Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components

Nuclear egress of herpesvirus capsids through the nuclear envelope is mediated by the multimeric nuclear egress complex (NEC). The human cytomegalovirus (HCMV) core NEC is defined by an interaction between the membrane- anchored pUL50 and its nuclear co-factor pUL53, tightly associated through heterodimeric corecruitment to the nuclear envelope. Cellular proteins, such as p32/gC1qR, emerin and protein kinase C (PKC), are recruited by direct interaction with pUL50 for the multimeric extension of the NEC. As a functionally important event, the recruitment of both viral and cellular protein kinases leads to site- specific lamin phosphorylation and nuclear lamina disassembly. In this study, interaction domains within pUL50 for its binding partners were defined by co-immunoprecipitation. The interaction domain for pUL53 is located within the pUL50 N-terminus (residues 10-169), interaction domains for p32/gC1qR (100-358) and PKC (100-280) overlap in the central part of pUL50, and the interaction domain for emerin is located in the C-terminus (265-397). Moreover, expression and formation of core NEC proteins at the nuclear rim were consistently detected in cells permissive for productive HCMV replication, including two trophoblast-cell lines. Importantly, regular nuclear-rim formation of the core NEC was blocked by inhibition of cyclin-dependent kinase (CDK) activity. In relation to the recently published crystal structure of the HCMV core NEC, our findings result in a refined view of NEC assembly. In particular, we suggest that CDKs may play an important regulatory role in NEC formation during HCMV replica

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

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

Charakterisierung eines multimeren Proteinkomplexes, der den nukleären Kapsidexport des humanen Cytomegalovirus steuert

