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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 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.
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.