Harald Granzow

Friedrich Loeffler Institute, Griefswald, Mecklenburg-Vorpommern, Germany

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Publications (102)393.81 Total impact

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    ABSTRACT: Nuclear egress of herpesvirus capsids is mediated by a conserved heterodimeric complex of two viral proteins, designated as pUL34 and pUL31 in Herpes simplex and Pseudorabies Virus (PrV). pUL34, a tail-anchored membrane protein, is targeted to the nuclear envelope and recruits pUL31 to the inner nuclear membrane (INM) to provide the docking and envelopment machinery for the nascent capsid. While the less conserved C-terminal part of pUL34 is required for correct positioning of the nuclear egress complex (NEC) at the INM, the conserved N-terminal part functions as docking site for pUL31. Since no crystal structure of the NEC is available yet, structure-function studies depend on mutational analyses with several approaches already performed for different herpesvirus NECs. Here, we extended our studies on PrV pUL34 identifying two asparagine residues (N75, N103) and a dileucine motif (LL166/167), adjacent to an ER retention signal, which are absolutely required for NEC function. While the pUL34-N75A substitution mutant is unable to interact with pUL31, pUL34-N103A is non-functional despite continuing complex formation. Surprisingly, mutant pUL34-G77A, which does not efficiently recruit pUL31 to the nuclear rim after cotransfection, nonetheless complements a UL34-deletion mutant indicating that the NEC may be stabilized by additional viral factors during infection. In the absence of a crystal structure of the nuclear egress complex (NEC) required for herpesvirus maturation, site-directed mutagenesis studies provide important information on critical amino acid residues. Here, we identify conserved amino acid residues in the membrane-bound component of the NEC which are relevant for its function.
    Journal of Virology 03/2014; · 5.08 Impact Factor
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    ABSTRACT: Herpesvirus capsid morphogenesis occurs in the nucleus, while final maturation takes place in the cytosol requiring translocation of capsids through the nuclear envelope. The nuclear egress complex consisting of homologs of herpes simplex virus pUL31 and pUL34, is required for efficient nuclear egress via primary envelopment-deenvelopment. Recently, we described an alternative mode of nuclear escape by fragmentation of the nuclear envelope induced by replication competent pUL31 and pUL34 deletion mutants of the alphaherpesvirus pseudorabies virus (PrV), which had been selected by serial passaging in cell culture. Both passaged viruses carry congruent mutations in seven genes including UL46 which encodes one of the major tegument proteins. Herpesvirus pUL46 homologs have recently been shown to activate the PI3K-Akt and ERK1/2 signaling pathways, which are involved in regulation of mitosis and apoptosis. Since in uninfected cells fragmentation of the nuclear envelope occurs during mitosis and apoptosis, we analyzed whether pUL46 of PrV is involved in signaling events impairing the integrity of the nuclear envelope. We show here that PrV pUL46 is able to induce phosphorylation of ERK1/2 and, thus, expression of ERK1/2 target genes, but fails to activate the PI3K-Akt pathway. Deletion of UL46 from PrV-ΔUL34Pass and PrV-ΔUL31Pass, or substitution by wild type UL46 resulted in enhanced nuclear envelope breakdown indicating that the mutations in pUL46 may limit the extent of NEBD. Thus, although pUL46 induces ERK1/2 phosphorylation, controlling the integrity of the nuclear envelope is independent of the ERK1/2 signaling pathway. Herpesvirus nucleocapsids can leave the nucleus by regulated, vesicle-mediated transport through the nuclear envelope designated as nuclear egress, or by inducing nuclear envelope breakdown (NEBD). The viral proteins involved in NEBD are unknown. We show here that the pseudorabies virus tegument protein pUL46 induces the ERK1/2 signaling pathway and modulates NEBD. However, these two processes are independent and ERK1/2 signaling induced by pUL46 is not involved in herpesvirus-induced NEBD.
