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-, ß- 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.

The peptidyl-prolyl cis/trans isomerase Pin1 interacts with three early regulatory proteins of human cytomegalovirus.

Human cytomegalovirus (HCMV) is a ubiquitous human pathogen of high clinical relevance. Despite intensive research of virus-host interaction, crucial details still remain unknown. In this study, the role of the cellular peptidyl-prolyl cis/trans isomerase Pin1 during HCMV infection was investigated. Pin1 is able to recognize phosphorylated serine/threonine-proline motifs and regulates the structural conformation, stability and function of its substrates. Concerning HCMV replication, our recent studies revealed that Pin1 plays an important role in viral nuclear egress by contributing to the depletion of the nuclear lamina at distinct sites through the cis/trans conversion of lamin proteins. Here, novel data illustrate the HCMV-induced upregulation of Pin1 including various cell types being permissive, semi-permissive or non-permissive for productive HCMV replication. Addressing the question of functional impact, Pin1 knock-out (KO) did not show a measurable effect on viral protein expression, at least when assessed by Western blot analysis. Applying highly sensitive methods of qPCR and plaque titration, a pharmacological inhibition of Pin1 activity, however, led to a significant decrease of viral genome equivalents and production of infectious virus, respectively. When focusing on the identification of viral proteins interacting with Pin1 by various coimmunoprecipitation (CoIP) settings, we obtained positive signals for (i) the core nuclear egress complex protein pUL50, (ii) the viral mRNA export factor pUL69 and (iii) the viral DNA polymerase processivity factor pUL44. Confocal immunofluorescence analysis focusing on partial colocalization between Pin1 and the coexpressed viral proteins pUL50, pUL69 or pUL44, respectively, was consistent with the CoIP experiments. Mapping experiments, using transient expression constructs for a series of truncated protein versions and specific replacement mutants, revealed a complex pattern of Pin1 interaction with these three early regulatory HCMV proteins. Data suggest a combination of different modes of Pin1 interactions, involving both classical phosphorylation-dependent Pin1 binding motifs and additional phosphorylation-independent binding sites. Combined, these results support the concept that Pin1 may play an important role in several stages of HCMV infection, thus determining viral replicative efficiency.

Patterns of Autologous and Nonautologous Interactions Between Core Nuclear Egress Complex (NEC) Proteins of α-, ß- and γ-Herpesviruses.

Nuclear egress is a regulated process shared by α-, ß- 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.

Phosphosite Analysis of the Cytomegaloviral mRNA Export Factor pUL69 Reveals Serines with Critical Importance for Recruitment of Cellular Proteins Pin1 and UAP56/URH49.

Human cytomegalovirus (HCMV) encodes the viral mRNA export factor pUL69, which facilitates the cytoplasmic accumulation of mRNA via interaction with the cellular RNA helicase UAP56 or URH49. We reported previously that pUL69 is phosphorylated by cellular CDKs and the viral CDK-like kinase pUL97. Here, we set out to identify phosphorylation sites within pUL69 and to characterize their importance. Mass spectrometry-based phosphosite mapping of pUL69 identified 10 serine/threonine residues as phosphoacceptors. Surprisingly, only a few of these sites localized to the N terminus of pUL69, which could be due to the presence of additional posttranslational modifications, like arginine methylation. As an alternative approach, pUL69 mutants with substitutions of putative phosphosites were analyzed by Phos-tag SDS-PAGE. This demonstrated that serines S46 and S49 serve as targets for phosphorylation by pUL97. Furthermore, we provide evidence that phosphorylation of these serines mediates cis/trans isomerization by the prolyl isomerase Pin1, thus forming a functional Pin1 binding motif. Surprisingly, while abrogation of the Pin1 motif did not affect the replication of recombinant cytomegaloviruses, mutation of serines next to the interaction site for UAP56/URH49 strongly decreased viral replication. This was correlated with a loss of UAP56/URH49 recruitment. Intriguingly, the critical serines S13 and S15 were located within a sequence resembling the UAP56 binding motif (UBM) of cellular mRNA adaptor proteins like REF and UIF. We propose that betaherpesviral mRNA export factors have evolved an extended UAP56/URH49 recognition sequence harboring phosphorylation sites to increase their binding affinities. This may serve as a strategy to successfully compete with cellular mRNA adaptor proteins for binding to UAP56/URH49.IMPORTANCE The multifunctional regulatory protein pUL69 of human cytomegalovirus acts as a viral RNA export factor with a critical role in efficient replication. Here, we identify serine/threonine phosphorylation sites for cellular and viral kinases within pUL69. We demonstrate that the pUL97/CDK phosphosites within alpha-helix 2 of pUL69 are crucial for its cis/trans isomerization by the cellular protein Pin1. Thus, we identified pUL69 as the first HCMV-encoded protein that is phosphorylated by cellular and viral serine/threonine kinases in order to serve as a substrate for Pin1. Furthermore, our study revealed that betaherpesviral mRNA export proteins contain extended binding motifs for the cellular mRNA adaptor proteins UAP56/URH49 harboring phosphorylated serines that are critical for efficient viral replication. Knowledge of the phosphorylation sites of pUL69 and the processes regulated by these posttranslational modifications is important in order to develop antiviral strategies based on a specific interference with pUL69 phosphorylation.

