Recent Submissions

  • The envelope protein of tick-borne encephalitis virus influences neuron entry, pathogenicity, and vaccine protection.

    Lindqvist, Richard; Rosendal, Ebba; Weber, Elvira; Asghar, Naveed; Schreier, Sarah; Lenman, Annasara; Johansson, Magnus; Dobler, Gerhard; Bestehorn, Malena; Kröger, Andrea; et al. (BMC, 2020-09-28)
    Background: Tick-borne encephalitis virus (TBEV) is considered to be the medically most important arthropod-borne virus in Europe. The symptoms of an infection range from subclinical to mild flu-like disease to lethal encephalitis. The exact determinants of disease severity are not known; however, the virulence of the strain as well as the immune status of the host are thought to be important factors for the outcome of the infection. Here we investigated virulence determinants in TBEV infection. Method: Mice were infected with different TBEV strains, and high virulent and low virulent TBEV strains were chosen. Sequence alignment identified differences that were cloned to generate chimera virus. The infection rate of the parental and chimeric virus were evaluated in primary mouse neurons, astrocytes, mouse embryonic fibroblasts, and in vivo. Neutralizing capacity of serum from individuals vaccinated with the FSME-IMMUN® and Encepur® or combined were evaluated. Results: We identified a highly pathogenic and neurovirulent TBEV strain, 93/783. Using sequence analysis, we identified the envelope (E) protein of 93/783 as a potential virulence determinant and cloned it into the less pathogenic TBEV strain Torö. We found that the chimeric virus specifically infected primary neurons more efficiently compared to wild-type (WT) Torö and this correlated with enhanced pathogenicity and higher levels of viral RNA in vivo. The E protein is also the major target of neutralizing antibodies; thus, genetic variation in the E protein could influence the efficiency of the two available vaccines, FSME-IMMUN® and Encepur®. As TBEV vaccine breakthroughs have occurred in Europe, we chose to compare neutralizing capacity from individuals vaccinated with the two different vaccines or a combination of them. Our data suggest that the different vaccines do not perform equally well against the two Swedish strains. Conclusions: Our findings show that two amino acid substitutions of the E protein found in 93/783, A83T, and A463S enhanced Torö infection of neurons as well as pathogenesis and viral replication in vivo; furthermore, we found that genetic divergence from the vaccine strain resulted in lower neutralizing antibody titers in vaccinated individuals.
  • OCIAD1 is a host mitochondrial substrate of the hepatitis C virus NS3-4A protease

    Tran, Huong T. L.; et al.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (PLOS, 2020-07-01)
    The hepatitis C virus (HCV) nonstructural protein 3-4A (NS3-4A) protease is a key component of the viral replication complex and the target of protease inhibitors used in current clinical practice. By cleaving and thereby inactivating selected host factors it also plays a role in the persistence and pathogenesis of hepatitis C. Here, we describe ovarian cancer immunoreactive antigen domain containing protein 1 (OCIAD1) as a novel cellular substrate of the HCV NS3-4A protease. OCIAD1 was identified by quantitative proteomics involving stable isotopic labeling using amino acids in cell culture coupled with mass spectrometry. It is a poorly characterized membrane protein believed to be involved in cancer development. OCIAD1 is cleaved by the NS3-4A protease at Cys 38, close to a predicted transmembrane segment. Cleavage was observed in heterologous expression systems, the replicon and cell culture-derived HCV systems, as well as in liver biopsies from patients with chronic hepatitis C. NS3-4A proteases from diverse hepacivirus species efficiently cleaved OCIAD1. The subcellular localization of OCIAD1 on mitochondria was not altered by NS3-4A-mediated cleavage. Interestingly, OCIAD2, a homolog of OCIAD1 with a cysteine residue in a similar position and identical subcellular localization, was not cleaved by NS3-4A. Domain swapping experiments revealed that the sequence surrounding the cleavage site as well as the predicted transmembrane segment contribute to substrate selectivity. Overexpression as well as knock down and rescue experiments did not affect the HCV life cycle in vitro, raising the possibility that OCIAD1 may be involved in the pathogenesis of hepatitis C in vivo.
  • 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.
  • Lactoferrin-Hexon Interactions Mediate CAR-Independent Adenovirus Infection of Human Respiratory Cells.

