Institute of Molecular Virology and Cell Biology

Greifswald, Germany

Institute of Molecular Virology and Cell Biology

Greifswald, Germany
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Bohm S.,Institute of Molecular Virology and Cell Biology | Eckroth E.,Institute of Molecular Virology and Cell Biology | Backovic M.,Institute Pasteur Paris | Klupp B.G.,Institute of Molecular Virology and Cell Biology | And 3 more authors.
Journal of Virology | Year: 2015

Enveloped viruses utilize membrane fusion for entry into, and release from, host cells. For entry, members of the Herpesviridae require at least three envelope glycoproteins: the homotrimeric gB and a heterodimer of gH and gL. The crystal structures of three gH homologues, including pseudorabies virus (PrV) gH, revealed four conserved domains. Domain II contains a planar β-sheet ("fence") and a syntaxin-like bundle of three α-helices (SLB), similar to those found in eukaryotic fusion proteins, potentially executing an important role in gH function. To test this hypothesis, we introduced targeted mutations into the PrV gH gene, which either disrupt the helices of the SLB by introduction of proline residues or covalently join them by artificial intramolecular disulfide bonds between themselves, to the adjacent fence region, or to domain III. Disruption of either of the three α-helices of the SLB (A250P, V275P, V298P) severely affected gH function in in vitro fusion assays and replication of corresponding PrV mutants. Considerable defects in fusion activity of gH, as well as in penetration kinetics and cell-to-cell spread of PrV mutants, were also observed after disulfide linkage of two α-helices within the SLB (A284C-S291C) or between SLB and domain III (H251C-L432C), as well as by insertions of additional cysteine pairs linking fence, SLB, and domain III. In vitro fusion activity of mutated gH could be partly restored by reduction of the artificial disulfide bonds. Our results indicate that the structure and flexibility of the SLB are relevant for the function of PrV gH in membrane fusion. © 2015, American Society for Microbiology.


Schroter C.,Institute of Molecular Virology and Cell Biology | Vallbracht M.,Institute of Molecular Virology and Cell Biology | Altenschmidt J.,Institute of Molecular Virology and Cell Biology | Altenschmidt J.,Dianova GmbH | And 5 more authors.
Journal of Virology | Year: 2016

Entry of herpesviruses depends on the combined action of viral glycoprotein B (gB) and the heterodimeric gH/gL complex, which are activated by binding of the virion to specific cellular receptors. While gB carries signatures of a bona fide fusion protein, efficient membrane fusion requires gH/gL. However, although gB and gH/gL are essential for entry, the alphaherpesvirus pseudorabies virus (PrV) is capable of limited cell-to-cell spread in the absence of gL. To understand gH/gL function in more detail, the limited spread of PrV-ΔgL was used for reversion analyses by serial cell culture passages. In a first experiment, an infectious gL-negative mutant in which gL function was replaced by generation of a gD-gH hybrid protein was isolated (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014 -3022, 1999). In a second, independent experiment PrV-ΔgLPassB4.1, which also replicated productively without gL, was isolated. Sequence analysis revealed mutations in gH but also in gB and gD. In a transfection- based fusion assay, two amino acid substitutions in the N-terminal part of gHB4.1 (L70P and W103R) were found to be sufficient to compensate for lack of gL, while mutations present in gBB4.1 enhanced fusogenicity. Coexpression of gBB4.1 with the homologous gHB4.1 resulted in strongly increased syncytium formation, which was further augmented by truncation of the gBB4.1 C-terminal 29 amino acids. Nevertheless, gH was still required for membrane fusion. Surprisingly, coexpression of gDB4.1 blocked syncytium formation in the fusion assays, which could be attributed to a V106A substitution within the ectodomain of gDB4.1. © 2016, American Society for Microbiology.