Differential upregulation of host cell protein kinases by the replication of α-, ß- and γ-herpesviruses provides a signature of virus-specific signalling.

Infections with human herpesviruses share several molecular characteristics, but the diversified medical outcomes are distinct to viral subfamilies and species. Notably, both clinical and molecular correlates of infection are a challenging field and distinct patterns of virus-host interaction have rarely been defined; this study therefore focuses on the search for virus-specific molecular indicators. As previous studies have demonstrated the impact of herpesvirus infections on changes in host signalling pathways, we illustrate virus-modulated expression levels of individual cellular protein kinases. Current data reveal (i) α-, ß- and γ-herpesvirus-specific patterns of kinase modulation as well as (ii) differential levels of up-/downregulated kinase expression and phosphorylation, which collectively suggest (iii) defined signalling patterns specific for the various viruses (VSS) that may prove useful for defining molecular indicators. Combined, the study confirms the correlation between herpesviral replication and modulation of signalling kinases, possibly exploitable for the in vitro characterization of viral infections.

Human cytomegalovirus overcomes SAMHD1 restriction in macrophages via pUL97.

The host restriction factor sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is an important component of the innate immune system. By regulating the intracellular nucleotide pool, SAMHD1 influences cell division and restricts the replication of viruses that depend on high nucleotide concentrations. Human cytomegalovirus (HCMV) is a pathogenic virus with a tropism for non-dividing myeloid cells, in which SAMHD1 is catalytically active. Here we investigate how HCMV achieves efficient propagation in these cells despite the SAMHD1-mediated dNTP depletion. Our analysis reveals that SAMHD1 has the capability to suppress HCMV replication. However, HCMV has evolved potent countermeasures to circumvent this block. HCMV interferes with SAMHD1 steady-state expression and actively induces SAMHD1 phosphorylation using the viral kinase pUL97 and by hijacking cellular kinases. These actions convert SAMHD1 to its inactive phosphorylated form. This mechanism of SAMHD1 inactivation by phosphorylation might also be used by other viruses to overcome intrinsic immunity.

A viral kinase counteracts in vivo restriction of murine cytomegalovirus by SAMHD1.

The deoxynucleotide triphosphate (dNTP) hydrolase SAMHD1 inhibits retroviruses in non-dividing myeloid cells. Although antiviral activity towards DNA viruses has also been demonstrated, the role of SAMHD1 during cytomegalovirus (CMV) infection remains unclear. To determine the impact of SAMHD1 on the replication of CMV, we used murine CMV (MCMV) to infect a previously established SAMHD1 knockout mouse model and found that SAMHD1 inhibits the replication of MCMV in vivo. By comparing the replication of MCMV in vitro in myeloid cells and fibroblasts from SAMHD1-knockout and control mice, we found that the viral kinase M97 counteracts SAMHD1 after infection by phosphorylating the regulatory residue threonine 603. The phosphorylation of SAMHD1 in infected cells correlated with a reduced level of dNTP hydrolase activity and the loss of viral restriction. Together, we demonstrate that SAMHD1 acts as a restriction factor in vivo and we identify the M97-mediated phosphorylation of SAMHD1 as a previously undescribed viral countermeasure.

Human cytomegalovirus utilises cellular dual-specificity tyrosine phosphorylation-regulated kinases during placental replication.

