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Currently, human infections with the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are accelerating the ongoing spread of the pandemic. Several innovative types of vaccines have already been developed, whereas effective options of antiviral treatments still await a scientific implementation. The development of novel anti-SARS-CoV-2 drug candidates demands skillful strategies and analysis systems. Promising results have been achieved with first generation direct-acting antivirals targeting the viral polymerase RdRp or the protease 3CLpro. Such recently approved or investigational drugs like remdesivir and GC376 represent a basis for further development and optimization. Here, we establish a multi-readout assay (MRA) system that enables the antiviral assessment and mechanistic characterization of novel test compounds, drug repurposing and combination treatments. Our SARS-CoV-2-specific MRA combines the quantitative measurement of several parameters of virus infection, such as the intracellular production of proteins and genomes, enzymatic activities and virion release, as well as the use of reporter systems. In this regard, the antiviral efficacy of remdesivir and GC376 has been investigated in human Caco-2 cells. The readouts included the use of spike- and double-strand RNA-specific monoclonal antibodies for in-cell fluorescence imaging, a newly generated recombinant SARS-CoV-2 reporter virus d6YFP, the novel 3CLpro-based FRET CFP::YFP and the previously reported FlipGFP reporter assays, as well as viral genome-specific RT-qPCR. The data produced by our MRA confirm the high antiviral potency of these two drugs in vitro. Combined, this MRA approach may be applied for broader analyses of SARS-CoV-2-specific antivirals, including compound screenings and the characterization of selected drug candidates.
Friedrich Hahn; Sigrun Häge; Alexandra Herrmann; Christina Wangen; Jintawee Kicuntod; Doris Jungnickl; Julia Tillmanns; Regina Müller; Kirsten Fraedrich; Klaus Überla; Hella Kohlhof; Armin Ensser; Manfred Marschall. Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2. Pathogens 2021, 10, 1076 .
AMA StyleFriedrich Hahn, Sigrun Häge, Alexandra Herrmann, Christina Wangen, Jintawee Kicuntod, Doris Jungnickl, Julia Tillmanns, Regina Müller, Kirsten Fraedrich, Klaus Überla, Hella Kohlhof, Armin Ensser, Manfred Marschall. Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2. Pathogens. 2021; 10 (9):1076.
Chicago/Turabian StyleFriedrich Hahn; Sigrun Häge; Alexandra Herrmann; Christina Wangen; Jintawee Kicuntod; Doris Jungnickl; Julia Tillmanns; Regina Müller; Kirsten Fraedrich; Klaus Überla; Hella Kohlhof; Armin Ensser; Manfred Marschall. 2021. "Methodological Development of a Multi-Readout Assay for the Assessment of Antiviral Drugs against SARS-CoV-2." Pathogens 10, no. 9: 1076.
Varicella zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. Here, the crystal structure of the VZV Orf24-Orf27 complex is described, representing the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled capsids from the nucleus. While previous studies have primarily emphasized the finding that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focusses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. CoIP and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of the NEC formation thermodynamic parameters of three prototypical α-, β- and γ herpesviruses, i.e. VZV, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) reveals highly similar binding affinities for the autologous interaction with some specific differences in the enthalpy and entropy terms. Computational alanine scanning and structural comparisons highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses. Combined, these data allow to explain the distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings might prove highly valuable when attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.
Johannes Schweininger; Mark Kriegel; Sigrun Häge; Marcus Conrad; Sewar Alkhashrom; Josephine Lösing; Sigrid Weiler; Julia Tillmanns; Claudia Egerer-Sieber; Andrea Decker; Tihana Lenac Roviš; Jutta Eichler; Heinrich Sticht; Manfred Marschall; Yves A. Muller. The crystal structure of the varicella zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity. 2021, 1 .
AMA StyleJohannes Schweininger, Mark Kriegel, Sigrun Häge, Marcus Conrad, Sewar Alkhashrom, Josephine Lösing, Sigrid Weiler, Julia Tillmanns, Claudia Egerer-Sieber, Andrea Decker, Tihana Lenac Roviš, Jutta Eichler, Heinrich Sticht, Manfred Marschall, Yves A. Muller. The crystal structure of the varicella zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity. . 2021; ():1.
Chicago/Turabian StyleJohannes Schweininger; Mark Kriegel; Sigrun Häge; Marcus Conrad; Sewar Alkhashrom; Josephine Lösing; Sigrid Weiler; Julia Tillmanns; Claudia Egerer-Sieber; Andrea Decker; Tihana Lenac Roviš; Jutta Eichler; Heinrich Sticht; Manfred Marschall; Yves A. Muller. 2021. "The crystal structure of the varicella zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity." , no. : 1.
The replication of human cytomegalovirus (HCMV) is characterized by a complex network of virus–host interaction. This involves the regulatory viral protein kinase pUL97, which represents a viral cyclin-dependent kinase ortholog (vCDK) combining typical structural and functional features of host CDKs. Notably, pUL97 interacts with the three human cyclin types T1, H and B1, whereby the binding region of cyclin T1 and the region conferring oligomerization of pUL97 were both assigned to amino acids 231–280. Here, we addressed the question of whether recombinant HCMVs harboring deletions in this region were impaired in cyclin interaction, kinase functionality or viral replication. To this end, recombinant HCMVs were generated by traceless BACmid mutagenesis and were phenotypically characterized using a methodological platform based on qPCR, coimmunoprecipitation, in vitro kinase assay (IVKA), Phos-tag Western blot and confocal imaging analysis. Combined data illustrate the following: (i) infection kinetics of all three recombinant HCMVs, i.e., ORF-UL97 ∆231–255, ∆256–280 and ∆231–280, showed impaired replication efficiency compared to the wild type, amongst which the largest deletion exhibited the most pronounced defect; (ii) specifically, this mutant ∆231–280 showed a loss of interaction with cyclin T1, as demonstrated by CoIP and confocal imaging; (iii) IVKA and Phos-tag analyses revealed strongly affected kinase activity for ∆231–280, with strong impairment of both autophosphorylation and substrate phosphorylation, but less pronounced impairments for ∆231–255 and ∆256–280; and (iv) a bioinformatic assessment of the pUL97–cyclin T1 complex led to the refinement of our current binding model. Thus, the results provide initial evidence for the functional importance of the pUL97–cyclin interaction concerning kinase activity and viral replication fitness.
