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PML nuclear bodies (PML-NBs) are dynamic macromolecular complexes that mediate intrinsic immunity against viruses of different families, including human cytomegalovirus (HCMV). Upon HCMV infection, PML-NBs target viral genomes entering the nucleus and restrict viral immediate–early gene expression by epigenetic silencing. Studies from several groups performed in human fibroblast cells have shown that the major PML-NB components PML, Daxx, Sp100 and ATRX contribute to this repression in a cooperative manner. Their role for HCMV restriction in endothelial cells, however, has not yet been characterized although infected endothelium is thought to play a crucial role for HCMV dissemination and development of vascular disease in vivo. Here, we use conditionally immortalized umbilical vein endothelial cells (HEC-LTT) as a cell culture model to elucidate the impact of PML-NB proteins on lytic HCMV infection. Depletion of individual PML-NB proteins by lentiviral transduction showed a particularly strong antiviral effect of PML in HEC-LTT, compared to human fibroblasts. A closer characterization of this antiviral function revealed that PML may not only effectively inhibit HCMV immediate-early gene expression but also act at later steps of the viral replication cycle. At contrast, we surprisingly noted an antiviral behavior of Daxx in complementary approaches: Depletion of Daxx resulted in decreased viral gene expression, while overexpression of Daxx promoted HCMV infection. In summary, our data demonstrate a cell type-specific effect of PML-NB components on lytic HCMV infection and suggest an important role of PML in the inhibition of HCMV dissemination through infected endothelial cells., publishedVersion

Human cytomegalovirus (HCMV) is the prototypic beta-herpesvirus and widespread throughout the human population. While infection is asymptomatic in healthy individuals, it can lead to high morbidity and mortality in immunocompromised persons. Importantly, HCMV evolved multiple strategies to interfere with immune cell function in order to establish latency in infected individuals. As mature DCs (mDCs) are antigen-presenting cells able to activate naïve T cells they play a crucial role during induction of effective antiviral immune responses. Interestingly, earlier studies demonstrated that the functionally important mDC surface molecule CD83 is down-regulated upon HCMV infection resulting in a reduced T cell stimulatory capacity of the infected cells. However, the viral effector protein and the precise mechanism of HCMV-mediated CD83 reduction remain to be discovered. Using flow cytometric analyses, we observed significant down-modulation of CD83 surface expression becoming significant already 12 h after HCMV infection. Moreover, Western bot analyses revealed that, in sharp contrast to previous studies, loss of CD83 is not restricted to the membrane-bound molecule, but also occurs intracellularly. Furthermore, inhibition of the proteasome almost completely restored CD83 surface expression during HCMV infection. Results of infection kinetics and cycloheximide-actinomycin D-chase experiments, strongly suggested that an HCMV immediate early gene product is responsible for the induction of CD83 down-modulation. Consequently, we were able to identify the major immediate early protein IE2 as the viral effector protein that induces proteasomal CD83 degradation.