    Journal of Virology 03/2014; · 5.08 Impact Factor
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    ABSTRACT: In autumn 2011, a novel species of the genus Orthobunyavirus of the Simbu serogroup was discovered close to the German/Dutch border and named Schmallenberg virus (SBV). Since then, SBV has caused a large epidemic in European livestock. Like other viruses of the Simbu serogroup, SBV is transmitted by insect vectors. Adult ruminants may show a mild transient disease, while an infection during a critical period of pregnancy can lead to severe congenital malformation, premature birth or stillbirth. The current knowledge about the virus, its diagnosis, the spread of the epidemic, the impact and the possibilities for preventing infections with SBV is described and discussed.
    Preventive Veterinary Medicine 01/2014; · 2.39 Impact Factor
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    ABSTRACT: Picornaviruses have been isolated from a variety of hosts, mainly mammals and birds. Here, we describe the sequence analysis of carp picornavirus 1 (CPV-1) F37/06 that was isolated from an organ pool (heart, brain, liver) of a common carp (Cyprinus carpio). This carp perished after an accidental discharge of liquid manure into a fish pond and presented without obvious clinical symptoms. Experimental intraperitoneal infection of young carps with CPV-1 revealed no clinical signs, but virus could be reisolated from various organs. Sequence analysis of almost the complete genome (7,632 nt excluding the poly-A tract) reveals a novel picornavirus clade. The polymerase sequence clusters in phylogenetic trees with parechoviruses, duck hepatitis A virus, eel picornavirus and aquamavirus A. The open reading frame includes 6,807 nucleotides and encodes a polyprotein of 2,269 amino acids. CPV-1 has a typical genome layout of picornaviruses except for the presence of two aphthovirus 2A-like NPGP sequence motifs: VPg+5'UTR[1AB-1C-1D-2A1npgp/2A2npgp-2B-2CATPase/3A-3BVPg-3Cpro-3Dpol]3'UTR-poly-A. 2A1npgp and 2A2npgp are separated by 133 amino acids. The proteins 2A2npgp, 2B, 3A and 3BVPg have no significant similarity to the corresponding proteins of other picornaviruses. Amino acid identities (aai) of the orthologous proteins P1, 2C, 3Cpro, 3Dpol range from 16.4 to 40.8 per cent in the eel picornavirus/CPV-1 comparison. 3Dpol shows closest similarity to eel picornavirus (40.8% aai), human parechovirus (36.5%), duck hepatitis A virus (32.7%) and swine pasivirus (29.3%). Both the unique genome organization and low sequence similarity support the assignment of CPV-1 to a novel picornavirus species within a novel genus.
    Journal of General Virology 10/2013; · 3.13 Impact Factor
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    ABSTRACT: A novel picornavirus was isolated from specimens of a diseased European eel (Anguilla anguilla). This virus induced a cytopathic effect in eel embryonic kidney cells and high mortality in a controlled transmission study using elvers. Eel picornavirus has a genome of 7,496 nucleotides that encodes a polyprotein of 2,259 amino acids. It has a typical picornavirus genome layout, but low similarity to known viral proteins suggests a novel species in the family Picornaviridae.
    Journal of Virology 07/2013; · 5.08 Impact Factor
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    ABSTRACT: Two isolates of a novel enveloped RNA virus were obtained from carp and koi carp with gill necrosis. Both isolates behaved identically and could be propagated in different cyprinid cell lines forming large syncytia. The virus was sensitive to lipid solvents and neither exhibited haemadsorption/haemagglutination nor reverse transcriptase activity. Mature virus particles displayed a spherical shape with diameter of 100-350 nm after negative staining and 100-300 nm in ultrathin sections, covered by short projections of 8-10 nm in length. Maturation of virus progeny was shown to occur by budding and envelopment of the filamentous helical nucleocapsids at the cell surface. A detailed comparison of ultrastructure and morphogenesis of the novel virus isolates with selected arena-, ortho- and paramyxoviruses as possible candidates for evaluation of taxonomic classification yielded no consistency in all phenotypic features. Thus, on the basis of ultrastructure the novel virus isolates could not be assigned unequivocally to any established virus family.
