Von Ratten und Menschen: Strukturelle und biophysikalische Charakterisierung der immediate-early 1 Proteine aus Ratten- und Humanem Cytomegalievirus

The human cytomegalovirus, HCMV, can inflict potentially life-threatening disease in immunocompromised patients and upon congenital infection. The seroprevalence of HCMV in the adult population is 50 to 99 %, and there is currently no vaccine available. Viral gene expression in infected cells is inhibited by the intrinsic immune defense mechanisms mediated by PML nuclear bodies (PML-NBs) in cell nuclei. HCMV has, however, developed mechanisms to counteract the host's intrinsic immune defense. In particular, the immediate-early protein 1 (IE1) targets the coiled-coil domain of PML (promyelocytic leukemia protein), the main component of PML-NBs, via its all-helical CORE domain and abrogates PML-NB formation by inhibiting de novo modification of PML with the small ubiquitin-like modifier (SUMO). The crystal structure of the first CMV IE1 protein, rhesIE1 from rhesus monkey CMV (RhCMV), was solved in 2014 and provided insight into the molecular architecture of the CMV IE1 CORE domain. It has also been shown that the function and presumably also the structure of IE1 is conserved in primates and primate virus variants, i.e. rhesIE1 or RhCMV are functional or infectious in human cells, respectively. However, subsequent investigations have shown that this is not true when host cells and viruses are less closely related, e.g. when human cells are infected by murine or rat CMV. These observations on functional similarities and differences between primate and rodent CMV variants and the associated IE1 proteins have raised questions regarding structural similarities and differences. In this work, the crystal structures of the CORE domains of human CMV IE1 (humIE1) and rat CMV IE1 (ratIE1) were solved. These structures were used to highlight the conserved structural determinants of the CMV IE1 CORE domain fold and to discuss distinct similarities and differences between primate and rodent virus protein variants. In addition, both proteins are biophysically characterized. Moreover, co-expression experiments with human PML and humIE1 have allowed studying the effect of humIE1 on PML auto-SUMOylation in vitro.Das humane Cytomegalievirus, HCMV, kann bei Patienten mit geschwächtem Immunsystem und nach kongenitaler Infektion potentiell lebensbedrohliche Erkrankungen hervorrufen. Die Seroprävalenz von HCMV beträgt in der erwachsenen Bevölkerung 50 bis 99 %, und es ist aktuell kein Impfstoff verfügbar. Die virale Genexpression in infizierten Zellen wird durch von PML nuclear bodies (PML-NBs) gesteuerte intrinsische Immunabwehrmechanismen inhibiert. HCMV hat allerdings Mechanismen entwickelt, um der intrinschen Immunabwehr des Wirtes entgegenzuwirken. Im Speziellen bindet das immediate-early protein 1 (IE1) die coiled-coil Domäne von PML (promyelocytic leukemia protein), der Hauptkomponente der PML-NBs, über seine komplett helikale CORE Domäne und verhindert die Bildung von PML-NBs durch Verhindern von de novo Modifikation von PML mit dem small ubiquitin-like modifier (SUMO). Die erste Kristallstruktur eines CMV IE1 Proteins, rhesIE1 aus Rhesusaffen Cytomegalievirus (RhCMV), wurde 2014 gelöst und lieferte Einblick in die molekulare Architektur der CMV IE1 CORE Domäne. Es wurde außerdem gezeigt, dass die Funktion und möglicherweise auch die Struktur von IE1 in Primaten und Primatenvirus-Varianten konserviert ist, das heißt, dass rhesIE1 und RhCMV in humanen Zellen funktional beziehungsweise infektiös sind. Spätere Untersuchen haben allerdings gezeigt, dass dies nicht der Fall ist, wenn Wirtszellen und Viren weniger nah verwandt sind, beispielsweise bei einer Infektion von humanen Zellen mit murinem oder Ratten-Cytomegalievirus. Diese Beobachtungen von Gemeinsamkeiten und Unterschieden zwischen Primaten- und Nager-CMV Varianten und der betreffenden IE1-Proteine haben Fragen bezüglich struktureller Gemeinsamkeiten und Unterschiede aufgeworfen. In dieser Arbeit wurden die Kristallstrukturen der CORE Domänen von HCMV IE1 (humIE1) und Ratten-CMV IE1 (ratIE1) gelöst. Diese Strukturen wurden verwendet, um die konservierten strukturellen Bestimmungsfaktoren der Faltung der CMV IE1 CORE Domäne hervorzuheben und deutliche Gemeinsamkeiten und Unterschiede zwischen den Proteinvarianten aus Primaten- und Nagerviren zu diskutieren. Zusätzlich wurden beide Proteine biophysikalisch charakterisiert. Außerdem erlaubten Koexpressionsexperimente mit humanem PML und humIE1 die Untersuchung des Einflusses von humIE1 auf die auto-SUMOylierung von PML in vitro

