A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus

Häge, Sigrun; Horsch, Deborah; Stilp, Anne-Charlotte; Kicuntod, Jintawee; Müller, Regina; Hamilton, Stuart T.; Egilmezer, Ece; Rawlinson, William D.; Stamminger, Thomas; Sonntag, Eric; Marschall, Manfred

doi:10.1016/j.jviromet.2020.113909

Cited by 15 publications

(36 citation statements)

References 25 publications

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“…BAC HB5/AD169-GFP [ 37 , 38 ] was used for the generation of HCMV UL97 deletion mutants ∆231–255, ∆256–280 and ∆231–280. Deletions were achieved by a two-step markerless Red recombination system as described previously [ 39 , 40 ]. The following primers were used: UL97∆231–280 for 5′-ggacgcggtgctcgaagaaaacgacgtggagctgcgcgcgaccacgtccatccgcggcctTAGGGATAACAGGGTAATCGATTT-3′; UL97∆231–280 rev 5′-acatacgcgggtcgcacgtaaggccgcggatggacgtggtcgcgcgcagctccacgtcgtGCCAGTGTTACAACCAATTAACC-3′; UL97∆256–280 for 5′-ccgcattccgcagccgctcagcggtagttccggggaggaaaccacgtccatccgcggcctTAGGGATAACAGGGTAATCGATTT-3′; UL97∆256–280 rev 5′-acatacgcgggtcgcacgtaaggccgcggatggacgtggtttcctccccggaactaccgcGCCAGTGTTACAACCAATTAACC-3′; UL97∆231–255 for 5′-ggacgcggtgctcgaagaaaacgacgtggagctgcgcgcgtccgccacggcggtggaggcTAGGGATAACAGGGTAATCGATTT-3′; UL97∆231–255 rev 5′-cgtcgtgtgacgtggagtcggcctccaccgccgtggcggacgcgcgcagctccacgtcgtGCCAGTGTTACAACCAATTAACC-3′.…”

Section: Methodsmentioning

confidence: 99%

Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97

Schütz

Steingruber

Socher

et al. 2021

Viruses

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The replication of human cytomegalovirus (HCMV) is characterized by a complex network of virus–host interaction. This involves the regulatory viral protein kinase pUL97, which represents a viral cyclin-dependent kinase ortholog (vCDK) combining typical structural and functional features of host CDKs. Notably, pUL97 interacts with the three human cyclin types T1, H and B1, whereby the binding region of cyclin T1 and the region conferring oligomerization of pUL97 were both assigned to amino acids 231–280. Here, we addressed the question of whether recombinant HCMVs harboring deletions in this region were impaired in cyclin interaction, kinase functionality or viral replication. To this end, recombinant HCMVs were generated by traceless BACmid mutagenesis and were phenotypically characterized using a methodological platform based on qPCR, coimmunoprecipitation, in vitro kinase assay (IVKA), Phos-tag Western blot and confocal imaging analysis. Combined data illustrate the following: (i) infection kinetics of all three recombinant HCMVs, i.e., ORF-UL97 ∆231–255, ∆256–280 and ∆231–280, showed impaired replication efficiency compared to the wild type, amongst which the largest deletion exhibited the most pronounced defect; (ii) specifically, this mutant ∆231–280 showed a loss of interaction with cyclin T1, as demonstrated by CoIP and confocal imaging; (iii) IVKA and Phos-tag analyses revealed strongly affected kinase activity for ∆231–280, with strong impairment of both autophosphorylation and substrate phosphorylation, but less pronounced impairments for ∆231–255 and ∆256–280; and (iv) a bioinformatic assessment of the pUL97–cyclin T1 complex led to the refinement of our current binding model. Thus, the results provide initial evidence for the functional importance of the pUL97–cyclin interaction concerning kinase activity and viral replication fitness.