    Journal of Fish Diseases 07/2013; · 1.59 Impact Factor
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    ABSTRACT: Herpesvirus nucleocapsids are assembled in the nucleus while maturation into infectious virions takes place in the cytosol. Since, due to their size, nucleocapsids cannot pass the nuclear pores they traverse the nuclear envelope by vesicle-mediated transport. Nucleocapsids bud at the inner nuclear membrane into the perinuclear space forming primary enveloped particles and are released into the cytosol after fusion of the primary envelope with the outer nuclear membrane. The nuclear egress complex (NEC), consisting of the conserved herpesvirus proteins (p)UL31 and pUL34, is required for this process, whereas viral glycoproteins gB and gH, which are essential for fusion during penetration, are not. We recently described herpesvirus-induced nuclear envelope breakdown (NEBD) as an alternative egress pathway used in the absence of the NEC. However, the molecular details of this pathway are still unknown. It had been speculated that glycoproteins involved in fusion during entry might play a role in NEBD. By deleting genes encoding glycoproteins gB and gH from the genome of NEBD-inducing pseudorabies viruses we demonstrate that these glycoproteins are not required for NEBD but are still necessary for syncytia formation again emphasizing fundamental differences in herpesvirus-induced alterations at the nuclear envelope and plasma membrane of infected cells.
    Journal of Virology 07/2013; · 5.08 Impact Factor
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    ABSTRACT: Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination.
    PLoS ONE 01/2013; 8(9):e72530. · 3.73 Impact Factor
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    ABSTRACT: Virus-like particles (VLPs) from hepatitis B and human papilloma viruses have been successfully used as preventative vaccines against these infectious agents. These VLPs consist of a self-associating capsid polymer formed from a single structure protein and are devoid of viral DNA. Since virions from herpesviruses consist of a large number of molecules of viral and cellular origin, generating VLPs from a subset of these would be a particularly arduous task. Therefore, we have adopted an alternative strategy that consists in producing DNA-free defective virus particles in a cell line infected by a herpesvirus mutant incapable of packaging DNA. We previously reported that an Epstein-Barr virus (EBV) mutant devoid of the terminal repeats (ΔTR) that act as packaging signals in herpesviruses produces substantial amounts of VLPs and of light particles (LPs). However, ΔTR virions retained some infectious genomes, and although these mutants had lost their transforming abilities, this poses potential concerns for clinical applications. Therefore, we have constructed a series of mutants that lack proteins involved in maturation and assessed their ability to produce viral DNA-free VLP/LPs. Some of the introduced mutations were deleterious for capsid maturation and virus production. However, deletion of BFLF1/BFRF1A or of BBRF1 resulted in the production of DNA-free VLPs/LPs. The ΔBFLF1/BFRF1A viruses elicited a potent CD4+ T cell response that was indistinguishable from the one obtained with wild type controls. In summary, the defective particles produced by the ΔBFLF1/BFRF1A mutant fulfill the criteria of efficacy and safety expected from a preventative vaccine.
    Journal of Virology 12/2012; · 5.08 Impact Factor
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    ABSTRACT: Herpesvirus capsids are assembled in the nucleus of infected cells whereas final maturation occurs in the cytosol. To access the final maturation compartment, intranuclear capsids have to cross the nuclear envelope which represents a formidable barrier. They do so by budding at the inner nuclear membrane, thereby forming a primary enveloped particle residing in the perinuclear cleft. Formation of primary envelopes is driven by a heterodimeric complex of two conserved herpesviral proteins, designated in the herpes simplex virus nomenclature as pUL34, a tail-anchored transmembrane protein located in the nuclear envelope, and pUL31. This nuclear egress complex (NEC) recruits viral and cellular kinases to soften the nuclear lamina and allowing access of capsids to the NEC. How capsids are recruited to the budding site and into the primary virus particle is still not completely understood, nor is the composition of the primary enveloped virion in the perinuclear cleft. Fusion of the primary envelope with the outer nuclear membrane then results in translocation of the capsid to the cytosol. This fusion event is clearly different from fusion during infectious entry of free virions into target cells in that it does not require the conserved essential core herpesvirus fusion machinery. Nuclear egress can thus be viewed as a vesicle (primary envelope)-mediated transport of cargo (capsids) through the nuclear envelope, a process which had been unique in cell biology. Only recently has a similar process been identified in Drosophila for nuclear egress of large ribonucleoprotein complexes. Thus, herpesviruses appear to subvert a hitherto cryptic cellular pathway for translocation of capsids from the nucleus to the cytosol.