    Persson, B David; Lenman, Annasara; Frängsmyr, Lars; Schmid, Markus; Ahlm, Clas; Plückthun, Andreas; Jenssen, Håvard; Arnberg, Niklas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (ASM, 2020-07-01)
    Virus entry into host cells is a complex process that is largely regulated by access to specific cellular receptors. Human adenoviruses (HAdVs) and many other viruses use cell adhesion molecules such as the coxsackievirus and adenovirus receptor (CAR) for attachment to and entry into target cells. These molecules are rarely expressed on the apical side of polarized epithelial cells, which raises the question of how adenoviruses-and other viruses that engage cell adhesion molecules-enter polarized cells from the apical side to initiate infection. We have previously shown that species C HAdVs utilize lactoferrin-a common innate immune component secreted to respiratory mucosa-for infection via unknown mechanisms. Using a series of biochemical, cellular, and molecular biology approaches, we mapped this effect to the proteolytically cleavable, positively charged, N-terminal 49 residues of human lactoferrin (hLF) known as human lactoferricin (hLfcin). Lactoferricin (Lfcin) binds to the hexon protein on the viral capsid and anchors the virus to an unknown receptor structure of target cells, resulting in infection. These findings suggest that HAdVs use distinct cell entry mechanisms at different stages of infection. To initiate infection, entry is likely to occur at the apical side of polarized epithelial cells, largely by means of hLF and hLfcin bridging HAdV capsids via hexons to as-yet-unknown receptors; when infection is established, progeny virions released from the basolateral side enter neighboring cells by means of hLF/hLfcin and CAR in parallel.IMPORTANCE Many viruses enter target cells using cell adhesion molecules as receptors. Paradoxically, these molecules are abundant on the lateral and basolateral side of intact, polarized, epithelial target cells, but absent on the apical side that must be penetrated by incoming viruses to initiate infection. Our study provides a model whereby viruses use different mechanisms to infect polarized epithelial cells depending on which side of the cell-apical or lateral/basolateral-is attacked. This study may also be useful to understand the biology of other viruses that use cell adhesion molecules as receptors.
  • Methylene Blue Treatment of Grafts During Cold Ischemia Time Reduces the Risk of Hepatitis C Virus Transmission.

    Helfritz, Fabian A; Bojkova, Denisa; Wanders, Verena; Kuklinski, Nina; Westhaus, Sandra; von Horn, Charlotte; Rauen, Ursula; Gallinat, Anja; Baba, Hideo A; Skyschally, Andreas; et al. (Oxford Academic, 2018-12-01)
    Background: Although organ shortage is a rising problem, organs from hepatitis C virus (HCV) ribonucleic acid (RNA)-positive donors are not routinely transplanted in HCV-negative individuals. Because HCV only infects hepatocytes, other organs such as kidneys are merely contaminated with HCV via the blood. In this study, we established a protocol to reduce HCV virions during the cold ischemic time. Methods: Standard virological assays were used to investigate the effect of antivirals, including methylene blue (MB), in different preservation solutions. Kidneys from mini pigs were contaminated with Jc1 or HCV RNA-positive human serum. Afterwards, organs were flushed with MB. Hypothermic machine perfusion was used to optimize reduction of HCV. Results: Three different antivirals were investigated for their ability to inactivate HCV in vitro. Only MB completely inactivated HCV in the presence of all perfusion solutions. Hepatitis C virus-contaminated kidneys from mini pigs were treated with MB and hypothermic machine perfusion without any negative effect on the graft. Human liver-uPA-SCID mice did not establish HCV infection after inoculation with flow through from these kidneys. Conclusions: This proof-of-concept study is a first step to reduce transmission of infectious HCV particles in the transplant setting and might serve as a model for other relevant pathogens.
  • Mesenchymal to epithelial transition driven by canine distemper virus infection of canine histiocytic sarcoma cells contributes to a reduced cell motility in vitro.

