V Suryanarayana; Weichert, Loreen; Hotop, Sven-Kevin; Klahn, Philipp; Mohr, Kathrin; Franz, Sergej; Banda, Dominic H; Blockus, Sebastian; et al. (ASM, 2019-10-30)
      To counteract the serious health threat posed by known and novel viral pathogens, drugs that target a variety of viruses through a common mechanism have attracted recent attention due to their potential in treating (re-)emerging infections, for which direct acting antivirals are not available. We found that labyrinthopeptins A1 and A2, the prototype congeners of carbacyclic lanthipeptides, inhibit the proliferation of diverse enveloped viruses, including Dengue virus, Zika virus, West Nile virus, Hepatitis C virus, Chikungunya virus, Karposi's Sarcoma-associated Herpes virus, Cytomegalovirus, and Herpes Simplex virus, in the low μM to nM range. Mechanistic studies on viral particles revealed that labyrinthopeptins induce a virolytic effect through binding to the viral membrane lipid phosphatidylethanolamine (PE). These effects are enhanced by a combined equimolar application of both labyrinthopeptins, and a clear synergism was observed across a concentration range corresponding to IC10-IC90 values of the compounds. Time-resolved experiments with large unilamellar vesicles (LUVs) reveal that membrane lipid raft compositions (PC/PE/Chol/SM (17:10:33:40)) are particularly sensitive to labyrinthopeptins compared to PC/PE (90:10) LUVs, even though the overall PE-amount remains constant. Labyrinthopeptins exhibited low cytotoxicity and had favorable pharmacokinetic properties in mice (t1/2= 10.0 h), which designates them as promising antiviral compounds acting by an unusual viral lipid targeting mechanism.Importance For many viral infections, current treatment options are insufficient. Because the development of each antiviral drug is time-consuming and expensive, the prospect of finding broad-spectrum antivirals that can fight multiple, diverse viruses - well-known as well as (re-)emerging species - has gained attention, especially for the treatment of viral co-infections. While most known broad spectrum agents address processes in the host cell, we found that targeting lipids of the free virus outside the host cell with the natural products labyrinthopeptin A1 and A2 is a viable strategy to inhibit the proliferation of a broad range of viruses from different families, including Chikungunya virus, Dengue virus, Zika virus, Karposi's Sarcoma-associated Herpes virus, or Cytomegalovirus. Labyrinthopeptins bind to viral phosphatidylethanolamine and induce virolysis without exerting cytotoxicity to host cells. This represents a novel and unusual mechanism to tackle medically relevant viral infections.
    • Langat virus infection affects hippocampal neuron morphology and function in mice without disease signs.

      Cornelius, Angela D A; Hosseini, Shirin; Schreier, Sarah; Fritzsch, David; Weichert, Loreen; Michaelsen-Preusse, Kristin; Fendt, Markus; Kröger, Andrea; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (BioMed Central, 2020-09-20)
      To compare the effect of low and high viral replication in the brain, wildtype and Irf-7-/- mice were infected with Langat virus (LGTV), which belongs to the TBEV-serogroup. The viral burden was analyzed in the olfactory bulb and the hippocampus. Open field, elevated plus maze, and Morris water maze experiments were performed to determine the impact on anxiety-like behavior, learning, and memory formation. Spine density of hippocampal neurons and activation of microglia and astrocytes were analyzed.
    • Maternal B Cell-Intrinsic MyD88 Signaling Mediates LPS-Driven Intrauterine Fetal Death.