Martin Schütz; Mirjam Steingruber; Eileen Socher; Regina Müller; Sabrina Wagner; Merle Kögel; Heinrich Sticht; Manfred Marschall. Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97. Viruses 2021, 13, 1248 .
AMA StyleMartin Schütz, Mirjam Steingruber, Eileen Socher, Regina Müller, Sabrina Wagner, Merle Kögel, Heinrich Sticht, Manfred Marschall. Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97. Viruses. 2021; 13 (7):1248.
Chicago/Turabian StyleMartin Schütz; Mirjam Steingruber; Eileen Socher; Regina Müller; Sabrina Wagner; Merle Kögel; Heinrich Sticht; Manfred Marschall. 2021. "Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97." Viruses 13, no. 7: 1248.
Nuclear egress is an essential process in the replication of human cytomegalovirus (HCMV), as it enables the migration of newly formed viral capsids from the nucleus into the cytoplasm. Inhibition of the HCMV core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, has been proposed as a potential new target for the treatment of HCMV infection and disease. Here, we present a new type of small molecule inhibitors of HCMV core NEC formation, which inhibit the pUL50-pUL53 interaction at nanomolar concentrations. These inhibitors, i.e., verteporfin and merbromin, were identified through the screening of the Prestwick Chemical Library® of approved drug compounds. The inhibitory effect of merbromin is both compound- and target-specific, as no inhibition was seen for other mercury-organic compounds. Furthermore, merbromin does not inhibit an unrelated protein–protein interaction either. More importantly, merbromin was found to inhibit HCMV infection of cells in three different assays, as well as to disrupt HCMV NEC nuclear rim formation. Thus, while not being an ideal drug candidate by itself, merbromin may serve as a blueprint for small molecules with high HCMV core NEC inhibitory potential, as candidates for novel anti-herpesviral drugs.
Sewar Alkhashrom; Jintawee Kicuntod; Sigrun Häge; Johannes Schweininger; Yves Muller; Peter Lischka; Manfred Marschall; Jutta Eichler. Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors. Viruses 2021, 13, 471 .
AMA StyleSewar Alkhashrom, Jintawee Kicuntod, Sigrun Häge, Johannes Schweininger, Yves Muller, Peter Lischka, Manfred Marschall, Jutta Eichler. Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors. Viruses. 2021; 13 (3):471.
Chicago/Turabian StyleSewar Alkhashrom; Jintawee Kicuntod; Sigrun Häge; Johannes Schweininger; Yves Muller; Peter Lischka; Manfred Marschall; Jutta Eichler. 2021. "Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors." Viruses 13, no. 3: 471.
Herpesviral nuclear egress is a regulated process shared by all family members, ensuring the efficient cytoplasmic release of viral capsids. In the case of human cytomegalovirus (HCMV), the core of the nuclear egress complex (NEC) consists of the pUL50-pUL53 heterodimer that builds hexameric lattices for capsid binding and multicomponent interaction, including NEC-associated host factors. A characteristic feature of NEC interaction is the N-terminal hook structure of pUL53 that binds to an alpha-helical groove of pUL50, thus termed as hook-into-groove interaction. This central regulatory element is essential for viral replication and shows structural–functional conservation, which has been postulated as a next-generation target of antiviral strategies. However, a solid validation of this concept has been missing. In the present study, we focused on the properties of oligomeric HCMV core NEC interaction and the antiviral activity of specifically targeted prototype inhibitors. Our data suggest the following: (i) transiently expressed, variably tagged versions of HCMV NEC proteins exert hook-into-groove complexes, putatively in oligomeric assemblies that are distinguishable from heterodimers, as shown by in vitro assembly and coimmunoprecipitation approaches; (ii) this postulated oligomeric binding pattern was further supported by the use of a pUL50::pUL53 fusion construct also showing a pronounced multi-interaction potency; (iii) using confocal imaging cellular NEC-associated proteins were found partly colocalized with the tagged core NECs; (iv) a small inhibitory molecule, recently identified by an in vitro binding inhibition assay, was likewise active in blocking pUL50–pUL53 oligomeric assembly and in exerting antiviral activity in HCMV-infected fibroblasts. In summary, the findings refine the previous concept of HCMV core NEC formation and nominate this drug-accessible complex as a validated antiviral drug target.
Jintawee Kicuntod; Sewar Alkhashrom; Sigrun Häge; Benedikt Diewald; Regina Müller; Friedrich Hahn; Peter Lischka; Heinrich Sticht; Jutta Eichler; Manfred Marschall. Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule. Viruses 2021, 13, 462 .
AMA StyleJintawee Kicuntod, Sewar Alkhashrom, Sigrun Häge, Benedikt Diewald, Regina Müller, Friedrich Hahn, Peter Lischka, Heinrich Sticht, Jutta Eichler, Manfred Marschall. Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule. Viruses. 2021; 13 (3):462.
Chicago/Turabian StyleJintawee Kicuntod; Sewar Alkhashrom; Sigrun Häge; Benedikt Diewald; Regina Müller; Friedrich Hahn; Peter Lischka; Heinrich Sticht; Jutta Eichler; Manfred Marschall. 2021. "Properties of Oligomeric Interaction of the Cytomegalovirus Core Nuclear Egress Complex (NEC) and Its Sensitivity to an NEC Inhibitory Small Molecule." Viruses 13, no. 3: 462.
Nuclear egress is a common herpesviral process regulating nucleocytoplasmic capsid release. For human cytomegalovirus (HCMV), the nuclear egress complex (NEC) is determined by the pUL50-pUL53 core that regulates multicomponent assembly with NEC-associated proteins and capsids. Recently, NEC crystal structures were resolved for α-, β- and γ-herpesviruses, revealing profound structural conservation, which was not mirrored, however, by primary sequence and binding properties. The NEC binding principle is based on hook-into-groove interaction through an N-terminal hook-like pUL53 protrusion that embraces an α-helical pUL50 binding groove. So far, pUL50 has been considered as the major kinase-interacting determinant and massive phosphorylation of pUL50-pUL53 was assigned to NEC formation and functionality. Here, we addressed the question of phenotypical changes of ORF-UL50-mutated HCMVs. Surprisingly, our analyses did not detect a predominant replication defect for most of these viral mutants, concerning parameters of replication kinetics (qPCR), viral protein production (Western blot/CoIP) and capsid egress (confocal imaging/EM). Specifically, only the ORF-UL50 deletion rescue virus showed a block of genome synthesis during late stages of infection, whereas all phosphosite mutants exhibited marginal differences compared to wild-type or revertants. These results (i) emphasize a rate-limiting function of pUL50 for nuclear egress, and (ii) demonstrate that mutations in all mapped pUL50 phosphosites may be largely compensated. A refined mechanistic concept points to a multifaceted nuclear egress regulation, for which the dependence on the expression and phosphorylation of pUL50 is discussed.