    Cellular Microbiology 10/2012; · 4.81 Impact Factor
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    ABSTRACT: Lyssavirus matrix proteins (M) support virus budding and have accessory functions that may contribute to host cell manipulation and adaptation to specific hosts. Here, we show that rabies virus (RABV) and European Bat Lyssavirus Type 1 (EBLV-1) M proteins differ in targeting and accumulation at cellular membranes. In contrast to RABV M, EBLV-1 M expressed from authentic EBLV-1 or chimeric RABV accumulated at the Golgi apparatus. Chimeric M proteins revealed that Golgi-association depends on the integrity of the entire EBLV-1 M protein. Since RABV and EBLV-1 M differ in the use of cellular membranes for particle formation (Finke et al., 2010), differential membrane targeting and transport of M might determine the site of virus production. Moreover, both RABV and EBLV-1 M were for the first time detected within the nucleus and in Negri body-like inclusions bodies. Whereas nuclear M may imply hitherto unknown functions of lyssavirus M in host cell manipulation, the presence of M in inclusion bodies may correlate with regulatory functions of M in virus RNA synthesis. The data strongly support a model in which targeting of lyssavirus M proteins to distinct intracellular sites is a key determinant of diverse features in lyssavirus replication, host adaptation and pathogenesis.
    Cellular Microbiology 10/2012; · 4.81 Impact Factor
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    ABSTRACT: Herpesvirus nucleocapsids are translocated from their assembly site in the nucleus to the cytosol by acquisition of a primary envelope at the inner nuclear membrane which subsequently fuses with the outer nuclear membrane. This transport through the nuclear envelope requires homologs of the conserved herpesviral pUL31 and pUL34 proteins which form the nuclear egress complex (NEC). In its absence, 1,000-fold less virus progeny is produced. We isolated a UL34-negative mutant of the alphaherpesvirus pseudorabies virus (PrV), PrV-ΔUL34Pass, which regained replication competence after serial passages in cell culture by inducing nuclear envelope breakdown (NEBD) (B. G. Klupp, H. Granzow, and T. C. Mettenleiter, J. Virol. 85:8285-8292, 2011). To test whether this phenotype is unique, passaging experiments were repeated with a UL31 deletion mutant. After 60 passages, the resulting PrV-ΔUL31Pass replicated similarly to wild-type PrV. Ultrastructural analyses confirmed escape from the nucleus via NEBD, indicating an inherent genetic disposition in herpesviruses. To identify the mutated viral genes responsible for this phenotype, the genome of PrV-ΔUL34Pass was sequenced and compared to the genomes of parental PrV-Ka and PrV-ΔUL34. Targeted sequencing of PrV-ΔUL31Pass disclosed congruent mutations comprising genes encoding tegument proteins (pUL49, pUL46, pUL21, pUS2), envelope proteins (gI, pUS9), and protease pUL26. To investigate involvement of cellular pathways, different inhibitors of cellular kinases were tested. While induction of apoptosis or inhibition of caspases had no specific effect on the passaged mutants, roscovitine, a cyclin-dependent kinase inhibitor, and U0126, an inhibitor of MEK1/2, specifically impaired replication of the passaged mutants, indicating involvement of mitosis-related processes in herpesvirus-induced NEBD.