Human cytomegalovirus (HCMV) has developed a replication strategy that is well adapted to conditions of the host cell. Notably, after nuclear capsid assembly, HCMV capsids traverse the nuclear envelope for nuclear egress. In this regard, the phosphorylation-mediated disassembly of the nuclear lamina is believed to be a prerequisite for the budding of viral capsids through the nuclear membrane. Although the interplay between the nuclear lamina-associated viral proteins pUL50 and pUL53 with viral and cellular protein kinases has been considered for several years, the exact molecular events for HCMV nuclear egress were still unclear. In this thesis, yeast two-hybrid and coimmunoprecipitation analyses were applied to detect the interaction of pUL50 with three proteins. Hereby, partly overlapping interaction domains could be identified and pUL50-associated protein complexes could be isolated using lysates of transiently transfected and HCMV-infected cells. As illustrated by immunofluorescence costaining analyses, the direct interaction of pUL50 with protein kinase C (PKC) could be confirmed and recruitment of PKC to the nuclear rim of transiently transfected cells was demonstrated. Interestingly, viral protein kinase pUL97 was also recruited by pUL50, although through a different mechanism lacking direct interaction. Experimental evidence pointed to an indirect bridging of pUL50-associated proteins mediated by the cellular adaptor p32. Combined, these data suggested the formation of a nuclear egress complex (NEC) which includes at least pUL50, pUL53, p32, the lamin B receptor and the two protein kinases pUL97 and PKC. Reports describing the ability of pUL97 and PKC to phosphorylate several types of lamins, coincided with the finding in this study that pUL97 and PKC had the potential to induce distinct, punctate lamina-depleted areas at the nuclear periphery in transiently transfected as well as HCMV-infected cells. Using a recombinant HCMV, the direct transition of GFP-labeled viral capsids through these areas could be visualized. To discover the molecular mechanism driving the NEC-mediated nuclear lamina destabilization, bioinformatical analyses were performed concerning the pUL97-dependent phosphorylation of lamin A/C at serine 22. As an important finding, phosphorylation at this specific serine generated a putative binding motif for the peptidyl-prolyl cis/trans isomerase Pin1. Interaction between Pin1 and lamin A was confirmed in HMCV-infected fibroblasts. Furthermore, the physiological localization of Pin1 was altered, leading to recruitment of Pin1 to the nuclear lamina late during infection. The local increase of Pin1 isomerase activity was suggested to promote conformational modulation of nuclear lamins. Taken together, this study strongly suggests the formation of a viral-cellular NEC which is required for the phosphorylation-triggered destabilization of the nuclear lamina during HCMV nuclear egress.Das humane Cytomegalovirus (HCMV) hat eine Replikationsstrategie entwickelt, die sehr gut an die regulatorischen Prozesse der Wirtszelle angepasst ist. Insbesondere ist es für den viralen Kernexport notwendig, dass die neuformierten Kapside die Kernmembran überwinden. Diesbezüglich wird die phosphorylierungsabhängige Destabilisierung der nukleären Lamina als Vorraussetzung für das Ausschleusen der viralen Kapside durch die Kernmembran betrachtet. Obwohl die Lamina-assoziierten viralen Proteine pUL50 und pUL53 über Jahre hinweg analysiert sowie deren Wechselwirkung mit viralen als auch zellulären Faktoren diskutiert worden waren, blieben Details der molekularen Vorgänge des HCMV-Kernexports im Unklaren. In der vorliegenden Arbeit, wurde die Interaktion von pUL50 mit drei Proteinen durch das Hefe-Zwei-Hybrid-System und Koimmunpräzipitationsanalysen nachgewiesen. Hierbei wurden überlappende Interaktionsbereiche für diese Bindungspartner identifiziert und pUL50-assoziierte Proteinkomplexe konnten sowohl in transfizierten als auch in HCMV-infizierten Zellen isoliert werden. Mit Hilfe von Immunfluoreszenz-Mehrfachfärbungen konnte die Interaktion zwischen pUL50 und der Proteinkinase C (PKC) bestätigt sowie eine Rekrutierung von PKC an die Zellkernperipherie demonstriert werden. Interessanterweise wurde die virale Proteinkinase pUL97 ebenfalls von pUL50 rekrutiert, wobei dies nicht durch eine direkte Interaktion vermittelt wurde. Experimentelle Hinweise deuteten auf eine indirekte Adaptorwirkung durch den zellulären Faktor p32 hin. Diese Daten legen die Bildung eines nukleären Egress-Komplexes (NEC) nahe, der pUL50, pUL53, p32, den Lamin B-Rezeptor und die Proteinkinasen pUL97 und PKC beinhaltet. Berichte über die Phosphorylierung verschiedener Lamintypen durch pUL97 und PKC, deckten sich mit der Beobachtung dieser Arbeit, dass beide Proteinkinasen in transfizierten als auch in HCMV-infizierten Zellen ausgeprägte, Lamin-freie Bereiche am Rande der Kernhülle induzieren können. Mit Hilfe eines rekombinanten Virus konnten GFP-markierte HCMV Kapside beim Durchqueren dieser Bereiche beobachtet werden. Um den molekularen Mechanismus der NEC-vermittelten Destabilisierung der nukleären Lamina aufzuklären, wurde die pUL97-abhängige Phosphorylierung von Serin 22 des Lamin A/C mittels Bioinformatik-Methoden untersucht. Ein wichtiger Befund bestand in der Entdeckung eines mutmaßlichen Bindemotivs für die Peptidyl-Prolyl-cis/trans-Isomerase Pin1 an diesem phosphorylierten Serin. Die Interaktion zwischen Pin1 und Lamin A konnte in HCMV-infizierten Fibroblasten erstmals demonstriert werden. Im Weiteren wurde die intrazelluläre Lokalisation von Pin1 so verändert, dass in der späten Phase der Infektion eine Rekrutierung von Pin1 an die nukleäre Lamina nachweisbar war. Die Pin1-Isomerase-Aktivität könnte somit für eine Restrukturierung der nukleären Lamine maßgeblich sein. Zusammenfassend liefert diese Studie einen deutlichen Hinweis für die Bildung eines viral-zellulären NEC, welcher für die phosphorylierungsabhängige Auflösung der nukleären Lamina während des HCMV-Kernexports notwendig ist