Sigrun Häge; Eric Sonntag; Adriana Svrlanska; Eva Maria Borst; Anne-Charlotte Stilp; Deborah Horsch; Regina Müller; Barbara Kropff; Jens Milbradt; Thomas Stamminger; Ursula Schlötzer-Schrehardt; Manfred Marschall. Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation. Viruses 2021, 13, 165 .
AMA StyleSigrun Häge, Eric Sonntag, Adriana Svrlanska, Eva Maria Borst, Anne-Charlotte Stilp, Deborah Horsch, Regina Müller, Barbara Kropff, Jens Milbradt, Thomas Stamminger, Ursula Schlötzer-Schrehardt, Manfred Marschall. Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation. Viruses. 2021; 13 (2):165.
Chicago/Turabian StyleSigrun Häge; Eric Sonntag; Adriana Svrlanska; Eva Maria Borst; Anne-Charlotte Stilp; Deborah Horsch; Regina Müller; Barbara Kropff; Jens Milbradt; Thomas Stamminger; Ursula Schlötzer-Schrehardt; Manfred Marschall. 2021. "Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation." Viruses 13, no. 2: 165.
Human cytomegalovirus (HCMV) is a human pathogenic herpesvirus associated with a variety of clinical symptoms. Current antiviral therapy is not always effective, so that improved drug classes and drug-targeting strategies are needed. Particularly host-directed antivirals, including pharmaceutical kinase inhibitors (PKIs), may help to overcome problems of drug resistance. Here, we focused on utilizing a selection of clinically relevant PKIs and determined their anticytomegaloviral efficacies. Particularly, PKIs directed to host or viral cyclin-dependent kinases, i.e., abemaciclib, LDC4297 and maribavir, exerted promising profiles against human and murine cytomegaloviruses. The anti-HCMV in vitro activity of the approved anti-cancer drug abemaciclib was confirmed in vivo using our luciferase-based murine cytomegalovirus (MCMV) animal model in immunocompetent mice. To assess drug combinations, we applied the Bliss independence checkerboard and Loewe additivity fixed-dose assays in parallel. Results revealed that (i) both affirmative approaches provided valuable information on anti-CMV drug efficacies and interactions, (ii) the analyzed combinations comprised additive, synergistic or antagonistic drug interactions consistent with the drugs’ antiviral mode-of-action, (iii) the selected PKIs, especially LDC4297, showed promising inhibitory profiles, not only against HCMV but also other α-, β- and γ-herpesviruses, and specifically, (iv) the combination treatment with LDC4297 and maribavir revealed a strong synergism against HCMV, which might open doors towards novel clinical options in the near future. Taken together, this study highlights the potential of therapeutic drug combinations of current developmental/preclinical PKIs.
Markus Wild; Jintawee Kicuntod; Lisa Seyler; Christina Wangen; Luca D. Bertzbach; Andelé M. Conradie; Benedikt B. Kaufer; Sabrina Wagner; Detlef Michel; Jan Eickhoff; Svetlana B. Tsogoeva; Tobias Bäuerle; Friedrich Hahn; Manfred Marschall. Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs). International Journal of Molecular Sciences 2021, 22, 575 .
AMA StyleMarkus Wild, Jintawee Kicuntod, Lisa Seyler, Christina Wangen, Luca D. Bertzbach, Andelé M. Conradie, Benedikt B. Kaufer, Sabrina Wagner, Detlef Michel, Jan Eickhoff, Svetlana B. Tsogoeva, Tobias Bäuerle, Friedrich Hahn, Manfred Marschall. Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs). International Journal of Molecular Sciences. 2021; 22 (2):575.
Chicago/Turabian StyleMarkus Wild; Jintawee Kicuntod; Lisa Seyler; Christina Wangen; Luca D. Bertzbach; Andelé M. Conradie; Benedikt B. Kaufer; Sabrina Wagner; Detlef Michel; Jan Eickhoff; Svetlana B. Tsogoeva; Tobias Bäuerle; Friedrich Hahn; Manfred Marschall. 2021. "Combinatorial Drug Treatments Reveal Promising Anticytomegaloviral Profiles for Clinically Relevant Pharmaceutical Kinase Inhibitors (PKIs)." International Journal of Molecular Sciences 22, no. 2: 575.
Naturally occurring polysaccharide sulfates are highly diverse, owning variations in the backbone structure, linkage pattern and stereochemistry, branching diversity, sulfate content and positions of sulfate group(s). These structural characteristics bring about diverse sulfated polymers with dissimilar negative charge densities and structure–activity relationships. Herein, we start with a short discussion of techniques needed for extraction, purification, chemical sulfation, and structural characterization of polysaccharides. Processes of isolation and sulfation of plant-derived polysaccharides are challenging and usually involve two steps. In this context, we describe an integrated extraction-sulfation procedure that produces polysaccharide sulfates from natural products in one step, thereby generating additional pharmacological activities. Finally, we provide examples of the spectrum of natural source-derived polysaccharides possessing specific features of bioactivity, in particular focusing on current aspects of antiviral drug development and drug–target interaction. Thus, the review presents a detailed view on chemically engineered polysaccharides, especially sulfated derivatives, and underlines their promising biomedical perspectives.
Bimalendu Ray; Martin Schütz; Shuvam Mukherjee; Subrata Jana; Sayani Ray; Manfred Marschall. Exploiting the Amazing Diversity of Natural Source-Derived Polysaccharides: Modern Procedures of Isolation, Engineering, and Optimization of Antiviral Activities. Polymers 2020, 13, 136 .
