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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.
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.
Herpesvirus genome replication, capsid assembly and packaging take place in the host cell nucleus. Matured capsids leave the nucleus through a unique envelopment-de-envelopment process at the nuclear membranes called nuclear egress. How assembled and DNA-containing herpesvirus capsids reach the sites of nuclear egress is however still controversially discussed, as host chromatin that marginalizes during infection might constitute a major barrier. For alphaherpesviruses, previous work has suggested that nuclear capsids use active transport mediated by nuclear filamentous actin (F-actin). However, direct evidence for nuclear capsid motility on nuclear F-actin was missing. Our subsequent work did not detect nuclear F-actin associated with motile capsids, but instead found evidence for chromatin remodeling to facilitate passive capsid diffusion. A recent report described that human cyto-megalovirus, a betaherpesvirus, induces nuclear F-actin and that the motor protein myosin V localizes to these structures. Direct evidence of capsid recruitment to these structures and motility on them was however missing. In this study, we tested the functional role of HCMV-induced, nuclear actin assemblies for capsid transport. We did not observe transport events along nuclear F-actin. Instead, reproduction of nuclear F-actin was only possible using strong overexpression of the fluorescent marker LifeAct-mCherry-NLS. Also, two alternative fluo-rescent F-actin markers did not detect F-actin in HCMV-infected cells. Furthermore, single particle tracking of nuclear HCMV capsids showed no indication for active transport, which is in line with previous work on alphaherpesviruses.
Felix Flomm; Eva Maria Borst; Thomas Günther; Rudolph Reimer; Laura De Vries; Carola Schneider; Adam Grundhoff; Kay Grünewald; Martin Messerle; Jens Bern-Hard Bosse. Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles. 2019, 641266 .
AMA StyleFelix Flomm, Eva Maria Borst, Thomas Günther, Rudolph Reimer, Laura De Vries, Carola Schneider, Adam Grundhoff, Kay Grünewald, Martin Messerle, Jens Bern-Hard Bosse. Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles. . 2019; ():641266.
Chicago/Turabian StyleFelix Flomm; Eva Maria Borst; Thomas Günther; Rudolph Reimer; Laura De Vries; Carola Schneider; Adam Grundhoff; Kay Grünewald; Martin Messerle; Jens Bern-Hard Bosse. 2019. "Human cytomegalovirus nuclear capsid motility is non-directed and independent of nuclear actin bundles." , no. : 641266.
Human cytomegalovirus (HCMV) genome encapsidation requires several essential viral proteins, among them pUL56, pUL89, and the recently described pUL51, which constitute the viral terminase. To gain insight into terminase complex assembly, we investigated interactions between the individual subunits. For analysis in the viral context, HCMV bacterial artificial chromosomes carrying deletions in the open reading frames encoding the terminase proteins were used. These experiments were complemented by transient-transfection assays with plasmids expressing the terminase components. We found that if one terminase protein was missing, the levels of the other terminase proteins were markedly diminished, which could be overcome by proteasome inhibition or providing the missing subunit in trans . These data imply that sequestration of the individual subunits within the terminase complex protects them from proteasomal turnover. The finding that efficient interactions among the terminase proteins occurred only when all three were present together is reminiscent of a folding-upon-binding principle leading to cooperative stability. Furthermore, whereas pUL56 was translocated into the nucleus on its own, correct nuclear localization of pUL51 and pUL89 again required all three terminase constituents. Altogether, these features point to a model of the HCMV terminase as a multiprotein complex in which the three players regulate each other concerning stability, subcellular localization, and assembly into the functional tripartite holoenzyme. IMPORTANCE HCMV is a major risk factor in immunocompromised individuals, and congenital CMV infection is the leading viral cause for long-term sequelae, including deafness and mental retardation. The current treatment of CMV disease is based on drugs sharing the same mechanism, namely, inhibiting viral DNA replication, and often results in adverse side effects and the appearance of resistant virus strains. Recently, the HCMV terminase has emerged as an auspicious target for novel antiviral drugs. A new drug candidate inhibiting the HCMV terminase, Letermovir, displayed excellent potency in clinical trials; however, its precise mode of action is not understood yet. Here, we describe the mutual dependence of the HCMV terminase constituents for their assembly into a functional terminase complex. Besides providing new basic insights into terminase formation, these results will be valuable when studying the mechanism of action for drugs targeting the HCMV terminase and developing additional substances interfering with viral genome encapsidation.