INTRODUCTION: Congenital cytomegalovirus (HCMV) infection may cause significant fetal malformation and in severe cases fetal and neonatal death. Fetal injury may be caused indirectly by the placental response to infection. Dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs) have recently been identified as critical kinases for HCMV replication. In this study we provide first evidence that DYRK1A and DYRK1B are utilised during HCMV placental replication. METHODS: DYRK expression was investigated in AD169- and Merlin-infected TEV-1 trophoblast cells, ex vivo placental explants and naturally infected clinical placentae by immunofluorescence, western blot, co-immunoprecipitation and RT-qPCR. RESULTS: HCMV-infected placental cells showed accumulation and re-localisation of DYRK1A and DYRK1B protein to areas of cytoplasmic virion assembly complexes and nuclear viral replication compartments, respectively. This accumulation was a result of upregulated DYRK1A/B protein expression with HCMV inducing up to a 5.3-fold increase in DYRK1A and up to a 4.7-fold increase in DYRK1B protein, relative to mock-infected TEV-1 cells (p < 0.0001). Increased DYRK protein expression was correlated with DYRK1A/B mRNA upregulation, with HCMV-infected cells showing up to a 3.7-fold increase and 2.9-fold increase in DYRK1A and DYRK1B mRNA levels respectively (p < 0.05). Protein-protein interactions were detected between DYRK1A/1B complexes and HCMV immediate early IE2p86, early pp65 and pUL44 and late pp150 proteins. Treatment of HCMV-infected TEV-1 cells and placental explants with DYRK inhibitors significantly inhibited HCMV replication (p < 0.05) indicating these cellular kinases are required during HCMV placental replication. CONCLUSION: HCMV modulates cellular DYRKs during placental replication which may have implications for congenital HCMV pathogenesis and represent promising antiviral targets.

Transmembrane Protein pUL50 of Human Cytomegalovirus Inhibits ISGylation by Downregulating UBE1L.

Interferon-stimulated gene 15 (ISG15) encodes a ubiquitin-like protein that can be conjugated to proteins via an enzymatic cascade involving the E1, E2, and E3 enzymes. ISG15 expression and protein ISGylation modulate viral infection; however, the viral mechanisms regulating the function of ISG15 and ISGylation are not well understood. We recently showed that ISGylation suppresses the growth of human cytomegalovirus (HCMV) at multiple steps of the virus life cycle and that the virus-encoded pUL26 protein inhibits protein ISGylation. In this study, we demonstrate that the HCMV UL50-encoded transmembrane protein, a component of the nuclear egress complex, also inhibits ISGylation. pUL50 interacted with UBE1L, an E1-activating enzyme for ISGylation, and (to a lesser extent) with ISG15, as did pUL26. However, unlike pUL26, pUL50 caused proteasomal degradation of UBE1L. The UBE1L level induced in human fibroblast cells by interferon beta treatment or virus infection was reduced by pUL50 expression. This activity of pUL50 involved the transmembrane (TM) domain within its C-terminal region, although pUL50 could interact with UBE1L in a manner independent of the TM domain. Consistently, colocalization of pUL50 with UBE1L was observed in cells treated with a proteasome inhibitor. Furthermore, we found that RNF170, an endoplasmic reticulum (ER)-associated ubiquitin E3 ligase, interacted with pUL50 and promoted pUL50-mediated UBE1L degradation via ubiquitination. Our results demonstrate a novel role for the pUL50 transmembrane protein of HCMV in the regulation of protein ISGylation.IMPORTANCE Proteins can be conjugated covalently by ubiquitin or ubiquitin-like proteins, such as SUMO and ISG15. ISG15 is highly induced in viral infection, and ISG15 conjugation, termed ISGylation, plays important regulatory roles in viral growth. Although ISGylation has been shown to negatively affect many viruses, including human cytomegalovirus (HCMV), viral countermeasures that might modulate ISGylation are not well understood. In the present study, we show that the transmembrane protein encoded by HCMV UL50 inhibits ISGylation by causing proteasomal degradation of UBE1L, an E1-activating enzyme for ISGylation. This pUL50 activity requires membrane targeting. In support of this finding, RNF170, an ER-associated ubiquitin E3 ligase, interacts with pUL50 and promotes UL50-mediated UBE1L ubiquitination and degradation. Our results provide the first evidence, to our knowledge, that viruses can regulate ISGylation by directly targeting the ISGylation E1 enzyme.

Human Cytomegalovirus Nuclear Capsids Associate with the Core Nuclear Egress Complex and the Viral Protein Kinase pUL97.