AMA StyleBimalendu Ray, Martin Schütz, Shuvam Mukherjee, Subrata Jana, Sayani Ray, Manfred Marschall. Exploiting the Amazing Diversity of Natural Source-Derived Polysaccharides: Modern Procedures of Isolation, Engineering, and Optimization of Antiviral Activities. Polymers. 2020; 13 (1):136.
Chicago/Turabian StyleBimalendu Ray; Martin Schütz; Shuvam Mukherjee; Subrata Jana; Sayani Ray; Manfred Marschall. 2020. "Exploiting the Amazing Diversity of Natural Source-Derived Polysaccharides: Modern Procedures of Isolation, Engineering, and Optimization of Antiviral Activities." Polymers 13, no. 1: 136.
The ongoing pandemic spread of the severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) demands skillful strategies for novel drug development, drug repurposing and cotreatments, in particular focusing on existing candidates of host-directed antivirals (HDAs). The developmental drug IMU-838, currently being investigated in a phase 2b trial in patients suffering from autoimmune diseases, represents an inhibitor of human dihydroorotate dehydrogenase (DHODH) with a recently proven antiviral activity in vitro and in vivo. Here, we established an analysis system for assessing the antiviral potency of IMU-838 and DHODH-directed back-up drugs in cultured cell-based infection models. By the use of SARS-CoV-2-specific immunofluorescence, Western blot, in-cell ELISA, viral yield reduction and RT-qPCR methods, we demonstrated the following: (i) IMU-838 and back-ups show anti-SARS-CoV-2 activity at several levels of viral replication, i.e., protein production, double-strand RNA synthesis, and release of infectious virus; (ii) antiviral efficacy in Vero cells was demonstrated in a micromolar range (IMU-838 half-maximal effective concentration, EC50, of 7.6 ± 5.8 µM); (iii) anti-SARS-CoV-2 activity was distinct from cytotoxic effects (half-cytotoxic concentration, CC50, >100 µM); (iv) the drug in vitro potency was confirmed using several Vero lineages and human cells; (v) combination with remdesivir showed enhanced anti-SARS-CoV-2 activity; (vi) vidofludimus, the active determinant of IMU-838, exerted a broad-spectrum activity against a selection of major human pathogenic viruses. These findings strongly suggest that developmental DHODH inhibitors represent promising candidates for use as anti-SARS-CoV-2 therapeutics.
Friedrich Hahn; Christina Wangen; Sigrun Häge; Antonia Sophia Peter; Gerhard Dobler; Brett Hurst; Justin Julander; Jonas Fuchs; Zsolt Ruzsics; Klaus Überla; Hans-Martin Jäck; Roger Ptak; Andreas Muehler; Manfred Gröppel; Daniel Vitt; Evelyn Peelen; Hella Kohlhof; Manfred Marschall. IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro. Viruses 2020, 12, 1394 .
AMA StyleFriedrich Hahn, Christina Wangen, Sigrun Häge, Antonia Sophia Peter, Gerhard Dobler, Brett Hurst, Justin Julander, Jonas Fuchs, Zsolt Ruzsics, Klaus Überla, Hans-Martin Jäck, Roger Ptak, Andreas Muehler, Manfred Gröppel, Daniel Vitt, Evelyn Peelen, Hella Kohlhof, Manfred Marschall. IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro. Viruses. 2020; 12 (12):1394.
Chicago/Turabian StyleFriedrich Hahn; Christina Wangen; Sigrun Häge; Antonia Sophia Peter; Gerhard Dobler; Brett Hurst; Justin Julander; Jonas Fuchs; Zsolt Ruzsics; Klaus Überla; Hans-Martin Jäck; Roger Ptak; Andreas Muehler; Manfred Gröppel; Daniel Vitt; Evelyn Peelen; Hella Kohlhof; Manfred Marschall. 2020. "IMU-838, a Developmental DHODH Inhibitor in Phase II for Autoimmune Disease, Shows Anti-SARS-CoV-2 and Broad-Spectrum Antiviral Efficacy In Vitro." Viruses 12, no. 12: 1394.
Human cytomegalovirus (HCMV) is a major human pathogen associated with severe pathology. Current options of antiviral therapy only partly satisfy the needs of a well-tolerated long-term treatment/prophylaxis free from drug-induced viral resistance. Recently, we reported the strong antiviral properties in vitro and in vivo of the broad-spectrum anti-infective drug artesunate and its optimized derivatives. NF-κB signaling was described as a targeting mechanism and additional target proteins have recently been identified. Here, we analyzed the autofluorescent hybrid compound BG95, which could be utilized for intracellular visualization by confocal imaging and a tracking analysis in virus-infected primary human fibroblasts. As an important finding, BG95 accumulated in mitochondria visualized by anti-prohibitin and MitoTracker staining, and induced statistically significant changes of mitochondrial morphology, distinct from those induced by HCMV infection. Notably, mitochondrial membrane potential was found substantially reduced by BG95, an effect apparently counteracting efficient HCMV replication, which requires active mitochondria and upregulated energy levels. This finding was consistent with binding properties of artesunate-like compounds to mitochondrial proteins and thereby suggested a new mechanistic aspect. Combined, the present study underlines an important role of mitochondria in the multifaceted, host-directed antiviral mechanism of this drug class, postulating a new mitochondria-specific mode of protein targeting.
Markus Wild; Friedrich Hahn; Benedikt Grau; Lars Herrmann; Aischa Niesar; Martin Schütz; Melanie M. Lorion; Lutz Ackermann; Svetlana B. Tsogoeva; Manfred Marschall. The Artemisinin-Derived Autofluorescent Compound BG95 Exerts Strong Anticytomegaloviral Activity Based on a Mitochondrial Targeting Mechanism. International Journal of Molecular Sciences 2020, 21, 5578 .
AMA StyleMarkus Wild, Friedrich Hahn, Benedikt Grau, Lars Herrmann, Aischa Niesar, Martin Schütz, Melanie M. Lorion, Lutz Ackermann, Svetlana B. Tsogoeva, Manfred Marschall. The Artemisinin-Derived Autofluorescent Compound BG95 Exerts Strong Anticytomegaloviral Activity Based on a Mitochondrial Targeting Mechanism. International Journal of Molecular Sciences. 2020; 21 (15):5578.