    Armando, Federico; Gambini, Matteo; Corradi, Attilio; Becker, Kathrin; Marek, Katarzyna; Pfankuche, Vanessa Maria; Mergani, Ahmed Elmonastir; Brogden, Graham; de Buhr, Nicole; Von Köckritz-Blickwede, Maren; et al. (Blackwell Publishing, 2020-07-06)
    Sarcomas especially of histiocytic origin often possess a poor prognosis and response to conventional therapies. Interestingly, tumours undergoing mesenchymal to epithelial transition (MET) are often associated with a favourable clinical outcome. This process is characterized by an increased expression of epithelial markers leading to a decreased invasion and metastatic rate. Based on the failure of conventional therapies, viral oncolysis might represent a promising alternative with canine distemper virus (CDV) as a possible candidate. This study hypothesizes that a CDV infection of canine histiocytic sarcoma cells (DH82 cells) triggers the MET process leading to a decreased cellular motility. Immunofluorescence and immunoblotting were used to investigate the expression of epithelial and mesenchymal markers followed by scratch assay and an invasion assay as functional confirmation. Furthermore, microarray data were analysed for genes associated with the MET process, invasion and angiogenesis. CDV-infected cells exhibited an increased expression of epithelial markers such as E-cadherin and cytokeratin 8 compared to controls, indicating a MET process. This was accompanied by a reduced cell motility and invasiveness. Summarized, these results suggest that CDV infection of DH82 cells triggers the MET process by an increased expression of epithelial markers resulting in a decreased cell motility in vitro.
  • The ATGL lipase cooperates with ABHD5 to mobilize lipids for hepatitis C virus assembly.

    Vieyres, Gabrielle; Reichert, Isabelle; Carpentier, Arnaud; Vondran, Florian W R; Pietschmann, Thomas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (PLOS, 2020-06-15)
    Lipid droplets are essential cellular organelles for storage of fatty acids and triglycerides. The hepatitis C virus (HCV) translocates several of its proteins onto their surface and uses them for production of infectious progeny. We recently reported that the lipid droplet-associated α/β hydrolase domain-containing protein 5 (ABHD5/CGI-58) participates in HCV assembly by mobilizing lipid droplet-associated lipids. However, ABHD5 itself has no lipase activity and it remained unclear how ABHD5 mediates lipolysis critical for HCV assembly. Here, we identify adipose triglyceride lipase (ATGL) as ABHD5 effector and new host factor involved in the hepatic lipid droplet degradation as well as in HCV and lipoprotein morphogenesis. Modulation of ATGL protein expression and lipase activity controlled lipid droplet lipolysis and virus production. ABHD4 is a paralog of ABHD5 unable to activate ATGL or support HCV assembly and lipid droplet lipolysis. Grafting ABHD5 residues critical for activation of ATGL onto ABHD4 restored the interaction between lipase and co-lipase and bestowed the pro-viral and lipolytic functions onto the engineered protein. Congruently, mutation of the predicted ABHD5 protein interface to ATGL ablated ABHD5 functions in lipid droplet lipolysis and HCV assembly. Interestingly, minor alleles of ABHD5 and ATGL associated with neutral lipid storage diseases in human, are also impaired in lipid droplet lipolysis and their pro-viral functions. Collectively, these results show that ABHD5 cooperates with ATGL to mobilize triglycerides for HCV infectious virus production. Moreover, viral manipulation of lipid droplet homeostasis via the ABHD5-ATGL axis, akin to natural genetic variation in these proteins, emerges as a possible mechanism by which chronic HCV infection causes liver steatosis.
  • Identification of a Human Respiratory Syncytial Virus Cell Entry Inhibitor by Using a Novel Lentiviral Pseudotype System.