The nuclear phase of herpesvirus replication is regulated through the formation of regulatory multi-component protein complexes. Viral genomic replication is followed by nuclear capsid assembly, DNA encapsidation and nuclear egress. The latter has been studied intensely pointing to the formation of a viral core nuclear egress complex (NEC) that recruits a multimeric assembly of viral and cellular factors for the reorganization of the nuclear envelope. To date, the mechanism of the association of human cytomegalovirus (HCMV) capsids with the NEC, which in turn initiates the specific steps of nuclear capsid budding, remains undefined. Here, we provide electron microscopy-based data demonstrating the association of both nuclear capsids and NEC proteins at nuclear lamina budding sites. Specifically, immunogold labelling of the core NEC constituent pUL53 and NEC-associated viral kinase pUL97 suggested an intranuclear NEC-capsid interaction. Staining patterns with phospho-specific lamin A/C antibodies are compatible with earlier postulates of targeted capsid egress at lamina-depleted areas. Important data were provided by co-immunoprecipitation and in vitro kinase analyses using lysates from HCMV-infected cells, nuclear fractions, or infectious virions. Data strongly suggest that nuclear capsids interact with pUL53 and pUL97. Combined, the findings support a refined concept of HCMV nuclear trafficking and NEC-capsid interaction.

Protein kinases responsible for the phosphorylation of the nuclear egress core complex of human cytomegalovirus.

Nuclear egress of herpesvirus capsids is mediated by a multi-component nuclear egress complex (NEC) assembled by a heterodimer of two essential viral core egress proteins. In the case of human cytomegalovirus (HCMV), this core NEC is defined by the interaction between the membrane-anchored pUL50 and its nuclear cofactor, pUL53. NEC protein phosphorylation is considered to be an important regulatory step, so this study focused on the respective role of viral and cellular protein kinases. Multiply phosphorylated pUL50 varieties were detected by Western blot and Phos-tag analyses as resulting from both viral and cellular kinase activities. In vitro kinase analyses demonstrated that pUL50 is a substrate of both PKCα and CDK1, while pUL53 can also be moderately phosphorylated by CDK1. The use of kinase inhibitors further illustrated the importance of distinct kinases for core NEC phosphorylation. Importantly, mass spectrometry-based proteomic analyses identified five major and nine minor sites of pUL50 phosphorylation. The functional relevance of core NEC phosphorylation was confirmed by various experimental settings, including kinase knock-down/knock-out and confocal imaging, in which it was found that (i) HCMV core NEC proteins are not phosphorylated solely by viral pUL97, but also by cellular kinases; (ii) both PKC and CDK1 phosphorylation are detectable for pUL50; (iii) no impact of PKC phosphorylation on NEC functionality has been identified so far; (iv) nonetheless, CDK1-specific phosphorylation appears to be required for functional core NEC interaction. In summary, our findings provide the first evidence that the HCMV core NEC is phosphorylated by cellular kinases, and that the complex pattern of NEC phosphorylation has functional relevance.

The human cytomegalovirus nuclear egress complex unites multiple functions: Recruitment of effectors, nuclear envelope rearrangement, and docking to nuclear capsids.

BACKGROUND: Nuclear replication represents a common hallmark of herpesviruses achieved by a number of sequentially unrolled regulatory processes. A rate-limiting step is provided by nucleo-cytoplasmic capsid export, for which a defined multiregulatory protein complex, namely, the nuclear egress complex (NEC), is assembled comprising both viral and cellular components. The NEC regulates at least 3 aspects of herpesviral nuclear replication: (1) multimeric recruitment of NEC-associated effector proteins, (2) reorganization of the nuclear lamina and membranes, and (3) the docking to nuclear capsids. Here, we review published data and own experimental work that characterizes the NEC of HCMV and other herpesviruses. METHODS: A systematic review of information on nuclear egress of HCMV compared to selected alpha-, beta-, and gamma-herpesviruses: proteomics-based approaches, high-resolution imaging techniques, and functional investigations. RESULTS: A large number of reports on herpesviral NECs have been published during the last two decades, focusing on protein-protein interactions, nuclear localization, regulatory phosphorylation, and functional validation. The emerging picture provides an illustrated example of well-balanced and sophisticated protein networking in virus-host interaction. CONCLUSIONS: Current evidence refined the view about herpesviral NECs. Datasets published for HCMV, murine CMV, herpes simplex virus, and Epstein-Barr virus illustrate the marked functional consistency in the way herpesviruses achieve nuclear egress. However, this compares with only limited sequence conservation of core NEC proteins and a structural conservation restricted to individual domains. The translational use of this information might help to define a novel antiviral strategy on the basis of NEC-directed small molecules.