Chicago/Turabian StyleMarkus Wild; Friedrich Hahn; Benedikt Grau; Lars Herrmann; Aischa Niesar; Martin Schütz; Melanie M. Lorion; Lutz Ackermann; Svetlana B. Tsogoeva; Manfred Marschall. 2020. "The Artemisinin-Derived Autofluorescent Compound BG95 Exerts Strong Anticytomegaloviral Activity Based on a Mitochondrial Targeting Mechanism." International Journal of Molecular Sciences 21, no. 15: 5578.
Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, β- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed.
Manfred Marschall; Sigrun Häge; Marcus Conrad; Sewar Alkhashrom; Jintawee Kicuntod; Johannes Schweininger; Mark Kriegel; Josephine Lösing; Julia Tillmanns; Frank Neipel; Jutta Eichler; Yves A. Muller; Heinrich Sticht. Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties. Viruses 2020, 12, 683 .
AMA StyleManfred Marschall, Sigrun Häge, Marcus Conrad, Sewar Alkhashrom, Jintawee Kicuntod, Johannes Schweininger, Mark Kriegel, Josephine Lösing, Julia Tillmanns, Frank Neipel, Jutta Eichler, Yves A. Muller, Heinrich Sticht. Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties. Viruses. 2020; 12 (6):683.
Chicago/Turabian StyleManfred Marschall; Sigrun Häge; Marcus Conrad; Sewar Alkhashrom; Jintawee Kicuntod; Johannes Schweininger; Mark Kriegel; Josephine Lösing; Julia Tillmanns; Frank Neipel; Jutta Eichler; Yves A. Muller; Heinrich Sticht. 2020. "Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties." Viruses 12, no. 6: 683.
Human cytomegalovirus (HCMV) infection is associated with serious pathology such as transplant rejection or embryonic developmental defects. Antiviral treatment with currently available drugs targeting viral enzymes is often accompanied with severe side effects and the occurrence of drug-resistant viruses. For this reason, novel ways of anti-HCMV therapy focusing on so far unexploited small molecules, targets and mechanisms are intensively studied. Recently, we described the pronounced antiviral activity of the artesunate-related class of trioxane compounds, comprising NF-κB/signaling inhibitors like the trimeric derivative TF27, which proved to be highly active in a nanomolar range both in vitro and in vivo. Here, we extend this analysis by presenting further TF27/artesunate-derived antiviral compounds designed for their specific use in target verification by click chemistry applied in fluorescence labeling and tag affinity strategies. Our main findings are as follows: (i) compounds TF27, BG95, AC98 and AC173 exert strong inhibitory activity against HCMV replication in cultured primary human cells, (ii) autofluorescence activity could be quantitatively detected for BG95 and AC98, and confocal fluorescence imaging revealed accumulation in mitochondria, (iii) postulated cellular targets including mitochondrial proteins were down-regulated upon TF27 treatment, (iv) a click chemistry-based protocol of target enrichment was established, and (v) mass spectrometry-based proteomic analysis, using proteins from HCMV-infected fibroblasts covalently interacting with AC173, revealed a refined list of targets. Combined, data strongly suggest a complex mode of antiviral drug-target interaction of artesunate-related compounds, now highlighting potential roles of mitochondrial, NF-κB pathway proteins, exportins and possibly more. This strategy may further promote antiviral drug development on the basis of pharmacologically optimized trioxane derivatives.
Friedrich Hahn; Aischa Niesar; Christina Wangen; Markus Wild; Benedikt Grau; Lars Herrmann; Aysun Capci; Annie Adrait; Yohann Couté; Svetlana B. Tsogoeva; Manfred Marschall. Target verification of artesunate-related antiviral drugs: Assessing the role of mitochondrial and regulatory proteins by click chemistry and fluorescence labeling. Antiviral Research 2020, 180, 104861 .
AMA StyleFriedrich Hahn, Aischa Niesar, Christina Wangen, Markus Wild, Benedikt Grau, Lars Herrmann, Aysun Capci, Annie Adrait, Yohann Couté, Svetlana B. Tsogoeva, Manfred Marschall. Target verification of artesunate-related antiviral drugs: Assessing the role of mitochondrial and regulatory proteins by click chemistry and fluorescence labeling. Antiviral Research. 2020; 180 ():104861.
Chicago/Turabian StyleFriedrich Hahn; Aischa Niesar; Christina Wangen; Markus Wild; Benedikt Grau; Lars Herrmann; Aysun Capci; Annie Adrait; Yohann Couté; Svetlana B. Tsogoeva; Manfred Marschall. 2020. "Target verification of artesunate-related antiviral drugs: Assessing the role of mitochondrial and regulatory proteins by click chemistry and fluorescence labeling." Antiviral Research 180, no. : 104861.
Nuclear egress is a rate-limiting step of herpesviral replication, restricting the nucleocytoplasmic transport of viral capsids. The process is regulated by two viral nuclear egress proteins (core NEC pUL50-pUL53), which recruit additional cellular and viral proteins. The multicomponent NEC mediates disassembly of the nuclear lamina barrier and the docking of nuclear capsids. The quantitation of nuclear egress has been accomplished by electron microscopic analysis, but is generally hampered by the low number of detectable cytoplasmic capsids. A newly established method for the quantitation of viral nuclear egress improves the characterization of viral mutants, host cell permissiveness and antiviral drug efficacy. In this study, various strains of human cytomegalovirus (HCMV) were used to measure the replication efficiencies in primary human fibroblasts, applying methods of cell fractionation, DNase digestion, sucrose cushions and quantitative PCR. Several stages of optimization led to a reliable quantitative assay that allowed the characterization of viral nuclear egress efficacy. Using this assay, recovery of the nuclear egress of a NEC-defective HCMV mutant was quantitatively assessed by applying an inducible NEC-expressing fibroblast culture for trans-complementation. This novel assay system can be further used to accurately quantitate and characterize the functionality of nuclear egress of HCMV or other herpesviruses.
Sigrun Häge; Deborah Horsch; Anne-Charlotte Stilp; Jintawee Kicuntod; Regina Müller; Stuart T. Hamilton; Ece Egilmezer; William D. Rawlinson; Thomas Stamminger; Eric Sonntag; Manfred Marschall. A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus. Journal of Virological Methods 2020, 283, 113909 .