    Haid, Sibylle; Grethe, Christina; Bankwitz, Dorothea; Grunwald, Thomas; Pietschmann, Thomas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (ASM, 2015-12-30)
    Lentiviral budding is governed by group-specific antigens (Gag proteins) and proceeds in the absence of cognate viral envelope proteins, which has been exploited to create pseudotypes incorporating envelope proteins from nonlentiviral families. Here, we report the generation of infectious lentiviral pseudoparticles incorporating human respiratory syncytial virus (hRSV) F protein alone (hRSV-Fpp) or carrying SH, G, and F proteins (hRSV-SH/G/Fpp). These particles recapitulate key infection steps of authentic hRSV particles, including utilization of glycosaminoglycans and low-pH-independent cell entry. Moreover, hRSV pseudoparticles (hRSVpp) can faithfully reproduce phenotypic resistance to a small-molecule fusion inhibitor in clinical development (BMS-433771) and a licensed therapeutic F protein-targeting antibody (palivizumab). Inoculation of several human cell lines from lung and liver revealed more than 30-fold differences in susceptibility to hRSVpp infection, suggesting differential expression of hRSV entry cofactors and/or restriction factors between these cell types. Moreover, we observed cell-type-dependent functional differences between hRSVpp carrying solely F protein or SH, G, and F proteins with regard to utilization of glycosaminoglycans. Using hRSVpp, we identified penta-O-galloyl-β-d-glucose (PGG) as a novel hRSV cell entry inhibitor. Moreover, we show that PGG also inhibits cell entry of hRSVpp carrying F proteins resistant to BMS-433771 or palivizumab. This work sheds new light on the mechanisms of hRSV cell entry, including possible strategies for antiviral intervention. Moreover, hRSVpp should prove valuable to dissect hRSV envelope protein functions, including the interaction with cell entry factors. Importance: Lentiviral pseudotypes are highly useful to specifically dissect the functions of viral and host factors in cell entry, which have been exploited for numerous viruses. Here, we successfully created hRSVpp and show that they faithfully recapitulate key characteristics of parental hRSV cell entry. Importantly, hRSVpp accurately mirror hRSV resistance to small-molecule fusion inhibitors and clinically approved therapeutic antibodies. Moreover, we observed highly different susceptibilities of cell lines to hRSVpp infection and also differences between hRSVpp types (with F protein alone or with SH, G, and F proteins) in regard to cell entry. This indicates differential expression of host factors determining hRSV cell entry between these cell lines and highlights the fact that the hRSVpp system is useful to explore the functional properties of hRSV envelope protein combinations. Therefore, this system will be highly useful to study hRSV cell entry and host factor usage and to explore antiviral strategies targeting hRSV cell entry.
  • Critical challenges and emerging opportunities in hepatitis C virus research in an era of potent antiviral therapy: Considerations for scientists and funding agencies.

    Bartenschlager, Ralf; Baumert, Thomas F; Bukh, Jens; Houghton, Michael; Lemon, Stanley M; Lindenbach, Brett D; Lohmann, Volker; Moradpour, Darius; Pietschmann, Thomas; Rice, Charles M; et al. (Elsevier, 2018-03-02)
    The development and clinical implementation of direct-acting antivirals (DAAs) has revolutionized the treatment of chronic hepatitis C. Infection with any hepatitis C virus (HCV) genotype can now be eliminated in more than 95% of patients with short courses of all-oral, well-tolerated drugs, even in those with advanced liver disease and liver transplant recipients. DAAs have proven so successful that some now consider HCV amenable to eradication, and continued research on the virus of little remaining medical relevance. However, given 400,000 HCV-related deaths annually important challenges remain, including identifying those who are infected, providing access to treatment and reducing its costs. Moreover, HCV infection rarely induces sterilizing immunity, and those who have been cured with DAAs remain at risk for reinfection. Thus, it is very unlikely that global eradication and elimination of the cancer risk associated with HCV infection can be achieved without a vaccine, yet research in that direction receives little attention. Further, over the past two decades HCV research has spearheaded numerous fundamental discoveries in the fields of molecular and cell biology, immunology and microbiology. It will continue to do so, given the unique opportunities afforded by the reagents and knowledge base that have been generated in the development and clinical application of DAAs. Considering these critical challenges and new opportunities, we conclude that funding for HCV research must be sustained.
  • Filovirus antiviral activity of cationic amphiphilic drugs is associated with lipophilicity and ability to induce phospholipidosis.