Passvogel L.,Institute of Molecular Virology and Cell Biology | Klupp B.G.,Institute of Molecular Virology and Cell Biology | Granzow H.,Institute of Infectology | Fuchs W.,Institute of Molecular Virology and Cell Biology | Mettenleiter T.C.,Institute of Molecular Virology and Cell Biology
Journal of Virology | Year: 2015

The herpesviral nuclear egress complex (NEC), consisting of pUL31 and pUL34 homologs, mediates efficient translocation of newly synthesized capsids from the nucleus to the cytosol. The tail-anchored membrane protein pUL34 is autonomously targeted to the nuclear envelope, while pUL31 is recruited to the inner nuclear membrane (INM) by interaction with pUL34. A nuclear localization signal (NLS) in several pUL31 homologs suggests importin-mediated translocation of the protein. Here we demonstrate that deletion or mutation of the NLS in pseudorabies virus (PrV) pUL31 resulted in exclusively cytosolic localization, indicating active nuclear export. Deletion or mutation of a predicted nuclear export signal (NES) in mutant constructs lacking a functional NLS resulted in diffuse nuclear and cytosolic localization, indicating that both signals are functional. pUL31 molecules lacking the complete NLS or NES were not recruited to the INM by pUL34, while site-specifically mutated proteins formed the NEC and partially complemented the defect of the UL31 deletion mutant. Our data demonstrate that the N terminus of pUL31, encompassing the NLS, is required for efficient nuclear targeting but not for pUL34 interaction, while the C terminus, containing the NES but not necessarily the NES itself, is required for complex formation and efficient budding of viral capsids at the INM. Moreover, pUL31-ΔNLS displayed a dominant negative effect on wild-type PrV replication, probably by diverting pUL34 to cytoplasmic membranes. © 2015, American Society for Microbiology.


Schroter C.,Institute of Molecular Virology and Cell Biology | Klupp B.G.,Institute of Molecular Virology and Cell Biology | Fuchs W.,Institute of Molecular Virology and Cell Biology | Gerhard M.,Institute of Molecular Virology and Cell Biology | And 3 more authors.
Journal of Virology | Year: 2014

Membrane fusion in herpesviruses requires viral glycoproteins (g) gB and gH/gL. While gB is considered the actual fusion protein but is nonfusogenic per se, the function of gH/gL remains enigmatic. Crystal structures for different gH homologs are strikingly similar despite only moderate amino acid sequence conservation. A highly conserved sequence motif comprises the residues serine-prolinecysteine corresponding to positions 437 to 439 in pseudorabies virus (PrV) gH. The PrV-gH structure shows that proline438 induces bending at the end of an alpha-helix, thereby placing cysteine404 and cysteine439 in juxtaposition to allow formation of a strictly conserved disulfide bond. However, PrV vaccine strain Bartha unexpectedly carries a serine at this conserved position. To test the influence of this substitution, we constructed different gH chimeras carrying proline or serine at position 438 in gH derived from either PrV strain Kaplan or strain Bartha. Mutants expressing gH with serine438 showed reduced fusion activity in transient-fusion assays and during infection, with delayed penetration kinetics and a small-plaque phenotype which indicates that proline438 is important for efficient fusion. A more drastic effect was observed when disulfide bond formation was completely blocked by mutation of cysteine404 to serine. Although PrV expressing gHC404S was viable, plaque size and penetration kinetics were drastically reduced. Alteration of serine438 to proline in gH of strain Bartha enhanced cell-to-cell spread and penetration kinetics, but restoration of full activity required additional alteration of aspartic acid to valine at position 59.


Vallbracht M.,Institute of Molecular Virology and Cell Biology | Rehwaldt S.,Institute of Molecular Virology and Cell Biology | Klupp B.G.,Institute of Molecular Virology and Cell Biology | Mettenleiter T.C.,Institute of Molecular Virology and Cell Biology | Fuchs W.,Institute of Molecular Virology and Cell Biology
Journal of Virology | Year: 2017