Exploring the Human Cytomegalovirus Core Nuclear Egress Complex as a Novel Antiviral Target: A New Type of Small Molecule Inhibitors

Nuclear egress is an essential process in the replication of human cytomegalovirus (HCMV), as it enables the migration of newly formed viral capsids from the nucleus into the cytoplasm. Inhibition of the HCMV core nuclear egress complex (core NEC), composed of viral proteins pUL50 and pUL53, has been proposed as a potential new target for the treatment of HCMV infection and disease. Here, we present a new type of small molecule inhibitors of HCMV core NEC formation, which inhibit the pUL50-pUL53 interaction at nanomolar concentrations. These inhibitors, i.e., verteporfin and merbromin, were identified through the screening of the Prestwick Chemical Library® of approved drug compounds. The inhibitory effect of merbromin is both compound- and target-specific, as no inhibition was seen for other mercury-organic compounds. Furthermore, merbromin does not inhibit an unrelated protein–protein interaction either. More importantly, merbromin was found to inhibit HCMV infection of cells in three different assays, as well as to disrupt HCMV NEC nuclear rim formation. Thus, while not being an ideal drug candidate by itself, merbromin may serve as a blueprint for small molecules with high HCMV core NEC inhibitory potential, as candidates for novel anti-herpesviral drugs

Cross-Species Analysis of Innate Immune Antagonism by Cytomegalovirus IE1 Protein

The human cytomegalovirus (CMV) immediate early 1 (IE1) protein has evolved as a multifunctional antagonist of intrinsic and innate immune mechanisms. In addition, this protein serves as a transactivator and potential genome maintenance protein. Recently, the crystal structures of the human and rat CMV IE1 (hIE1, rIE1) core domain were solved. Despite low sequence identity, the respective structures display a highly similar, all alpha-helical fold with distinct variations. To elucidate which activities of IE1 are either species-specific or conserved, this study aimed at a comparative analysis of hIE1 and rIE1 functions. To facilitate the quantitative evaluation of interactions between IE1 and cellular proteins, a sensitive NanoBRET assay was established. This confirmed the species-specific interaction of IE1 with the cellular restriction factor promyelocytic leukemia protein (PML) and with the DNA replication factor flap endonuclease 1 (FEN1). To characterize the respective binding surfaces, helix exchange mutants were generated by swapping hIE1 helices with the corresponding rIE1 helices. Interestingly, while all mutants were defective for PML binding, loss of FEN1 interaction was confined to the exchange of helices 1 and 2, suggesting that FEN1 binds to the stalk region of IE1. Furthermore, our data reveal that both hIE1 and rIE1 antagonize human STAT2; however, distinct regions of the respective viral proteins mediated the interaction. Finally, while PML, FEN1, and STAT2 binding were conserved between primate and rodent proteins, we detected that rIE1 lacks a chromatin tethering function suggesting that this activity is dispensable for rat CMV. In conclusion, our study revealed conserved and distinct functions of primate and rodent IE1 proteins, further supporting the concept that IE1 proteins underwent a narrow co-evolution with their respective hosts to maximize their efficacy in antagonizing innate immune mechanisms and supporting viral replication

Cytomegalovirus immediate-early 1 proteins form a structurally distinct protein class with adaptations determining cross-species barriers