    Gunesch, Antonia P; Zapatero-Belinchon, Francisco J; Pinkert, Lukas; Steinmann, Eike; Manns, Michael P; Schneider, Gisbert; Pietschmann, Thomas; Brönstrup, Mark; von Hahn, Thomas; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (ASM, 2020-06-08)
    Several cationic amphiphilic drugs (CADs) have been found to inhibit cell entry of filoviruses and other enveloped viruses. Structurally unrelated CADs may have antiviral activity, yet the underlying common mechanism and structure-activity relationship are incompletely understood.We aimed to understand how widespread antiviral activity is among CADs and which structural and physico-chemical properties are linked to entry inhibition.We measured inhibition of Marburg virus pseudoparticle (MARVpp) cell entry by 45 heterogeneous and mostly FDA-approved CADs and cytotoxicity in EA.hy926 cells. We analysed correlation of antiviral activity with four chemical properties: pKa, ClogP, molecular weight and distance between the basic group and hydrophobic ring structures. Additionally, we quantified drug-induced phospholipidosis (DIPL) of a CAD subset by flow cytometry. Structurally similar compounds (derivatives) and those with similar chemical properties but unrelated structure (analogues) to strong inhibitors were obtained by two in silico similarity search approaches and tested for antiviral activity. Overall 11 out of 45 (24 %) CADs inhibited MARVpp by 40 % or more. The strongest antiviral compounds were dronedarone, triparanol and quinacrine. Structure-activity relationship studies revealed highly significant correlations between antiviral activity, hydrophobicity (ClogP>4), and DIPL. Moreover, pKa and intra-molecular distance between hydrophobic and hydrophilic moieties correlated with antiviral activity, but to a lesser extent. We also showed that in contrast to analogues, derivatives had similar antiviral activity as the seed compound dronedarone. Overall, one quarter of CADs inhibits MARVpp entry in vitro and antiviral activity of CADs mostly relies on their hydrophobicity, yet is promoted by the individual structure.
  • Add on the next level-the time point of the type I IFN response orchestrates the immune response.

    Wedekind, Angela; Fritzsch, David; Kröger, Andrea; HZI,Helmholtz-Zentrum für Infektionsforschung GmbH, Inhoffenstr. 7,38124 Braunschweig, Germany. (Springer Nature, 2020-04-28)
    [No abstract available]
  • Labyrinthopeptins as virolytic inhibitors of respiratory syncytial virus cell entry.

    Blockus, Sebastian; Sake, Svenja M; Wetzke, Martin; Grethe, Christina; Graalmann, Theresa; Pils, Marina; Le Goffic, Ronan; Galloux, Marie; Prochnow, Hans; Rox, Katharina; et al. (Elsevier, 2020-03-18)
    Acute lower respiratory tract infections (ALRI) caused by respiratory syncytial virus (RSV) are associated with a severe disease burden among infants and elderly patients. Treatment options are limited. While numerous drug candidates with different viral targets are under development, the utility of RSV entry inhibitors is challenged by a low resistance barrier and by single mutations causing cross-resistance against a wide spectrum of fusion inhibitor chemotypes. We developed a cell-based screening assay for discovery of compounds inhibiting infection with primary RSV isolates. Using this system, we identified labyrinthopeptin A1 and A2 (Laby A1/A2), lantibiotics isolated from Actinomadura namibiensis, as effective RSV cell entry inhibitors with IC50s of 0.39 μM and 4.97 μM, respectively, and with favourable therapeutic index (>200 and > 20, respectively). Both molecules were active against multiple RSV strains including primary isolates and their antiviral activity against RSV was confirmed in primary human airway cells ex vivo and a murine model in vivo. Laby A1/A2 were antiviral in prophylactic and therapeutic treatment regimens and displayed synergistic activity when applied in combination with each other. Mechanistic studies showed that Laby A1/A2 exert virolytic activity likely by binding to phosphatidylethanolamine moieties within the viral membrane and by disrupting virus particle membrane integrity. Probably due to its specific mode of action, Laby A1/A2 antiviral activity was not affected by common resistance mutations to known RSV entry inhibitors. Taken together, Laby A1/A2 represent promising candidates for development as RSV inhibitors. Moreover, the cell-based screening system with primary RSV isolates described here should be useful to identify further antiviral agents.
  • Efficient acute and chronic infection of stem cell-derived hepatocytes by hepatitis C virus.