AMA StyleSigrun Häge, Deborah Horsch, Anne-Charlotte Stilp, Jintawee Kicuntod, Regina Müller, Stuart T. Hamilton, Ece Egilmezer, William D. Rawlinson, Thomas Stamminger, Eric Sonntag, Manfred Marschall. A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus. Journal of Virological Methods. 2020; 283 ():113909.
Chicago/Turabian StyleSigrun Häge; Deborah Horsch; Anne-Charlotte Stilp; Jintawee Kicuntod; Regina Müller; Stuart T. Hamilton; Ece Egilmezer; William D. Rawlinson; Thomas Stamminger; Eric Sonntag; Manfred Marschall. 2020. "A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus." Journal of Virological Methods 283, no. : 113909.
The assembly of human cytomegalovirus (HCMV) virions is an orchestrated process that requires, as an essential prerequisite, the complex crosstalk between viral structural proteins. Currently, however, the mechanisms governing the successive steps in the constitution of virion protein complexes remain elusive. Protein phosphorylation is a key regulator determining the sequential changes in the conformation, binding, dynamics, and stability of proteins in the course of multiprotein assembly. In this review, we present a comprehensive map of the HCMV virion proteome, including a refined view on the virion phosphoproteome, based on previous publications supplemented by new results. Thus, a novel dataset of viral and cellular proteins contained in HCMV virions is generated, providing a basis for future analyses of individual phosphorylation steps and sites involved in the orchestrated assembly of HCMV virion-specific multiprotein complexes. Finally, we present the current knowledge on the activity of pUL97, the HCMV-encoded and virion-associated kinase, in phosphorylating viral and host proteins.
Yohann Couté; Alexandra Kraut; Christine Zimmermann; Nicole Büscher; Anne-Marie Hesse; Christophe Bruley; Marco De Andrea; Christina Wangen; Friedrich Hahn; Manfred Marschall; Bodo Plachter. Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus. Microorganisms 2020, 8, 1 .
AMA StyleYohann Couté, Alexandra Kraut, Christine Zimmermann, Nicole Büscher, Anne-Marie Hesse, Christophe Bruley, Marco De Andrea, Christina Wangen, Friedrich Hahn, Manfred Marschall, Bodo Plachter. Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus. Microorganisms. 2020; 8 (6):1.
Chicago/Turabian StyleYohann Couté; Alexandra Kraut; Christine Zimmermann; Nicole Büscher; Anne-Marie Hesse; Christophe Bruley; Marco De Andrea; Christina Wangen; Friedrich Hahn; Manfred Marschall; Bodo Plachter. 2020. "Mass Spectrometry-Based Characterization of the Virion Proteome, Phosphoproteome, and Associated Kinase Activity of Human Cytomegalovirus." Microorganisms 8, no. 6: 1.
Human cytomegalovirus (HCMV) is a ubiquitous human pathogen of high clinical relevance. Despite intensive research of virus-host interaction, crucial details still remain unknown. In this study, the role of the cellular peptidyl-prolyl cis/trans isomerase Pin1 during HCMV infection was investigated. Pin1 is able to recognize phosphorylated serine/threonine-proline motifs and regulates the structural conformation, stability and function of its substrates. Concerning HCMV replication, our recent studies revealed that Pin1 plays an important role in viral nuclear egress by contributing to the depletion of the nuclear lamina at distinct sites through the cis-trans conversion of lamin proteins. Here, novel data illustrate the HCMV-induced upregulation of Pin1 including various cell types being permissive, semi-permissive or non-permissive for productive HCMV replication. Addressing the question of functional impact, Pin1 knock-out (KO) did not show a measurable effect on viral protein expression, at least when assessed by Western blot analysis. Applying highly sensitive methods of qPCR and plaque titration, a pharmacological inhibition of Pin1 activity, however, led to a significant decrease of viral genome equivalents and production of infectious virus, respectively. When focusing on the identification of viral proteins interacting with Pin1 by various coimmunoprecipitation (CoIP) settings, we obtained positive signals for (i) the core nuclear egress complex protein pUL50, (ii) the viral mRNA export factor pUL69 and (iii) the viral DNA polymerase processivity factor pUL44. Confocal immunofluorescence analysis focusing on partial colocalization between Pin1 and the coexpressed viral proteins pUL50, pUL69 or pUL44, respectively, was consistent with the CoIP experiments. Mapping experiments, using transient expression constructs for a series of truncated protein versions and specific replacement mutants, revealed a complex pattern of Pin1 interaction with these three early regulatory HCMV proteins. Data suggest a combination of different modes of Pin1 interactions, involving both classical phosphorylation-dependent Pin1 binding motifs and additional phosphorylation-independent binding sites. Combined, these results support the concept that Pin1 may play an important role in several stages of HCMV infection, thus determining viral replicative efficiency.
Martin Schütz; Marco Thomas; Christina Wangen; Sabrina Wagner; Luisa Rauschert; Theresa Errerd; Melissa Kießling; Heinrich Sticht; Jens Milbradt; Manfred Marschall. The peptidyl-prolyl cis/trans isomerase Pin1 interacts with three early regulatory proteins of human cytomegalovirus. Virus Research 2020, 285, 198023 .
AMA StyleMartin Schütz, Marco Thomas, Christina Wangen, Sabrina Wagner, Luisa Rauschert, Theresa Errerd, Melissa Kießling, Heinrich Sticht, Jens Milbradt, Manfred Marschall. The peptidyl-prolyl cis/trans isomerase Pin1 interacts with three early regulatory proteins of human cytomegalovirus. Virus Research. 2020; 285 ():198023.
Chicago/Turabian StyleMartin Schütz; Marco Thomas; Christina Wangen; Sabrina Wagner; Luisa Rauschert; Theresa Errerd; Melissa Kießling; Heinrich Sticht; Jens Milbradt; Manfred Marschall. 2020. "The peptidyl-prolyl cis/trans isomerase Pin1 interacts with three early regulatory proteins of human cytomegalovirus." Virus Research 285, no. : 198023.
