Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation
| Autor: | Häge, Sigrun, Sonntag, Eric, Svrlanska, Adriana, Borst, Eva Maria, Stilp, Anne-Charlotte, Horsch, Deborah, Müller, Regina, Kropff, Barbara, Milbradt, Jens, Stamminger, Thomas, Schlötzer-Schrehardt, Ursula, Marschall, Manfred |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
Cell Nucleus
Nuclear Envelope viruses lcsh:QR1-502 Cytomegalovirus differential functional relevance Virus Replication Article lcsh:Microbiology Viral Proteins Capsid HEK293 Cells human cytomegalovirus Mutation core nuclear egress complex pUL50 phosphosite mutants Humans ddc:610 Phosphorylation ORF-UL50 deletion Virus Release HeLa Cells phenotypical changes |
| Zdroj: | Viruses, Vol 13, Iss 165, p 165 (2021) Viruses Volume 13 Issue 2 |
| ISSN: | 1999-4915 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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