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein

Update on the management of gestational trophoblastic neoplasia

<p>MRC-5 cells were untreated or transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54B), UL97 (siUL97A), and UL122/123 (siUL122B) and infected with HCMV AD169 (0.001 pfu/cell) at 24 hours post siRNA transfection. Cells were harvested 1, 4 and 7 days post infection and HCMV immediate-early (IE1p72), early (pp65), and early-late (gB) protein expression measured using immunofluorescence and quantitative image analysis. Data presented relative to HCMV-infected untreated cells and represent mean of 10 fields of view ± SD from duplicate biological experiments.</p

siRNA efficacy at inhibiting plasmid-derived HCMV protein expression.

<p>293T cells were transfected with 50 µg of plasmid expressing the HCMV siRNA target transcript 24 hours post siRNA transfection. (A) Cells were harvested 72 hours post plasmid transfection and protein expression measured using western blot. (B) Protein expression in siRNA treated and plasmid transfected cells relative to cells treated with control scrambled siRNA (siSc) measured using densitometry. Results are from duplicate biological experiments and densitometry results presented as mean ± SD.</p

siRNA efficacy at inhibiting HCMV protein expression during a single round of HCMV replication at high multiplicity of infection.

<p>MRC-5 cells were transfected with 50 nM of non-specific scrambled control siRNA (siSc) or siRNAs targeting HCMV transcripts UL54 (siUL54A/B), UL97 (siUL97A/B), and UL122/123 (siUL122A/B) followed by infection with HCMV (1 pfu/cell) at 24 hours post transfection. (A) Cells were harvested 24, 48 and 72 hours post infection and HCMV immediate-early (IE1p72 and IE2p86), early (pp65), early-late (pUL97) and true late (MCP) protein expression measured using western blot. (B) Protein expression in UL54A/B, UL97A/B and UL122A/B siRNA treated and HCMV-infected cells relative to cells treated with scrambled siRNA (siSc) measured using densitometry. Results derived from two representative samples from triplicate biological experiments and densitometry results presented as mean ± SD.</p

Human Cytomegalovirus Nuclear Egress Proteins Ectopically Expressed in the Heterologous Environment of Plant Cells are Strictly Targeted to the Nuclear Envelope

In all eukaryotic cells, the nucleus forms a prominent cellular compartment containing the cell’s nuclear genome. Although structurally similar, animal and plant nuclei differ substantially in details of their architecture. One example is the nuclear lamina, a layer of tightly interconnected filament proteins (lamins) underlying the nuclear envelope of metazoans. So far no orthologous lamin genes could be detected in plant genomes and putative lamin-like proteins are only poorly described in plants. To probe for potentially conserved features of metazoan and plant nuclear envelopes, we ectopically expressed the core nuclear egress proteins of human cytomegalovirus pUL50 and pUL53 in plant cells. pUL50 localizes to the inner envelope of metazoan nuclei and recruits the nuclear localized pUL53 to it, forming heterodimers. Upon expression in plant cells, a very similar localization pattern of both proteins could be determined. Notably, pUL50 is specifically targeted to the plant nuclear envelope in a rim-like fashion, a location to which coexpressed pUL53 becomes strictly corecruited from its initial nucleoplasmic distribution. Using pUL50 as bait in a yeast two-hybrid screening, the cytoplasmic re-initiation supporting protein RISP could be identified. Interaction of pUL50 and RISP could be confirmed by coexpression and coimmunoprecipitation in mammalian cells and by confocal laser scanning microscopy in plant cells, demonstrating partial pUL50-RISP colocalization in areas of the nuclear rim and other intracellular compartments. Thus, our study provides strong evidence for conserved structural features of plant and metazoan nuclear envelops and identifies RISP as a potential pUL50-interacting plant protein