Sebastian Neuber; Karen Wagner; Thomas Goldner; Peter Lischka; Lars Steinbrueck; Martin Messerle; Eva Maria Borst. Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation. Journal of Virology 2017, 91, e02384-16 .
AMA StyleSebastian Neuber, Karen Wagner, Thomas Goldner, Peter Lischka, Lars Steinbrueck, Martin Messerle, Eva Maria Borst. Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation. Journal of Virology. 2017; 91 (12):e02384-16.
Chicago/Turabian StyleSebastian Neuber; Karen Wagner; Thomas Goldner; Peter Lischka; Lars Steinbrueck; Martin Messerle; Eva Maria Borst. 2017. "Mutual Interplay between the Human Cytomegalovirus Terminase Subunits pUL51, pUL56, and pUL89 Promotes Terminase Complex Formation." Journal of Virology 91, no. 12: e02384-16.
Several essential viral proteins are proposed to participate in genome encapsidation of human cytomegalovirus (HCMV), among them pUL77 and pUL93, which remain largely uncharacterized. To gain insight into their properties, we generated an HCMV mutant expressing a pUL77-monomeric enhanced green fluorescent protein (mGFP) fusion protein and a pUL93-specific antibody. Immunoblotting demonstrated that both proteins are incorporated into capsids and virions. Conversely to data suggesting internal translation initiation sites within the UL93 open reading frame (ORF), we provide evidence that pUL93 synthesis commences at the first start codon. In infected cells, pUL77-mGFP was found in nuclear replication compartments and dot-like structures, colocalizing with capsid proteins. Immunogold labeling of nuclear capsids revealed that pUL77 is present on A, B, and C capsids. Pulldown of pUL77-mGFP revealed copurification of pUL93, indicating interaction between these proteins, which still occurred when capsid formation was prevented. Correct subnuclear distribution of pUL77-mGFP required pUL93 as well as the major capsid protein (and thus probably the presence of capsids), but not the tegument protein pp150 or the encapsidation protein pUL52, demonstrating that pUL77 nuclear targeting occurs independently of the formation of DNA-filled capsids. When pUL77 or pUL93 was missing, generation of unit-length genomes was not observed, and only empty B capsids were produced. Taken together, these results show that pUL77 and pUL93 are capsid constituents needed for HCMV genome encapsidation. Therefore, the task of pUL77 seems to differ from that of its alphaherpesvirus orthologue pUL25, which exerts its function subsequent to genome cleavage-packaging. IMPORTANCE The essential HCMV proteins pUL77 and pUL93 were suggested to be involved in viral genome cleavage-packaging but are poorly characterized both biochemically and functionally. By producing a monoclonal antibody against pUL93 and generating an HCMV mutant in which pUL77 is fused to a fluorescent protein, we show that pUL77 and pUL93 are capsid constituents, with pUL77 being similarly abundant on all capsid types. Each protein is required for genome encapsidation, as the absence of either pUL77 or pUL93 results in a genome packaging defect with the formation of empty capsids only. This distinguishes pUL77 from its alphaherpesvirus orthologue pUL25, which is enriched on DNA-filled capsids and exerts its function after the viral DNA is packaged. Our data for the first time describe an HCMV mutant with a fluorescent capsid and provide insight into the roles of pUL77 and pUL93, thus contributing to a better understanding of the HCMV encapsidation network.
