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Friedrich-Wilhelm-Lübke-Koog, Germany

Mettenleiter T.C.,Institute of Molecular Virology and Cell Biology
Journal of Molecular Biology | Year: 2016

Many DNA and a few RNA viruses use the host cell nucleus for virion formation and/or genome replication. To this end, the nuclear envelope (NE) barrier has to be overcome for entry into and egress from the intranuclear replication compartment. Different virus families have devised ingenious ways of entering and leaving the nucleus usurping cellular transport pathways through the nuclear pore complex but also translocating directly through both membranes of the NE. This intriguing diversity in nuclear entry and egress of viruses also highlights different ways nucleocytoplasmic transport can occur. Thus, the study of interactions between viruses and the NE also helps to unravel hitherto unknown cellular pathways such as vesicular nucleocytoplasmic transfer. © 2015 Elsevier Ltd.


Zuhlsdorf M.,University of Greifswald | Werten S.,University of Greifswald | Klupp B.G.,Institute of Molecular Virology and Cell Biology | Palm G.J.,University of Greifswald | And 2 more authors.
PLoS Pathogens | Year: 2015

Herpesviruses encode a characteristic serine protease with a unique fold and an active site that comprises the unusual triad Ser-His-His. The protease is essential for viral replication and as such constitutes a promising drug target. In solution, a dynamic equilibrium exists between an inactive monomeric and an active dimeric form of the enzyme, which is believed to play a key regulatory role in the orchestration of proteolysis and capsid assembly. Currently available crystal structures of herpesvirus proteases correspond either to the dimeric state or to complexes with peptide mimetics that alter the dimerization interface. In contrast, the structure of the native monomeric state has remained elusive. Here, we present the three-dimensional structures of native monomeric, active dimeric, and diisopropyl fluorophosphate-inhibited dimeric protease derived from pseudorabies virus, an alphaherpesvirus of swine. These structures, solved by X-ray crystallography to respective resolutions of 2.05, 2.10 and 2.03 Å, allow a direct comparison of the main conformational states of the protease. In the dimeric form, a functional oxyanion hole is formed by a loop of 10 amino-acid residues encompassing two consecutive arginine residues (Arg136 and Arg137); both are strictly conserved throughout the herpesviruses. In the monomeric form, the top of the loop is shifted by approximately 11 Å, resulting in a complete disruption of the oxyanion hole and loss of activity. The dimerization-induced allosteric changes described here form the physical basis for the concentration-dependent activation of the protease, which is essential for proper virus replication. Small-angle X-ray scattering experiments confirmed a concentration-dependent equilibrium of monomeric and dimeric protease in solution. © 2015 Zühlsdorf et al.


Fuchs W.,Institute of Molecular Virology and Cell Biology | Granzow H.,Institute of Infectology | Dauber M.,Friedrich Loeffler Institute | Fichtner D.,Institute of Infectology | Mettenleiter T.C.,Institute of Molecular Virology and Cell Biology
Archives of Virology | Year: 2014

As a prerequisite for development of improved vaccines and diagnostic tools for control of the fish pathogen koi herpesvirus, or cyprinid herpesvirus 3 (CyHV-3), we have started to identify putative viral envelope and capsid proteins. The complete or partial CyHV-3 open reading frames ORF25, ORF65, ORF92, ORF99, ORF136, ORF138, ORF146, ORF148, and ORF149 were expressed as bacterial fusion proteins, which were then used for preparation of monospecific rabbit antisera. All of the sera that were obtained detected their target proteins in cells transfected with the corresponding eukaryotic expression plasmids. However, only the type I membrane proteins pORF25, pORF65, pORF99, pORF136 and pORF149 and the major capsid protein pORF92 were sufficiently abundant and immunogenic to permit unambiguous detection in CyHV-3-infected cells. In indirect immunofluorescence tests (IIFT), sera from naturally or experimentally CyHV-3-infected carp and koi predominantly reacted with cells transfected with expression plasmids encoding pORF25, pORF65, pORF148, and pORF149, which represent a family of related CyHV-3 membrane proteins. Moreover, several neutralizing monoclonal antibodies raised against CyHV-3 virions proved to be specific for pORF149 in IIFT of transfected cells and in immunoelectron microscopic analysis of CyHV-3 particles. Since pORF149 appears to be an immunorelevant envelope protein of CyHV-3, a recombinant baculovirus was generated for its expression in insect cells, and pORF149 was shown to be incorporated into pseudotyped baculovirus particles, which might be suitable as diagnostic tools or subunit vaccines. © 2014, Springer-Verlag Wien.


Schulz K.S.,Institute of Molecular Virology and Cell Biology | Klupp B.G.,Institute of Molecular Virology and Cell Biology | Granzow H.,Institute of Infectology | Passvogel L.,Institute of Molecular Virology and Cell Biology | Mettenleiter T.C.,Institute of Molecular Virology and Cell Biology
Virus Research | Year: 2015

Herpesvirus replication takes place in the nucleus and in the cytosol. After entering the cell, nucleocapsids are transported to nuclear pores where viral DNA is released into the nucleus. After gene expression and DNA replication new nucleocapsids are assembled which have to exit the nucleus for virion formation in the cytosol. Since nuclear pores are not wide enough to allow passage of the nucleocapsid, nuclear egress occurs by vesicle-mediated transport through the nuclear envelope. To this end, nucleocapsids bud at the inner nuclear membrane (INM) recruiting a primary envelope which then fuses with the outer nuclear membrane (ONM). In the absence of this regulated nuclear egress, mutants of the alphaherpesvirus pseudorabies virus have been described that escape from the nucleus after virus-induced nuclear envelope breakdown. Here we review these exit pathways and demonstrate that both can occur simultaneously under appropriate conditions. © 2015 Elsevier B.V.


Eckardt M.,German Federal Institute for Risk Assessment | Eckardt M.,Humboldt University of Berlin | Freuling C.,Institute of Molecular Virology and Cell Biology | Muller T.,Institute of Molecular Virology and Cell Biology | Selhorst T.,German Federal Institute for Risk Assessment
Geospatial Health | Year: 2015

Aiming to achieve new insights into rabies dynamics, this paper is the first to investigate fox rabies in Germany from a space-time pattern perspective. Based on a locally restricted dataset covering a fourteen month period, our findings indicate a strongly aggregated spatiotemporal point pattern resulting from an inhomogeneous stochastic process. In contrast to spatial or temporal approaches or cellular automata, our analysis focuses on the disease dynamics in time and space in a continuous time domain. Our findings confirm existing theories regarding fox rabies control highlighting the potential risk of urban areas and the need for effective rabies vaccination. © Copyright M. Eckardt et al.

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