Several envelope glycoproteins are involved in herpesvirus entry into cells, direct cell-to-cell spread, and induction of cell fusion. The membrane fusion protein glycoprotein B (gB) and the presumably gB-activating heterodimer gH/gL are essential for these processes and conserved throughout the Herpesviridae. However, after extended cell culture passage of gL-negative mutants of the alphaherpesvirus pseudorabies virus (PrV), phenotypic revertants could be isolated which had acquired spontaneous mutations affecting the gL-interacting N-terminal part of the gH ectodomain (gDH and gHB4.1) (B. G. Klupp and T. C. Mettenleiter, J Virol 73:3014- 3022, 1999; C. Schröter, M. Vallbracht, J. Altenschmidt, S. Kargoll, W. Fuchs, B. G. Klupp, and T. C. Mettenleiter, J Virol 90:2264 -2272, 2016). To investigate the functional relevance of this part of gH in more detail, we introduced an in-frame deletion of 66 codons at the 5= end of the plasmid-cloned gH gene (gH32/98). The N-terminal signal peptide was retained, and the deletion did not affect expression or processing of gH but abrogated its function in in vitro fusion assays. Insertion of the engineered gH gene into the PrV genome resulted in a defective mutant (pPrV-gH32/98K), which was incapable of entry and spread. Interestingly, in vitro activity of mutated gH32/98 was restored when it was coexpressed with hyperfusogenic gBB4.1, obtained from a passaged gL deletion mutant of PrV. Moreover, the entry and spread defects of pPrV-gH32/98K were compensated by the mutations in gBB4.1 in cis, as well as in trans, independent of gL. Thus, PrV gL and the gL-interacting domain of gH are not strictly required for function. © 2017 American Society for Microbiology.


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.


Henning A.-K.,Institute of Molecular Virology and Cell Biology | Albrecht D.,University of Greifswald | Riedel K.,University of Greifswald | Mettenleiter T.C.,Institute of Molecular Virology and Cell Biology | Karger A.,Institute of Molecular Virology and Cell Biology
Proteomics | Year: 2015

Serum proteome analysis is severely hampered by the extreme dynamic range of protein concentrations, but tools for the specific depletion of highly abundant serum proteins lack for most farm and companion animals. A well-established alternative strategy to reduce the dynamic range of plasma protein concentrations, treatment with combinatorial peptide ligand libraries (CPLL), is generally applicable but requires large amounts of sample. Therefore, additional depletion/enrichment protocols for plasma and serum samples from animals are desirable. In this respect, we have tested a protein precipitate that formed after withdrawal of salt from human, bovine, or porcine serum at pH 4.2. The bovine sample was composed of over 300 proteins making it a potential source for biomarker discovery. Precipitation was highly reproducible and the concentrations of albumin and other highly abundant serum proteins were strongly reduced. In comparison to the CPLL treatment, precipitation did not introduce any selection bias based on hydrophathy or pI. However, the composition of both preparations was partially complementary. Salt withdrawal at pH 4.2 is suggested as additional depletion/enrichment strategy for serum samples. Also, we point out that the removal of precipitates from serum samples under the described conditions bears the risk of losing a valuable protein fraction. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


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.


Within the past few years identification of bacteria by MALDI-TOF MS has become a standard technique in bacteriological laboratories for good reasons. MALDI-TOF MS identification is rapid, robust, automatable, and the per-sample costs are low. Yet, the spectra are very informative and the reliable identification of bacterial species is usually possible. Recently, new MS-based approaches for the identification of bacteria are emerging that are based on the detailed analysis of the bacterial proteome by high-resolution MS. These “proteotyping” approaches are highly discriminative and outperform MALDI-TOF MS-based identification in terms of specificity, but require a laborious proteomic workflow and far more expertise and sophisticated instrumentation than identification on basis of MALDI-TOF MS spectra, which can be obtained with relative simple and uncostly linear MALDI-TOF mass spectrometers. Thus MALDI-TOF MS identification of bacteria remains an attractive option for routine diagnostics. Additionally, MALDI-TOF MS identification protocols have been extended and improved in many respects making linear MALDI-TOF MS a versatile tool that can be useful beyond the identification of a bacterial species, e.g. for the characterization of leucocytes and arthropod vectors of infectious diseases. This review focuses on such improvements and extensions of the typical MALDI-TOF MS workflow in the field of infectious diseases. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim


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.

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