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Section: Methodsmentioning

confidence: 99%

Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97

Schütz

Steingruber

Socher

et al. 2021

Viruses

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“…Primary human foreskin fibroblasts (HFFs, own repository of primary cell cultures) and HFF-UL50 [19] were propagated as described previously [8,19]. HeLa and 293T cells were cultivated at 37 • C, 5% CO 2 and 80% humidity using Dulbecco's modified Eagle's medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1× GlutaMAX™ (35050038, Thermo Fisher Scientific), 10 µg/mL gentamicin (22185.03, SERVA, Heidelberg, Germany) and 10% fetal bovine serum (FBS, F7524, Sigma Aldrich, St. Louis, MO, USA).…”

Section: Cell Culture Virus Infection and Transient Transfectionmentioning

confidence: 99%

“…HeLa and 293T cells were cultivated at 37 • C, 5% CO 2 and 80% humidity using Dulbecco's modified Eagle's medium (DMEM, 11960044, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1× GlutaMAX™ (35050038, Thermo Fisher Scientific), 10 µg/mL gentamicin (22185.03, SERVA, Heidelberg, Germany) and 10% fetal bovine serum (FBS, F7524, Sigma Aldrich, St. Louis, MO, USA). HCMV strain AD169 variant UK, HCMV ∆UL50 (described in [19]) and phosphosite mutants generated in this study were propagated as stocks and titrated in the respective cell lines as described previously [20,21]. Infection experiments were performed at indicated multiplicity of infection (MOI) using parental or recombinant HCMVs.…”

Section: Cell Culture Virus Infection and Transient Transfectionmentioning

confidence: 99%

“…For the generation of HCMV UL50 phosphosite mutations, the BAC HB15/AD169∆UL50 (described in [19]) (Figure S1A) was used for the insertion of UL50 phosphosite UTCs by a two-step markerless Red recombination system as described previously (Figure S1C; [19,26]). In a first step the pUL50 phosphosite UTCs were inserted into HB15/AD169∆UL50 as described before and in antisense direction (Figure S1C; [22,26,27]).…”

Section: Generation Of Recombinant Hcmvmentioning

confidence: 99%

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Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation

Häge

Sonntag

Svrlanska

et al. 2021

Viruses

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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.

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“…Cell culture medium was supplemented with 1x GlutaMAX TM (35050038, ThermoFisher Scientific), 10 μg/ml gentamicin and 10 % fetal bovine serum (FBS, F7524, Sigma-Aldrich). Primary human foreskin fibroblasts (HFFs, own repository of primary cell cultures) were propagated as described previously (25,52) and infectious stocks of HCMV strain AD169-GFP and VZV strain Oka-GFP were produced on HFFs and used for infection experiments according to standard procedures (53)(54)(55).…”

Section: Cell Culture Virus Stocks and Infection Experimentsmentioning

confidence: 99%

The crystal structure of the varicella zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

Schweininger

Kriegel

Häge

et al. 2021

Preprint

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Varicella zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. Here, the crystal structure of the VZV Orf24-Orf27 complex is described, representing the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled capsids from the nucleus. While previous studies have primarily emphasized the finding that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focusses on subfamily-specific structural and functional features that help explain the differences in the autologous versus nonautologous interaction patterns observed for NEC formation across herpesviruses. CoIP and confocal imaging data show that Orf24-Orf27 complex formation displays some promiscuity in a herpesvirus subfamily-restricted manner. At the same time, analysis of the NEC formation thermodynamic parameters of three prototypical α-, β- and γ-herpesviruses, i.e. VZV, human cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) reveals highly similar binding affinities for the autologous interaction with some specific differences in the enthalpy and entropy terms. Computational alanine scanning and structural comparisons highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses. Combined, these data allow to explain the distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings might prove highly valuable when attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates.

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A quantitative nuclear egress assay to investigate the nucleocytoplasmic capsid release of human cytomegalovirus

Cited by 15 publications

References 25 publications

Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97

Functional Relevance of the Interaction between Human Cyclins and the Cytomegalovirus-Encoded CDK-Like Protein Kinase pUL97

Phenotypical Characterization of the Nuclear Egress of Recombinant Cytomegaloviruses Reveals Defective Replication upon ORF-UL50 Deletion but Not pUL50 Phosphosite Mutation

The crystal structure of the varicella zoster Orf24-Orf27 nuclear egress complex spotlights multiple determinants of herpesvirus subfamily specificity

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