Inhibitors of dual-specificity tyrosine phosphorylation-regulated kinases (DYRK) exert a strong anti-herpesviral activity.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of (val)ganciclovir therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy of antiviral treatment is based on the exploitation of cell-directed signaling, e. g. pathways with a known relevance for carcinogenesis and tumor drug development. Here we describe a principle for putative antiviral drugs based on targeting dual-specificity tyrosine phosphorylation-regulated kinases (DYRKs). DYRKs constitute an evolutionarily conserved family of protein kinases with key roles in the control of cell proliferation and differentiation. Members of the DYRK family are capable of phosphorylating a number of substrate proteins, including regulators of the cell cycle, e.g. DYRK1B can induce cell cycle arrest, a critical step for the regulation of HCMV replication. Here we provide first evidence for a critical role of DYRKs during viral replication and the high antiviral potential of DYRK inhibitors (SC84227, SC97202 and SC97208, Harmine and AZ-191). Using established replication assays for laboratory and clinically relevant strains of HCMV, concentration-dependent profiles of inhibition were obtained. Mean inhibitory concentrations (EC50) of 0.98 ± 0.08 µM/SC84227, 0.60 ± 0.02 µM/SC97202, 6.26 ± 1.64 µM/SC97208, 0.71 ± 0.019 µM/Harmine and 0.63 ± 0.23 µM/AZ-191 were determined with HCMV strain AD169-GFP for the infection of primary human fibroblasts. A first analysis of the mode of antiviral action suggested a block of viral replication at the early-late stage of HCMV gene expression. Moreover, rhesus macaque cytomegalovirus (RhCMV), varicella-zoster virus (VZV) and herpes simplex virus (HSV-1) showed a similarly high sensitivity to these compounds. Thus, we conclude that DYRK signaling represents a promising target pathway for the development of novel anti-herpesviral strategies.

The Prolyl Isomerase Pin1 Promotes the Herpesvirus-Induced Phosphorylation-Dependent Disassembly of the Nuclear Lamina Required for Nucleocytoplasmic Egress.

The nuclear lamina lines the inner nuclear membrane providing a structural framework for the nucleus. Cellular processes, such as nuclear envelope breakdown during mitosis or nuclear export of large ribonucleoprotein complexes, are functionally linked to the disassembly of the nuclear lamina. In general, lamina disassembly is mediated by phosphorylation, but the precise molecular mechanism is still not completely understood. Recently, we suggested a novel mechanism for lamina disassembly during the nuclear egress of herpesviral capsids which involves the cellular isomerase Pin1. In this study, we focused on mechanistic details of herpesviral nuclear replication to demonstrate the general importance of Pin1 for lamina disassembly. In particular, Ser22-specific lamin phosphorylation consistently generates a Pin1-binding motif in cells infected with human and animal alpha-, beta-, and gammaherpesviruses. Using nuclear magnetic resonance spectroscopy, we showed that binding of Pin1 to a synthetic lamin peptide induces its cis/trans isomerization in vitro. A detailed bioinformatic evaluation strongly suggests that this structural conversion induces large-scale secondary structural changes in the lamin N-terminus. Thus, we concluded that a Pin1-induced conformational change of lamins may represent the molecular trigger responsible for lamina disassembly. Consistent with this concept, pharmacological inhibition of Pin1 activity blocked lamina disassembly in herpesvirus-infected fibroblasts and consequently impaired virus replication. In addition, a phospho-mimetic Ser22Glu lamin mutant was still able to form a regular lamina structure and overexpression of a Ser22-phosphorylating kinase did not induce lamina disassembly in Pin1 knockout cells. Intriguingly, this was observed in absence of herpesvirus infection proposing a broader importance of Pin1 for lamina constitution. Thus, our results suggest a functional model of similar events leading to disassembly of the nuclear lamina in response to herpesviral or inherent cellular stimuli. In essence, Pin1 represents a regulatory effector of lamina disassembly that promotes the nuclear pore-independent egress of herpesviral capsids.

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 replication.