    Journal of Virology 04/2012; 86(12):6512-21. · 5.08 Impact Factor
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    ABSTRACT: pUL11 is a highly conserved, small, acylated, membrane-associated tegument protein of herpesviruses. It is involved in final envelopment of nascent virions in the cytoplasm, although the precise mechanism is still unknown. By screening of mouse monoclonal antibodies (mAb) raised against purified particles of infectious laryngotracheitis virus (ILTV) of chickens (Veits et al., 2003a), we identified two mAb recognizing the 15 kDa UL11 protein (pUL11) of this avian alphaherpesvirus. These mAb permitted detection and precise localization of pUL11 in mature ILT virions, as well as in the cytoplasm of infected chicken cells by Western blot analyses, indirect immunofluorescence tests, and immunoelectron microscopy. For investigation of gene function UL11-deleted ILTV recombinants were generated. Like its homologues in several other alphaherpesviruses, ILTV-pUL11 was shown to be nonessential for productive virus replication. However, compared to wild-type and UL11 rescued ILTV the deletion mutants exhibited significantly reduced virus yields and moderately impaired spread in cell culture. In the absence of pUL11, electron microscopy of infected cells revealed accumulations of tegument proteins with nucleocapsids, and marked distortions of Golgi membranes in the cytoplasm, which obviously inhibited the formation of mature, enveloped virus particles. Taken together, our results demonstrate that pUL11 is relevant for secondary envelopment of ILTV, and confirm functional conservation of this protein in herpesviruses. The now available unique pUL11-specific mAb will help to further analyze this function, which is presumably mediated by physical interactions with other viral gene products, in cultured cells and in the natural animal host of ILTV.
    Virus Research 02/2012; 163(2):599-608. · 2.75 Impact Factor
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    ABSTRACT: Herpesvirus proteins pUL34 and pUL31 form a complex at the inner nuclear membrane (INM) which is necessary for efficient nuclear egress. Pseudorabies virus (PrV) pUL34 is a type II membrane protein of 262 amino acids (aa). The transmembrane region (TM) is predicted to be located between aa 245 and 261, leaving only one amino acid in the C terminus that probably extends into the perinuclear space. It is targeted to the nuclear envelope in the absence of other viral proteins, pointing to intrinsic localization motifs, and shows structural similarity to cellular INM proteins like lamina-associated polypeptide (Lap) 2ß and Emerin. To investigate which domains of pUL34 are relevant for localization and function, we constructed chimeric proteins by replacing parts of pUL34 with regions of cellular INM proteins. First the 18 C-terminal amino acids encompassing the TM were exchanged with TM regions and C-terminal domains of Lap2ß and Emerin or with the first TM region of the polytopic lamin B receptor (LBR), including the nine following amino acids. All resulting chimeric proteins complemented the replication defect of PrV-ΔUL34, demonstrating that the substitution of the TM and the extension of the C-terminal domain does not interfere with the function of pUL34. Complementation was reduced but not abolished when the C-terminal 50 aa were replaced by corresponding Lap2ß sequences (pUL34-LapCT50). However, replacing the C-terminal 100 aa (pUL34-LapCT100) resulted in a nonfunctional protein despite continuing pUL31 binding, pointing to an important functional role of this region. The replacement of the N-terminal 100 aa (pUL34-LapNT100) had no effect on nuclear envelope localization but abrogated pUL31 binding and function.
    Journal of Virology 12/2011; 86(4):2079-88. · 5.08 Impact Factor
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    ABSTRACT: This report describes the generation of a new recombinant Orf virus (ORFV; Parapoxvirus) expressing the major capsid protein VP1 (VP60) of the calicivirus, rabbit hemorrhagic disease virus (RHDV). Authentic expression of VP1 could be demonstrated in cells infected with the recombinant D1701-V-VP1 without the need for production of infectious ORFV progeny. Notably, infected cells also released empty calicivirus-like particles (VLPs). Challenge experiments showed that even a single immunization with ≥10(5) PFU of D1701-V-VP1 protected rabbits against lethal RHDV infection. ELISA tests indicated that the protective immunity mediated by D1701-V-VP1 did not strictly depend on the presence of detectable RHDV-specific serum antibodies. The induction of interleukin-2 found only in the sera of rabbits immunized with the D1701-V-VP1, but not in sera of rabbits immunized with the inactivated commercial vaccine RIKA-VACC, might indicate also some involvement of T-cells in protection. Collectively, this work adds another example of the successful use of the ORFV vector system for the generation of a recombinant vaccine, and demonstrates its potential as an alternative vaccine to protect rabbits against RHDV infection.