Eva Maria Borst; Rudolf Bauerfeind; Anne Binz; Thomas Min Stephan; Sebastian Neuber; Karen Wagner; Lars Steinbrück; Beate Sodeik; Tihana Lenac Roviš; Stipan Jonjić; Martin Messerle. The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation. Journal of Virology 2016, 90, 5860 -5875.
AMA StyleEva Maria Borst, Rudolf Bauerfeind, Anne Binz, Thomas Min Stephan, Sebastian Neuber, Karen Wagner, Lars Steinbrück, Beate Sodeik, Tihana Lenac Roviš, Stipan Jonjić, Martin Messerle. The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation. Journal of Virology. 2016; 90 (13):5860-5875.
Chicago/Turabian StyleEva Maria Borst; Rudolf Bauerfeind; Anne Binz; Thomas Min Stephan; Sebastian Neuber; Karen Wagner; Lars Steinbrück; Beate Sodeik; Tihana Lenac Roviš; Stipan Jonjić; Martin Messerle. 2016. "The Essential Human Cytomegalovirus Proteins pUL77 and pUL93 Are Structural Components Necessary for Viral Genome Encapsidation." Journal of Virology 90, no. 13: 5860-5875.
Human cytomegalovirus (HCMV) has a large 240 kb genome that may encode more than 700 gene products with many of them remaining uncharacterized. Mutagenesis of bacterial artificial chromosome (BAC)-cloned CMV genomes has greatly facilitated the analysis of viral gene functions. However, the roles of essential proteins often remain particularly elusive because their investigation requires the cumbersome establishment of suitable complementation systems. Here, we show that HCMV genomes can be introduced into cells with unprecedented efficiency by applying a transfection protocol based on replication-defective, inactivated adenovirus particles (adenofection). Upon adenofection of several permissive cell types with HCMV genomes carrying mutations in essential genes, transfection rates of up to 60% were observed and viral proteins of all kinetic classes were found expressed. This enabled further analyses of the transfected cells by standard biochemical techniques. Remarkably, HCMV genomes lacking elements essential for viral DNA replication, such as the lytic origin of replication, still expressed several late proteins. In conclusion, adenofection allows the study of essential HCMV genes directly in BAC-transfected cells without the need for sophisticated complementation strategies.
Endrit Elbasani; Ildar Gabaev; Lars Steinbrück; Martin Messerle; Eva Maria Borst. Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes. Viruses 2014, 6, 354 -370.
AMA StyleEndrit Elbasani, Ildar Gabaev, Lars Steinbrück, Martin Messerle, Eva Maria Borst. Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes. Viruses. 2014; 6 (1):354-370.
Chicago/Turabian StyleEndrit Elbasani; Ildar Gabaev; Lars Steinbrück; Martin Messerle; Eva Maria Borst. 2014. "Analysis of Essential Viral Gene Functions after Highly Efficient Adenofection of Cells with Cloned Human Cytomegalovirus Genomes." Viruses 6, no. 1: 354-370.
For mutagenesis of BAC-cloned herpesvirus genomes, we have adapted a two-step replacement procedure originally described by O’Connor et al. (1) for the manipulation of large DNA segments in Escherichia coli (E. coli). In principle, the mutant allele to be introduced is provided on a so-called shuttle plasmid. The mutant allele has to be flanked by regions homologous to the desired integration site of the mutation in the BAC (regions A and B, see Fig. 1, step 1). By homologous recombination in E. coli, the shuttle plasmid will completely insert into the BAC, leading to a cointegrate (see Fig. 1, step 2). As a consequence, the cointegrate carries the wild-type, as well as a mutant allele, and a duplication of the homologous sequences flanking the mutation and the wild-type locus. The cointegrate can spontaneously resolve by homologous recombination via regions A or B giving rise to either the wild-type herpesvirus BAC plasmid or a mutant BAC, depending on which of the homologous regions (A or B) is used for recombination (see Fig. 1, step 3). The propensity for resolution of the cointegrate goes up with increasing sizes of the homologous sequences. However, even if homologous sequences of 2 or 3 kbp are provided, resolution of the cointegrates remains a rather rare event. Therefore, we have introduced a negative selection marker into the shuttle plasmid (the sacB gene) that allows to select against the bacteria still containing a nonresolved cointegrate (see Fig. 1, step 4) and to identify bacterial clones harboring the mutant or the parental BAC. Fig. 1. Two-step replacement procedure for mutagenesis of the BAC-cloned CMV genome by homologous recombination in E. coli. Step 1: The shuttle plasmid carrying the desired mutation plus flanking homologies (A and B) is transformed into bacteria that already contain the BAC. Step 2: Through homologous recombination via region A or B the shuttle plasmid is completely integrated into the viral BAC genome, leading to a cointegrate. Bacteria containing the cointegrate are selected by incubation at 43°C. Step 3: Resolution of the cointegrate via A or B leads to either the wildtype or the mutant BAC. Step 4: Bacteria harboring a resolved cointegrate are selected at 30°C on agar plates containing 5% sucrose.