    Carpentier, Arnaud; Sheldon, Julie; Vondran, Florian W R; Brown, Richard Jp; Pietschmann, Thomas; TWINCORE, Zentrum für Experimentelle und Klinische Infektionsforschung GmbH, Feodor-Lynen-Str. 7, 30625 Hannover, Germany. (BMJ Publishing Group, 2020-02-29)
    Transcriptional profiling revealed that HLCs constitutively express messenger RNA of RLRs, and members of the IFN pathway. Moreover, HLCs upregulated IFNs and canonical interferon-regulated genes (IRGs) upon transfection with the double-stranded RNA mimic poly(I:C). Infection of HLCs with Jc1-HCVcc produced only limited viral progeny. In contrast, infection with p100, a Jc1-derived virus population with enhanced replication fitness and partial resistance to IFN, resulted in robust yet transient viraemia. Viral titres declined concomitant with a peak of IRG induction. Addition of ruxolitinib, a JAK/STAT inhibitor, permitted chronic infection and raised p100 infectious virus titres to 1×105 FFU/mL. IRGs expression profiling in infected HLCs revealed a landscape of HCV-dependent transcriptional changes similar to HCV-infected primary human hepatocytes, but distinct from Huh-7.5 cells. Withdrawal of ruxolitinib restored innate immune responses and resulted in HCV clearance.
  • Extracellular Traps: An Ancient Weapon of Multiple Kingdoms.

    Neumann, Ariane; Brogden, Graham; von Köckritz-Blickwede, Maren; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MDPI, 2020-02-18)
    The discovery, in 2004, of extracellular traps released by neutrophils has extended our understanding of the mode of action of various innate immune cells. This fascinating discovery demonstrated the extracellular trapping and killing of various pathogens by neutrophils. During the last decade, evidence has accumulated showing that extracellular traps play a crucial role in the defence mechanisms of various cell types present in vertebrates, invertebrates, and plants. The aim of this review is to summarise the relevant literature on the evolutionary history of extracellular traps used as a weapon in various kingdoms of life.
  • The Antiviral Activity of the Cellular Glycoprotein LGALS3BP/90K Is Species Specific.