    Vaccine 11/2011; 29(49):9256-64. · 3.77 Impact Factor
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    ABSTRACT: Herpesvirus nucleocapsids assemble in the nucleus but mature to infectious virions in the cytoplasm. To gain access to this cellular compartment, nucleocapsids are translocated to the cytoplasm by primary envelopment at the inner nuclear membrane and subsequent fusion of the primary envelope with the outer nuclear membrane. The conserved viral pUL34 and pUL31 proteins play a crucial role in this process. In their absence, viral replication is strongly impaired but not totally abolished. We used the residual infectivity of a pUL34-deleted mutant of the alphaherpesvirus pseudorabies virus (PrV) for reversion analysis. To this end, PrV-ΔUL34 was serially passaged in rabbit kidney cells until final titers of the mutant virus PrV-ΔUL34Pass were comparable to those of wild-type PrV. PrV-ΔUL34Pass produced infectious progeny independently of the pUL34/pUL31 nuclear egress complex and the pUS3 protein kinase. Ultrastructural analyses demonstrated that this effect was due to virus-induced disintegration of the nuclear envelope, thereby releasing immature and mature capsids into the cytosol for secondary envelopment. Our data indicate that nuclear egress primarily serves to transfer capsids through the intact nuclear envelope. Immature and mature intranuclear capsids are competent for further virion maturation once they reach the cytoplasm. However, nuclear egress exhibits a strong bias for nucleocapsids, thereby also functioning as a quality control checkpoint which is abolished by herpesvirus-induced nuclear envelope breakdown.
    Journal of Virology 06/2011; 85(16):8285-92. · 5.08 Impact Factor
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    ABSTRACT: Bovine viral diarrhea virus (BVDV) belongs to the genus Pestivirus within the family Flaviviridae. The lipid membrane of the virions is supposed to contain the three glycosylated envelope proteins E(rns), E1 and E2, but detailed studies of virus assembly are complicated because no efficient purification method for pestiviruses has been described so far. In this study, we generated infectious BVDV with N-terminally FLAG-tagged E(rns) or E2 proteins, respectively. The expression of the epitope-tagged E(rns) and E2 proteins could be shown by immunofluorescence and Western blot experiments. Furthermore, an affinity tag purification protocol for the isolation and concentration of infectious BVDV was established. In the preparation with a titre of 10(8.75) TCID(50) ml(-1), spherical particles with a diameter of 43-58 nm (mean diameter: 48 nm) could be detected by negative staining electron microscopy, and immunogold labelling located both E(rns) and E2 proteins at the virus membrane.
    Journal of General Virology 02/2011; 92(Pt 6):1352-7. · 3.13 Impact Factor
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    ABSTRACT: Compared with many well-studied enveloped viruses, herpesviruses use a more sophisticated molecular machinery to induce fusion of viral and cellular membranes during cell invasion. This essential function is carried out by glycoprotein B (gB), a class III viral fusion protein, together with the heterodimer of glycoproteins H and L (gH/gL). In pseudorabies virus (PrV), a porcine herpesvirus, it was shown that gH/gL can be substituted by a chimeric fusion protein gDgH, containing the receptor binding domain (RBD) of glycoprotein D fused to a truncated version of gH lacking its N-terminal domain. We report here the 2.1-Å resolution structure of the core fragment of gH present in this chimera, bound to the Fab fragment of a PrV gH-specific monoclonal antibody. The structure strongly complements the information derived from the recently reported structure of gH/gL from herpes simplex virus type 2 (HSV-2). Together with the structure of Epstein-Barr virus (EBV) gH/gL reported in parallel, it provides insight into potentially functional conserved structural features. One feature is the presence of a syntaxin motif, and the other is an extended "flap" masking a conserved hydrophobic patch in the C-terminal domain, which is closest to the viral membrane. The negative electrostatic surface potential of this domain suggests repulsive interactions with the lipid heads. The structure indicates the possible unmasking of an extended hydrophobic patch by movement of the flap during a receptor-triggered conformational change of gH, exposing a hydrophobic surface to interact with the viral membrane during the fusion process.