Eva-Maria Borst; György Pósfai; Frank Pogoda; Martin Messerle; Shaying Zhao; Marvin Stodolsky. Mutagenesis of Herpesvirus BACs by Allele Replacement. Bacterial Artificial Chromosomes 2004, 256, 269 -280.
AMA StyleEva-Maria Borst, György Pósfai, Frank Pogoda, Martin Messerle, Shaying Zhao, Marvin Stodolsky. Mutagenesis of Herpesvirus BACs by Allele Replacement. Bacterial Artificial Chromosomes. 2004; 256 ():269-280.
Chicago/Turabian StyleEva-Maria Borst; György Pósfai; Frank Pogoda; Martin Messerle; Shaying Zhao; Marvin Stodolsky. 2004. "Mutagenesis of Herpesvirus BACs by Allele Replacement." Bacterial Artificial Chromosomes 256, no. : 269-280.
Herpesviruses form a family of DNA viruses that are of considerable medical importance (1). Although the genomes of many members of the herpesviruses have been completely sequenced, our knowledge on the function of the majority of herpesvirus genes is still quite insufficient. This is especially true for the members of the β-herpesviruses, i.e., the cytomegaloviruses (CMVs), because their slow replication kinetics, cell association, and large genome size (230 kb) made the construction of viral mutants a difficult and tedious procedure (2). Conventional mutagenesis protocols for herpesviruses are based on the insertion of marker genes into the viral genome, which allows to disrupt or delete viral genes (3–5). Unfortunately, the method has certain limitations. The protocols rely on recombination events in eukaryotic cells that are relatively rare and difficult to control. Accordingly, adventitious deletions, rearrangements, and illegitimate recombination events in the viral genomes have frequently been observed. It is especially cumbersome that the verification of the mutant genome structure can only be done at the very end of the lengthy isolation procedures of the mutant virus. Generation of viral mutants requires an obligatory selection process against the wild-type virus. In the end, the recombinant virus has to be plaque-purified and separated from the wild-type virus. This makes the isolation of viral mutants with growth disadvantages a difficult task.
Eva-Maria Borst; Irena Crnkovic-Mertens; Martin Messerle; Shaying Zhao; Marvin Stodolsky. Cloning of β-Herpesvirus Genomes as Bacterial Artificial Chromosomes. Bacterial Artificial Chromosomes 2004, 256, 221 -240.
AMA StyleEva-Maria Borst, Irena Crnkovic-Mertens, Martin Messerle, Shaying Zhao, Marvin Stodolsky. Cloning of β-Herpesvirus Genomes as Bacterial Artificial Chromosomes. Bacterial Artificial Chromosomes. 2004; 256 ():221-240.
Chicago/Turabian StyleEva-Maria Borst; Irena Crnkovic-Mertens; Martin Messerle; Shaying Zhao; Marvin Stodolsky. 2004. "Cloning of β-Herpesvirus Genomes as Bacterial Artificial Chromosomes." Bacterial Artificial Chromosomes 256, no. : 221-240.