      Busse, Mandy; Plenagl, Susanne; Campe, Norina Kim Jutta; Müller, Andreas J; Tedford, Kerry; Schumacher, Anne; Zenclussen, Ana Claudia; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (MDPI, 2021-10-08)
      Immunological networks balance tolerance towards paternal alloantigens during pregnancy with normal immune response to pathogens. Subclinical infections can impact this balance and lead to preterm birth or even intrauterine fetal death (IUFD). We recently showed that loss of maternal B cells renders murine fetuses susceptible to IUFD after LPS exposure. Since the signaling pathway involved in this B-cell mediated response remains unclear, we aimed to understand the participation of MyD88 in this response using B-cell-specific MyD88-deficient (BMyD88-/-) mice. B cells isolated from wild-type (WT), BMyD88-/-, CD19-/- and MyD88-/- dams on gestational day (gd) 10 responded differently to LPS concerning cytokine secretion. In vivo LPS challenge on gd 10 provoked IUFD in CD19-/- mothers with functional MyD88, while fetuses from BMyD88-/- and MyD88-/- mice were protected. These outcomes were associated with altered cytokine levels in the maternal serum and changes in CD4+ T-cell responses. Overall, the loss of MyD88 signaling in maternal B cells prevents the activation of cytokine release that leads to IUFD. Thus, while MyD88 signaling in maternal B cells protects the mother from infection, it ultimately kills the fetus. Understanding the cellular mechanisms underlying infection-driven pregnancy complications is the first step to designing powerful therapeutic strategies in the future.
    • NUDT2 initiates viral RNA degradation by removal of 5'-phosphates.

      Laudenbach, Beatrice T; Krey, Karsten; Emslander, Quirin; Andersen, Line Lykke; Reim, Alexander; Scaturro, Pietro; Mundigl, Sarah; Dächert, Christopher; Manske, Katrin; Moser, Markus; et al. (Springer Nature, 2021-11-25)
      While viral replication processes are largely understood, comparably little is known on cellular mechanisms degrading viral RNA. Some viral RNAs bear a 5'-triphosphate (PPP-) group that impairs degradation by the canonical 5'-3' degradation pathway. Here we show that the Nudix hydrolase 2 (NUDT2) trims viral PPP-RNA into monophosphorylated (P)-RNA, which serves as a substrate for the 5'-3' exonuclease XRN1. NUDT2 removes 5'-phosphates from PPP-RNA in an RNA sequence- and overhang-independent manner and its ablation in cells increases growth of PPP-RNA viruses, suggesting an involvement in antiviral immunity. NUDT2 is highly homologous to bacterial RNA pyrophosphatase H (RppH), a protein involved in the metabolism of bacterial mRNA, which is 5'-tri- or diphosphorylated. Our results show a conserved function between bacterial RppH and mammalian NUDT2, indicating that the function may have adapted from a protein responsible for RNA turnover in bacteria into a protein involved in the immune defense in mammals.
    • OTUB1 prevents lethal hepatocyte necroptosis through stabilization of c-IAP1 during murine liver inflammation.

      Koschel, Josephin; Nishanth, Gopala; Just, Sissy; Harit, Kunjan; Kröger, Andrea; Deckert, Martina; Naumann, Michael; Schlüter, Dirk; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2021-03-12)
      In bacterial and sterile inflammation of the liver, hepatocyte apoptosis is, in contrast to necroptosis, a common feature. The molecular mechanisms preventing hepatocyte necroptosis and the potential consequences of hepatocyte necroptosis are largely unknown. Apoptosis and necroptosis are critically regulated by the ubiquitination of signaling molecules but especially the regulatory function of deubiquitinating enzymes (DUBs) is imperfectly defined. Here, we addressed the role of the DUB OTU domain aldehyde binding-1 (OTUB1) in hepatocyte cell death upon both infection with the hepatocyte-infecting bacterium Listeria monocytogenes (Lm) and D-Galactosamine (DGal)/Tumor necrosis factor (TNF)-induced sterile inflammation. Combined in vivo and in vitro experiments comprising mice lacking OTUB1 specifically in liver parenchymal cells (OTUB1LPC-KO) and human OTUB1-deficient HepG2 cells revealed that OTUB1 prevented hepatocyte necroptosis but not apoptosis upon infection with Lm and DGal/TNF challenge. Lm-induced necroptosis in OTUB1LPC-KO mice resulted in increased alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) release and rapid lethality. Treatment with the receptor-interacting serine/threonine-protein kinase (RIPK) 1 inhibitor necrostatin-1s and deletion of the pseudokinase mixed lineage kinase domain-like protein (MLKL) prevented liver damage and death of infected OTUB1LPC-KO mice. Mechanistically, OTUB1 reduced K48-linked polyubiquitination of the cellular inhibitor of apoptosis 1 (c-IAP1), thereby diminishing its degradation. In the absence of OTUB1, c-IAP1 degradation resulted in reduced K63-linked polyubiquitination and increased phosphorylation of RIPK1, RIPK1/RIPK3 necrosome formation, MLKL-phosphorylation and hepatocyte death. Additionally, OTUB1-deficiency induced RIPK1-dependent extracellular-signal-regulated kinase (ERK) activation and TNF production in Lm-infected hepatocytes. Collectively, these findings identify OTUB1 as a novel regulator of hepatocyte-intrinsic necroptosis and a critical factor for survival of bacterial hepatitis and TNF challenge.
    • The role of the poly(A) tract in the replication and virulence of tick-borne encephalitis virus.