    Lodermeyer, Veronika; Ssebyatika, George; Passos, Vânia; Ponnurangam, Aparna; Malassa, Angelina; Ewald, Ellen; Stürzel, Christina M; Kirchhoff, Frank; Rotger, Margalida; Falk, Christine S; et al. (American Society for Microbiology (ASM), 2018-07-15)
    Cellular antiviral proteins interfere with distinct steps of replication cycles of viruses. The galectin 3 binding protein (LGALS3BP, also known as 90K) was previously shown to lower the infectivity of nascent human immunodeficiency virus type 1 (HIV-1) virions when expressed in virus-producing cells. This antiviral effect was accompanied by impaired gp160Env processing and reduced viral incorporation of mature Env glycoproteins. Here, we examined the ability of 90K orthologs from primate species to reduce the particle infectivity of distinct lentiviruses. We show that 90K's ability to diminish the infectivity of lentiviral particles is conserved within primate species, with the notable exception of 90K from rhesus macaque. Comparison of active and inactive 90K orthologs and variants uncovered the fact that inhibition of processing of the HIV-1 Env precursor and reduction of cell surface expression of HIV-1 Env gp120 are required, but not sufficient, for 90K-mediated antiviral activity. Rather, 90K-mediated reduction of virion-associated gp120 coincided with antiviral activity, suggesting that 90K impairs the incorporation of HIV-1 Env into budding virions. We show that a single "humanizing" amino acid exchange in the BTB (broad-complex, tramtrack, and bric-à-brac)/POZ (poxvirus and zinc finger) domain is sufficient to fully rescue the antiviral activity of a shortened version of rhesus macaque 90K, but not that of the full-length protein. Comparison of the X-ray structures of the BTB/POZ domains of 90K from rhesus macaques and humans point toward a slightly larger hydrophobic patch at the surface of the rhesus macaque BTB domain that may modulate a direct interaction with either a second 90K domain or a different protein.IMPORTANCE The cellular 90K protein has been shown to diminish the infectivity of nascent HIV-1 particles. When produced in 90K-expressing cells, particles bear smaller amounts of the HIV-1 Env glycoprotein, which is essential for attaching to and entering new target cells in the subsequent infection round. However, whether the antiviral function of 90K is conserved across primates is unknown. Here, we found that 90K orthologs from most primate species, but, surprisingly, not from rhesus macaques, inhibit HIV-1. The introduction of a single amino acid exchange into a short version of the rhesus macaque 90K protein, consisting of the two intermediate domains of 90K, resulted in full restoration of antiviral activity. Structural elucidation of the respective domain suggests that the absence of antiviral activity in the rhesus macaque factor may be linked to a subtle change in protein-protein interaction.
  • Hepatitis E virus replication and interferon responses in human placental cells.

    Knegendorf, Leonard; Drave, Svenja A; Dao Thi, Viet Loan; Debing, Yannick; Brown, Richard J P; Vondran, Florian W R; Resner, Kathrin; Friesland, Martina; Khera, Tanvi; Engelmann, Michael; et al. (Wiley-Blackwell, 2018-01-01)
    Hepatitis E virus (HEV) is a member of the genus Orthohepevirus in the family Hepeviridae and the causative agent of hepatitis E in humans. HEV is a major health problem in developing countries, causing mortality rates up to 25% in pregnant women. However, these cases are mainly reported for HEV genotype (gt)1, while gt3 infections are usually associated with subclinical courses of disease. The pathogenic mechanisms of adverse maternal and fetal outcome during pregnancy in HEV-infected pregnant women remain elusive. In this study, we observed that HEV is capable of completing the full viral life cycle in placental-derived cells (JEG-3). Following transfection of JEG-3 cells, HEV replication of both HEV gts could be observed. Furthermore, determination of extracellular and intracellular viral capsid levels, infectivity, and biophysical properties revealed production of HEV infectious particles with similar characteristics as in liver-derived cells. Viral entry was analyzed by infection of target cells and detection of either viral RNA or staining for viral capsid protein by immunofluorescence. HEV gt1 and gt3 were efficiently inhibited by ribavirin in placental as well as in human hepatoma cells. In contrast, interferon-α sensitivity was lower in the placental cells compared to liver cells for gt1 but not gt3 HEV. Simultaneous determination of interferon-stimulated gene expression levels demonstrated an efficient HEV-dependent restriction in JEG-3. Conclusion: We showed differential tissue-specific host responses to HEV genotypes, adding to our understanding of the mechanisms contributing to fatal outcomes of HEV infections during pregnancy. Using this cell-culture system, new therapeutic options for HEV during pregnancy can be identified and evaluated. (Hepatology Communications 2018;2:173-187).
  • Robust hepatitis E virus infection and transcriptional response in human hepatocytes.