    Proceedings of the National Academy of Sciences 12/2010; 107(52):22635-40. · 9.74 Impact Factor
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    ABSTRACT: After primary replication at the site of entry into the host, alphaherpesviruses infect and establish latency in neurons. To this end, they are transported within axons retrograde from the periphery to the cell body for replication and in an anterograde direction to synapses for infection of higher-order neurons or back to the periphery. Retrograde transport of incoming nucleocapsids is well documented. In contrast, there is still significant controversy on the mode of anterograde transport. By high-resolution transmission electron microscopy of primary neuronal cultures from embryonic rat superior cervical ganglia infected by pseudorabies virus (PrV), we observed the presence of enveloped virions in axons within vesicles supporting the "married model" of anterograde transport of complete virus particles within vesicles (C. Maresch, H. Granzow, A. Negatsch, B.G. Klupp, W. Fuchs, J.P. Teifke, and T.C. Mettenleiter, J. Virol. 84:5528-5539, 2010). We have now extended these analyses to the related human herpes simplex virus type 1 (HSV-1). We have demonstrated that in neurons infected by HSV-1 strains HFEM, 17+ or SC16, approximately 75% of virus particles observed intraaxonally or in growth cones late after infection constitute enveloped virions within vesicles, whereas approximately 25% present as naked capsids. In general, the number of HSV-1 particles in the axons was significantly less than that observed after PrV infection.
    Journal of Virology 10/2010; 84(24):13031-5. · 5.08 Impact Factor
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    ABSTRACT: Homologs of the UL17 and UL25 gene products of herpes simplex virus 1 (HSV-1) are conserved throughout the Herpesviridae and essential for virus replication. However, their exact function is still unknown. Although both proteins form a complex on DNA-containing C-capsids defects observed in the absence of either protein differ. Absence of pUL17 from HSV-1 or the related alphaherpesvirus pseudorabies virus (PrV) precludes cleavage and packaging of newly replicated viral DNA, whereas in the absence of pUL25 genomic DNA is encapsidated but nuclear egress of capsids to the cytosol is abolished. HSV-1 pUL25 partially complemented the defect in a PrV UL25 deletion mutant indicating overlapping functions. However, reciprocal complementation did not ensue, and the present study demonstrates that UL17-deleted HSV-1 or PrV mutants are also not rescued by heterologous pUL17. To analyze whether simultaneous substitution of both complex partners may allow or increase trans-complementation we generated rabbit kidney cell lines co-expressing either PrV or HSV-1 pUL17 and pUL25, and respective HSV-1 and PrV double deletion mutants. Whereas the defects of both double mutants were trans-complemented by cell lines co-expressing the homologous complex partners, productive replication was not restored by heterologous pUL17 and pUL25. Thus, the protein complexes of PrV and HSV-1 either possess distinct functions, or require interactions with other viral proteins which are impaired in a heterologous context.
    Virus Research 10/2010; 153(1):20-8. · 2.75 Impact Factor

Publication Stats

3k Citations
206 Downloads
393.81 Total Impact Points

Institutions

  • 1997–2014
    • Friedrich Loeffler Institute
      • • Institute of Infectology
      • • Institute of Molecular Biology
      • • Institute of Diagnostic Virology
      Griefswald, Mecklenburg-Vorpommern, Germany
  • 2002
    • Princeton University
      • Department of Molecular Biology
      Princeton, New Jersey, United States