Restriction factors are potent antiviral proteins that constitute a first line of intracellular defense by blocking viral replication and spread. During co-evolution, however, viruses have developed antagonistic proteins to modulate or degrade the restriction factors of their host. To ensure the success of lytic replication, the herpesvirus human cytomegalovirus (HCMV) expresses the immediate-early protein IE1, which acts as an antagonist of antiviral, subnuclear structures termed PML nuclear bodies (PML-NBs). IE1 interacts directly with PML, the key protein of PML-NBs, through its core domain and disrupts the dot-like multiprotein complexes thereby abrogating the antiviral effects. Here we present the crystal structures of the human and rat cytomegalovirus core domain (IE1CORE). We found that IE1CORE domains, also including the previously characterized IE1CORE of rhesus CMV, form a distinct class of proteins that are characterized by a highly similar and unique tertiary fold and quaternary assembly. This contrasts to a marked amino acid sequence diversity suggesting that strong positive selection evolved a conserved fold, while immune selection pressure may have fostered sequence divergence of IE1. At the same time, we detected specific differences in the helix arrangements of primate versus rodent IE1CORE structures. Functional characterization revealed a conserved mechanism of PML-NB disruption, however, primate and rodent IE1 proteins were only effective in cells of the natural host species but not during cross-species infection. Remarkably, we observed that expression of HCMV IE1 allows rat cytomegalovirus replication in human cells. We conclude that cytomegaloviruses have evolved a distinct protein tertiary structure of IE1 to effectively bind and inactivate an important cellular restriction factor. Furthermore, our data show that the IE1 fold has been adapted to maximize the efficacy of PML targeting in a species-specific manner and support the concept that the PML-NBs-based intrinsic defense constitutes a barrier to cross-species transmission of HCMV

Nuclear Egress Complexes of HCMV and Other Herpesviruses: Solving the Puzzle of Sequence Coevolution, Conserved Structures and Subfamily-Spanning Binding Properties

Herpesviruses uniquely express two essential nuclear egress-regulating proteins forming a heterodimeric nuclear egress complex (core NEC). These core NECs serve as hexameric lattice-structured platforms for capsid docking and recruit viral and cellular NEC-associated factors that jointly exert nuclear lamina as well as membrane-rearranging functions (multicomponent NEC). The regulation of nuclear egress has been profoundly analyzed for murine and human cytomegaloviruses (CMVs) on a mechanistic basis, followed by the description of core NEC crystal structures, first for HCMV, then HSV-1, PRV and EBV. Interestingly, the highly conserved structural domains of these proteins stand in contrast to a very limited sequence conservation of the key amino acids within core NEC-binding interfaces. Even more surprising, although a high functional consistency was found when regarding the basic role of NECs in nuclear egress, a clear specification was identified regarding the limited, subfamily-spanning binding properties of core NEC pairs and NEC multicomponent proteins. This review summarizes the evolving picture of the relationship between sequence coevolution, structural conservation and properties of NEC interaction, comparing HCMV to α-, β- and γ-herpesviruses. Since NECs represent substantially important elements of herpesviral replication that are considered as drug-accessible targets, their putative translational use for antiviral strategies is discussed

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

Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses 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. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation 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 thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein–Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates

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

Abstract Varicella-zoster virus (VZV) is a human pathogen from the α-subfamily of herpesviruses. The VZV Orf24-Orf27 complex represents the essential viral core nuclear egress complex (NEC) that orchestrates the egress of the preassembled virus capsids from the nucleus. While previous studies have primarily emphasized that the architecture of core NEC complexes is highly conserved among herpesviruses, the present report focuses 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. Here, we describe the crystal structure of the Orf24-Orf27 complex at 2.1 Å resolution. Coimmunoprecipitation 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 thermodynamic parameters of NEC formation of three prototypical α-, β-, and γ herpesviruses, i.e., VZV, human cytomegalovirus (HCMV), and Epstein-Barr virus (EBV), revealed highly similar binding affinities for the autologous interaction with specific differences in enthalpy and entropy. Computational alanine scanning, structural comparisons, and mutational data highlight intermolecular interactions shared among α-herpesviruses that are clearly distinct from those seen in β- and γ-herpesviruses, including a salt bridge formed between Orf24-Arg167 and Orf27-Asp126. This interaction is located outside of the hook-into-groove interface and contributes significantly to the free energy of complex formation. Combined, these data explain distinct properties of specificity and permissivity so far observed in herpesviral NEC interactions. These findings will prove valuable in attempting to target multiple herpesvirus core NECs with selective or broad-acting drug candidates