    Todt, Daniel; Friesland, Martina; Moeller, Nora; Praditya, Dimas; Kinast, Volker; Brüggemann, Yannick; Knegendorf, Leonard; Burkard, Thomas; Steinmann, Joerg; Burm, Rani; et al. (National Academy of Sciences, 2020-01-02)
    Hepatitis E virus (HEV) is the causative agent of hepatitis E in humans and the leading cause for acute viral hepatitis worldwide. The virus is classified as a member of the genus Orthohepevirus A within the Hepeviridae family. Due to the absence of a robust cell culture model for HEV infection, the analysis of the viral life cycle, the development of effective antivirals and a vaccine is severely limited. In this study, we established a protocol based on the HEV genotype 3 p6 (Kernow C-1) and the human hepatoma cell lines HepG2 and HepG2/C3A with different media conditions to produce intracellular HEV cell culture-derived particles (HEVcc) with viral titers between 105 and 106 FFU/mL. Viral titers could be further enhanced by an HEV variant harboring a mutation in the RNA-dependent RNA polymerase. These HEVcc particles were characterized in density gradients and allowed the trans-complementation of subgenomic reporter HEV replicons. In addition, in vitro produced intracellular-derived particles were infectious in liver-humanized mice with high RNA copy numbers detectable in serum and feces. Efficient infection of primary human and swine hepatocytes using the developed protocol could be observed and was inhibited by ribavirin. Finally, RNA sequencing studies of HEV-infected primary human hepatocytes demonstrated a temporally structured transcriptional defense response. In conclusion, this robust cell culture model of HEV infection provides a powerful tool for studying viral-host interactions that should facilitate the discovery of antiviral drugs for this important zoonotic pathogen.
  • Hepatitis C Virus Entry: Protein Interactions and Fusion Determinants Governing Productive Hepatocyte Invasion.

    Gerold, Gisa; Moeller, Rebecca; Pietschmann, Thomas; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (Cold Spring Harbor Laboratory Press, 2019-08-19)
    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 endosomal 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.
  • No Evidence of Mosquito Involvement in the Transmission of Equine Hepacivirus (Flaviviridae) in an Epidemiological Survey of Austrian Horses

    Badenhorst, Marcha; de Heus, Phebe; Auer, Angelika; Rümenapf, Till; Tegtmeyer, Birthe; Kolodziejek, Jolanta; Nowotny, Norbert; Steinmann, Eike; Cavalleri, Jessika M.V.; TWINCORE, Zentrum für experimentelle und klinische Infektionsforschung GmbH,Feodor-Lynen Str. 7, 30625 Hannover, Germany. (MPDI, 2019-11-01)
    Prevalence studies have demonstrated a global distribution of equine hepacivirus (EqHV), a member of the family Flaviviridae. However, apart from a single case of vertical transmission, natural routes of EqHV transmission remain elusive. Many known flaviviruses are horizontally transmitted between hematophagous arthropods and vertebrate hosts. This study represents the first investigation of potential EqHV transmission by mosquitoes. More than 5000 mosquitoes were collected across Austria and analyzed for EqHV ribonucleic acid (RNA) by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Concurrently, 386 serum samples from horses in eastern Austria were analyzed for EqHV-specific antibodies by luciferase immunoprecipitation system (LIPS) and for EqHV RNA by RT-qPCR. Additionally, liver-specific biochemistry parameters were compared between EqHV RNA-positive horses and EqHV RNA-negative horses. Phylogenetic analysis was conducted in comparison to previously published sequences from various origins. No EqHV RNA was detected in mosquito pools. Serum samples yielded an EqHV antibody prevalence of 45.9% (177/386) and RNA prevalence of 4.15% (16/386). EqHV RNA-positive horses had significantly higher glutamate dehydrogenase (GLDH) levels (p = 0.013) than control horses. Phylogenetic analysis showed high similarity between nucleotide sequences of EqHV in Austrian horses and EqHV circulating in other regions. Despite frequently detected evidence of EqHV infection in Austrian horses, no viral RNA was found in mosquitoes. It is therefore unlikely that mosquitoes are vectors of this flavivirus.
  • 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.

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