Human cytomegalovirus (HCMV) expresses a variety of viral regulatory proteins that undergo close interaction with host factors including viral-cellular multiprotein complexes. The HCMV protein kinase pUL97 represents a viral cyclin-dependent kinase ortholog (vCDK) that determines the efficiency of HCMV replication via phosphorylation of viral and cellular substrates. A hierarchy of functional importance of individual pUL97-mediated phosphorylation events has been discussed; however, the most pronounced pUL97-dependent phenotype could be assigned to viral nuclear egress, as illustrated by deletion of the UL97 gene or pharmacological pUL97 inhibition. Despite earlier data pointing to a cyclin-independent functionality, experimental evidence increasingly emphasized the role of pUL97-cyclin complexes. Consequently, the knowledge about pUL97 involvement in host interaction, viral nuclear egress and additional replicative steps led to the postulation of pUL97 as an antiviral target. Indeed, validation experiments in vitro and in vivo confirmed the sustainability of this approach. Consequently, current investigations of pUL97 in antiviral treatment go beyond the known pUL97-mediated ganciclovir prodrug activation and henceforward include pUL97-specific kinase inhibitors. Among a number of interesting small molecules analyzed in experimental and preclinical stages, maribavir is presently investigated in clinical studies and, in the near future, might represent a first kinase inhibitor applied in the field of antiviral therapy.
Mirjam Steingruber; Manfred Marschall. The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting. Microorganisms 2020, 8, 515 .
AMA StyleMirjam Steingruber, Manfred Marschall. The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting. Microorganisms. 2020; 8 (4):515.
Chicago/Turabian StyleMirjam Steingruber; Manfred Marschall. 2020. "The Cytomegalovirus Protein Kinase pUL97: Host Interactions, Regulatory Mechanisms and Antiviral Drug Targeting." Microorganisms 8, no. 4: 515.
Human cytomegalovirus (HCMV) causes serious and even life-threatening diseases, particularly upon congenital or post-transplant infection. Treatment of HCMV infections with currently available drugs targeting viral enzymes is often limited by severe side effects and the emergence of drug-resistant viruses. To avoid this problem, novel therapeutic options directed to host proteins involved in virus replication are being investigated. Recently, we described the pronounced antiherpesviral activity of the trimeric artesunate derivative TF27 at low nanomolar concentrations in vitro and in vivo. In the present study, we report first data on the prophylactic efficacy of TF27 against human and murine CMV and the oncogenic avian alphaherpesvirus Marek's disease virus (MDV). The main findings of this study are (i) a pronounced activity of the experimental drug TF27 against alpha- and betaherpesviruses in vitro upon prophylactic treatment and (ii) a therapeutic and prophylactic efficacy upon oral treatment in an immunocompetent mouse model. Moreover, our data highlight (iii) the tolerability of orally administered TF27 free of compound-associated adverse events and further confirm (iv) the suitability of cellular factors as primary antiviral targets. Thus, we provide evidence for therapeutic and prophylactic antiherpesviral efficacy of TF27 upon oral treatment in immunocompetent hosts and thereby underline its potential for future antiviral drug development.
Markus Wild; Luca D. Bertzbach; Pierre Tannig; Christina Wangen; Regina Müller; Lars Herrmann; Tony Fröhlich; Svetlana B. Tsogoeva; Benedikt B. Kaufer; Manfred Marschall; Friedrich Hahn. The trimeric artesunate derivative TF27 exerts strong anti-cytomegaloviral efficacy: Focus on prophylactic efficacy and oral treatment of immunocompetent mice. Antiviral Research 2020, 178, 104788 .
AMA StyleMarkus Wild, Luca D. Bertzbach, Pierre Tannig, Christina Wangen, Regina Müller, Lars Herrmann, Tony Fröhlich, Svetlana B. Tsogoeva, Benedikt B. Kaufer, Manfred Marschall, Friedrich Hahn. The trimeric artesunate derivative TF27 exerts strong anti-cytomegaloviral efficacy: Focus on prophylactic efficacy and oral treatment of immunocompetent mice. Antiviral Research. 2020; 178 ():104788.
Chicago/Turabian StyleMarkus Wild; Luca D. Bertzbach; Pierre Tannig; Christina Wangen; Regina Müller; Lars Herrmann; Tony Fröhlich; Svetlana B. Tsogoeva; Benedikt B. Kaufer; Manfred Marschall; Friedrich Hahn. 2020. "The trimeric artesunate derivative TF27 exerts strong anti-cytomegaloviral efficacy: Focus on prophylactic efficacy and oral treatment of immunocompetent mice." Antiviral Research 178, no. : 104788.
Nuclear egress is a regulated process shared by α-, β- and γ-herpesviruses. The core nuclear egress complex (NEC) is composed of the membrane-anchored protein homologs of human cytomegalovirus (HCMV) pUL50, murine cytomegalovirus (MCMV) pM50, Epstein–Barr virus (EBV) BFRF1 or varicella zoster virus (VZV) Orf24, which interact with the autologous NEC partners pUL53, pM53, BFLF2 or Orf27, respectively. Their recruitment of additional proteins leads to the assembly of a multicomponent NEC, coordinately regulating viral nucleocytoplasmic capsid egress. Here, the functionality of VZV, HCMV, MCMV and EBV core NECs was investigated by coimmunoprecipitation and confocal imaging analyses. Furthermore, a recombinant MCMV, harboring a replacement of ORF M50 by UL50, was analyzed both in vitro and in vivo. In essence, core NEC interactions were strictly limited to autologous NEC pairs and only included one measurable nonautologous interaction between the homologs of HCMV and MCMV. A comparative analysis of MCMV-WT versus MCMV-UL50-infected murine fibroblasts revealed almost identical phenotypes on the levels of protein and genomic replication kinetics. In infected BALB/c mice, virus spread to lung and other organs was found comparable between these viruses, thus stating functional complementarity. In conclusion, our study underlines that herpesviral core NEC proteins are functionally conserved regarding complementarity of core NEC interactions, which were found either virus-specific or restricted within subfamilies.
Sigrun Häge; Eric Sonntag; Eva Maria Borst; Pierre Tannig; Lisa Seyler; Tobias Bäuerle; Susanne M. Bailer; Chung-Pei Lee; Regina Müller; Christina Wangen; Jens Milbradt; Manfred Marschall. Patterns of Autologous and Nonautologous Interactions between Core Nuclear Egress Complex (NEC) Proteins of α-, β- and γ-Herpesviruses. Viruses 2020, 12, 303 .