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.

New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK orthologue pUL97.

Cyclin-dependent kinases (CDKs) are multifaceted regulators involved in the replication of human cytomegalovirus. Recently, we demonstrated an interaction of CDK9-cyclin T1 as well as viral CDK orthologue pUL97 with the viral regulator pUL69, thereby leading to pUL69-activating phosphorylation. Here, we demonstrate that colocalization and direct pUL69-cyclin T1 interaction is independent of viral strains and host cell types. In vitro phosphorylation of pUL69 by CDK9 or pUL97 did not occur in a single site-specific manner, but at multiple sites. The previously described fine-speckled nuclear aggregation of pUL69 was assigned to the late phase of viral replication. CDK inhibitors, including a novel inhibitor of the CDK-activating kinase CDK7, massively intensified this fine-speckled accumulation. Interestingly, we also observed spontaneous pUL69 accumulation in the absence of inhibitors at a lower frequency. These findings provide new insight into pUL69 kinase interregulation and emphasize the importance of pUL69 phosphorylation for correct intranuclear localization.

The broad-spectrum antiinfective drug artesunate interferes with the canonical nuclear factor kappa B (NF-κB) pathway by targeting RelA/p65.

Infection with human cytomegalovirus (HCMV) is a serious medical problem, particularly in immunocompromised individuals and neonates. The success of standard antiviral therapy is hampered by low drug compatibility and induction of viral resistance. A novel strategy is based on the exploitation of cell-directed signaling inhibitors. The broad antiinfective drug artesunate (ART) offers additional therapeutic options such as oral bioavailability and low levels of toxic side-effects. Here, novel ART-derived compounds including dimers and trimers were synthesized showing further improvements over the parental drug. Antiviral activity and mechanistic aspects were determined leading to the following statements: (i) ART exerts antiviral activity towards human and animal herpesviruses, (ii) no induction of ART-resistant HCMV mutants occurred in vitro, (iii) chemically modified derivatives of ART showed strongly enhanced anti-HCMV efficacy, (iv) NF-κB reporter constructs, upregulated during HCMV replication, could be partially blocked by ART treatment, (v) ART activity analyzed in stable reporter cell clones indicated an inhibition of stimulated NF-κB but not CREB pathway, (vi) solid-phase immobilized ART was able to bind to NF-κB RelA/p65, and (vii) peptides within NF-κB RelA/p65 represent candidates of ART binding as analyzed by in silico docking and mass spectrometry. These novel findings open new prospects for the future medical use of ART and ART-related drug candidates.

Crystal Structure of the Human Cytomegalovirus pUL50-pUL53 Core Nuclear Egress Complex Provides Insight into a Unique Assembly Scaffold for Virus-Host Protein Interactions.

Nuclear replication of cytomegalovirus relies on elaborate mechanisms of nucleocytoplasmic egress of viral particles. Thus, the role of two essential and conserved viral nuclear egress proteins, pUL50 and pUL53, is pivotal. pUL50 and pUL53 heterodimerize and form a core nuclear egress complex (NEC), which is anchored to the inner nuclear membrane and provides a scaffold for the assembly of a multimeric viral-cellular NEC. Here, we report the crystal structure of the pUL50-pUL53 heterodimer (amino acids 1-175 and 50-292, respectively) at 2.44 Å resolution. Both proteins adopt a globular fold with mixed α and ß secondary structure elements. pUL53-specific features include a zinc-binding site and a hook-like N-terminal extension, the latter representing a hallmark element of the pUL50-pUL53 interaction. The hook-like extension (amino acids 59-87) embraces pUL50 and contributes 1510 Å(2) to the total interface area (1880 Å(2)). The pUL50 structure overall resembles the recently published NMR structure of the murine cytomegalovirus homolog pM50 but reveals a considerable repositioning of the very C-terminal α-helix of pUL50 upon pUL53 binding. pUL53 shows structural resemblance with the GHKL domain of bacterial sensory histidine kinases. A close examination of the crystal structure indicates partial assembly of pUL50-pUL53 heterodimers to hexameric ring-like structures possibly providing additional scaffolding opportunities for NEC. In combination, the structural information on pUL50-pUL53 considerably improves our understanding of the mechanism of HCMV nuclear egress. It may also accelerate the validation of the NEC as a unique target for developing a novel type of antiviral drug and improved options of broad-spectrum antiherpesviral therapy.