      Asghar, Naveed; Lee, Yi-Ping; Nilsson, Emma; Lindqvist, Richard; Melik, Wessam; Kröger, Andrea; Överby, Anna K; Johansson, Magnus; Helmholtz Centre for infection research. Inhoffenstr. 7. 38124 Braunschweig, Germany. (2016-12-16)
      The tick-borne encephalitis virus (TBEV) is a flavivirus transmitted to humans, usually via tick bites. The virus causes tick-borne encephalitis (TBE) in humans, and symptoms range from mild flu-like symptoms to severe and long-lasting sequelae, including permanent brain damage. It has been suggested that within the population of viruses transmitted to the mammalian host, quasispecies with neurotropic properties might become dominant in the host resulting in neurological symptoms. We previously demonstrated the existence of TBEV variants with variable poly(A) tracts within a single blood-fed tick. To characterize the role of the poly(A) tract in TBEV replication and virulence, we generated infectious clones of Torö-2003 with the wild-type (A)3C(A)6 sequence (Torö-6A) or with a modified (A)3C(A)38 sequence (Torö-38A). Torö-38A replicated poorly compared to Torö-6A in cell culture, but Torö-38A was more virulent than Torö-6A in a mouse model of TBE. Next-generation sequencing of TBEV genomes after passaging in cell culture and/or mouse brain revealed mutations in specific genomic regions and the presence of quasispecies that might contribute to the observed differences in virulence. These data suggest a role for quasispecies development within the poly(A) tract as a virulence determinant for TBEV in mice.
    • SARS-CoV-2 neutralizing human recombinant antibodies selected from pre-pandemic healthy donors binding at RBD-ACE2 interface.

      Bertoglio, Federico; Meier, Doris; Langreder, Nora; Steinke, Stephan; Rand, Ulfert; Simonelli, Luca; Heine, Philip Alexander; Ballmann, Rico; Schneider, Kai-Thomas; Roth, Kristian Daniel Ralph; et al. (NPG, 2021-03-11)
      COVID-19 is a severe acute respiratory disease caused by SARS-CoV-2, a new recently emerged sarbecovirus. This virus uses the human ACE2 enzyme as receptor for cell entry, recognizing it with the receptor binding domain (RBD) of the S1 subunit of the viral spike protein. We present the use of phage display to select anti-SARS-CoV-2 spike antibodies from the human naïve antibody gene libraries HAL9/10 and subsequent identification of 309 unique fully human antibodies against S1. 17 antibodies are binding to the RBD, showing inhibition of spike binding to cells expressing ACE2 as scFv-Fc and neutralize active SARS-CoV-2 virus infection of VeroE6 cells. The antibody STE73-2E9 is showing neutralization of active SARS-CoV-2 as IgG and is binding to the ACE2-RBD interface. Thus, universal libraries from healthy human donors offer the advantage that antibodies can be generated quickly and independent from the availability of material from recovering patients in a pandemic situation.