AMA StyleSigrun Häge, Eric Sonntag, Eva Maria Borst, Pierre Tannig, Lisa Seyler, Tobias Bäuerle, Susanne M. Bailer, Chung-Pei Lee, Regina Müller, Christina Wangen, Jens Milbradt, Manfred Marschall. Patterns of Autologous and Nonautologous Interactions between Core Nuclear Egress Complex (NEC) Proteins of α-, β- and γ-Herpesviruses. Viruses. 2020; 12 (3):303.
Chicago/Turabian StyleSigrun Häge; Eric Sonntag; Eva Maria Borst; Pierre Tannig; Lisa Seyler; Tobias Bäuerle; Susanne M. Bailer; Chung-Pei Lee; Regina Müller; Christina Wangen; Jens Milbradt; Manfred Marschall. 2020. "Patterns of Autologous and Nonautologous Interactions between Core Nuclear Egress Complex (NEC) Proteins of α-, β- and γ-Herpesviruses." Viruses 12, no. 3: 303.
Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric basic structure of the nuclear egress complex (core NEC). These core NECs serve as a hexameric lattice-structured platform for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina- and membrane-rearranging functions (multicomponent NEC). Here, we report the X-ray structures of β- and γ-herpesvirus core NECs obtained through an innovative recombinant expression strategy based on NEC-hook::NEC-groove protein fusion constructs. This approach yielded the first structure of γ-herpesviral core NEC, namely the 1.56 Å structure of Epstein-Barr virus (EBV) BFRF1–BFLF2, as well as an increased resolution 1.48 Å structure of human cytomegalovirus (HCMV) pUL50-pUL53. Detailed analysis of these structures revealed that the prominent hook segment is absolutely required for core NEC formation and contributes approximately 80% of the interaction surface of the globular domains of NEC proteins. Moreover, using HCMV::EBV hook domain swap constructs, computational prediction of the roles of individual hook residues for binding, and quantitative binding assays with synthetic peptides presenting the HCMV- and EBV-specific NEC hook sequences, we characterized the unique hook-into-groove NEC interaction at various levels. Although the overall physicochemical characteristics of the protein interfaces differ considerably in these β- and γ-herpesvirus NECs, the binding free energy contributions of residues displayed from identical positions are similar. In summary, the results of our study reveal critical details of the molecular mechanism of herpesviral NEC interactions and highlight their potential as an antiviral drug target.
Yves A. Muller; Sigrun Häge; Sewar Alkhashrom; Tobias Höllriegl; Sebastian Weigert; Simon Dolles; Kerstin Hof; Sascha A. Walzer; Claudia Egerer-Sieber; Marcus Conrad; Stephanie Holst; Josephine Lösing; Eric Sonntag; Heinrich Sticht; Jutta Eichler; Manfred Marschall. High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity. Journal of Biological Chemistry 2020, 295, 3189 -3201.
AMA StyleYves A. Muller, Sigrun Häge, Sewar Alkhashrom, Tobias Höllriegl, Sebastian Weigert, Simon Dolles, Kerstin Hof, Sascha A. Walzer, Claudia Egerer-Sieber, Marcus Conrad, Stephanie Holst, Josephine Lösing, Eric Sonntag, Heinrich Sticht, Jutta Eichler, Manfred Marschall. High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity. Journal of Biological Chemistry. 2020; 295 (10):3189-3201.
Chicago/Turabian StyleYves A. Muller; Sigrun Häge; Sewar Alkhashrom; Tobias Höllriegl; Sebastian Weigert; Simon Dolles; Kerstin Hof; Sascha A. Walzer; Claudia Egerer-Sieber; Marcus Conrad; Stephanie Holst; Josephine Lösing; Eric Sonntag; Heinrich Sticht; Jutta Eichler; Manfred Marschall. 2020. "High-resolution crystal structures of two prototypical β- and γ-herpesviral nuclear egress complexes unravel the determinants of subfamily specificity." Journal of Biological Chemistry 295, no. 10: 3189-3201.
Nucleoside analogues have been the cornerstone of clinical treatment of herpesvirus infections since the 1970s. However, severe side effects and emergence of drug resistant viruses raise the need for alternative treatment options. We recently investigated the broad and strong antiherpesviral activity of the optimized artesunate derivative TF27 in vitro. TF27 efficiently inhibited replication of the highly oncogenic Marek's disease virus (MDV), a virus that infects chickens, causes deadly lymphomas and threatens poultry populations worldwide. In this study, we used this natural virus-host model for herpesvirus-induced cancer by infecting chickens with MDV, and evaluated the protective efficacy of TF27 and the nucleoside analogue valganciclovir (VGCV) on virus replication and tumorigenesis. We could demonstrate that both drugs reduced viral load in the blood and prevented tumor development in a large portion of the animals. Antiviral treatment also had a positive impact on body weight gain, while no negative compound-associated side effects were observed. This research provides the first evidence that the artesunate derivative TF27 and VGCV can be used in avian species and that they inhibit MDV replication and tumorigenesis. In addition, our study paves the way for promising approaches in future antiherpesviral drug development.
Luca D. Bertzbach; Andelé M. Conradie; Friedrich Hahn; Markus Wild; Manfred Marschall; Benedikt B. Kaufer. Artesunate derivative TF27 inhibits replication and pathogenesis of an oncogenic avian alphaherpesvirus. Antiviral Research 2019, 171, 104606 .
AMA StyleLuca D. Bertzbach, Andelé M. Conradie, Friedrich Hahn, Markus Wild, Manfred Marschall, Benedikt B. Kaufer. Artesunate derivative TF27 inhibits replication and pathogenesis of an oncogenic avian alphaherpesvirus. Antiviral Research. 2019; 171 ():104606.
Chicago/Turabian StyleLuca D. Bertzbach; Andelé M. Conradie; Friedrich Hahn; Markus Wild; Manfred Marschall; Benedikt B. Kaufer. 2019. "Artesunate derivative TF27 inhibits replication and pathogenesis of an oncogenic avian alphaherpesvirus." Antiviral Research 171, no. : 104606.