Protection against viruses is provided by the innate, adaptive, and cellular intrinsic immunity. Recently, a subnuclear structure known as nuclear domain 10 (ND10), which represents accumulations of the cellular proteins PML, hDaxx, and Sp100, was shown to mediate intrinsic resistance against infections with human cytomegalovirus (HCMV). The immediate-early protein IE1 of HCMV efficiently antagonizes this antiviral defense mechanism by inducing disruption of the ND10 structures. It has been proposed that this event involves de SUMOylation of the PML protein by IE1, since SUMO-modified forms of PML are essential for ND10 formation. The exact mechanism, however, remains unclear. In order to address the question whether IE1 affects additional proteins besides PML to disrupt ND10 integrity, primary human fibroblasts stably expressing FLAG-tagged versions of SUMO-1 or SUMO-3 were generated. Expression of IE1 in these cells, followed by Western blot analyses, revealed that not only PML but also Sp100 is a specific target of IE1-mediated de SUMOylation. Furthermore, it was observed that overexpression of FLAG-SUMO-1 and FLAG-SUMO-3 promotes HCMV replication. These findings indicate that, besides de-SUMOylation of cellular proteins, HCMV has evolved additional mechanisms to manipulate the SUMO conjugation pathway for its own benefit. In order to gain insight into the structural basis of ND10 disruption, determination of the three-dimensional structure of IE1 was performed. The crystal structure of a central, 45 kDa subdomain of the rhesus macaque cytomegalovirus (RhCMV) IE1 protein revealed an unusual all alpha-helical fold that displays no homology to known protein structures. In vitro and in vivo approaches demonstrated that function and structure are conserved between the IE1 proteins of HCMV and RhCMV. Infection experiments with a recombinant HCMV, expressing the truncated IE1 variant, revealed that the IE1 subdomain is sufficient to induce de-SUMOylation of PML. Intruigingly, this resulted in the dispersal of ND10-associated proteins but not of PML itself, suggesting that ND10 disruption by IE1 is an at least two-step process mediated by distinct domains of the IE1 protein. Finally, the crystal structure of IE1 allowed for the generation and initial characterization of mutant IE1 proteins and, thus, represents a valuable tool to further elucidate the mechanism by which IE1 counteracts ND10-based intrinsic immunity. Das angeborene, erworbene und intrinsische Immunsystem bieten Schutz vor viralen Infektionen. Kürzlich wurde gezeigt, dass eine als „nukleäre Domäne 10“ (ND10) bezeichnete Struktur im Zellkern, die aus Ansammlungen der zellulären Proteine PML, hDaxx und Sp100 besteht, intrinsische Immunität gegen Infektionen mit humanem Cytomegalovirus (HCMV) vermittelt. Das IE1 Protein von HCMV wirkt diesem antiviralen Abwehrmechanismus effizient entgegen, indem es die ND10-Domänen auflöst. Man vermutet, dass diesem Vorgang eine de-SUMOylierung von PML zugrunde liegt, da SUMO-modifizierte Formen von PML für die ND10-Bildung essentiell sind. Der genaue Mechanismus ist jedoch noch unklar. Um die Frage zu beantworten, ob IE1 neben PML weitere Proteine beeinflusst, um die Integrität von ND10 zu zerstören, wurden primäre humane Fibroblasten generiert, die FLAG-markierte Versionen von SUMO-1 oder SUMO-3 stabil exprimierten. Die Expression von IE1 in diesen Zellen, gefolgt von Western Blot-Analysen, ergab, dass nicht nur PML, sondern auch Sp100 ein spezifisches Zielprotein der IE1-vermittelten de-SUMOylierung ist. Weiterhin wurde beobachtet, dass die Überexpression von FLAG-SUMO-1 und FLAG-SUMO-3 die HCMV-Replikation fördert. Dieser Befund weist darauf hin, dass HCMV, zusätzlich zu der de-SUMOylierung zellulärer Proteine, zusätzliche Mechanismen entwickelt hat, um das SUMO-System zu seinem Vorteil zu beeinflussen. Um Hinweise auf die strukturelle Grundlage der ND10-Auflösung zu erhalten, wurde die drei-dimensionale Struktur von IE1 bestimmt. Die Kristallstruktur einer zentralen, 45 kDa großen Unterdomäne von IE1 des Rhesusaffen-Cytomegalovirus (RhCMV) zeigte eine ungewöhnliche alpha-helikale Faltung, die keine Ähnlichkeiten zu bekannten Proteinstrukturen aufweist. In vitro- und in vivo-Versuche ergaben, dass Funktion und Struktur der IE1-Proteine von HCMV und RhCMV konserviert sind. Infektionsexperimente mit einem rekombinanten HCMV, das die verkürzte IE1-Variante exprimierte, zeigten, dass die Unterdomäne von IE1 ausreicht, um PML zu de-SUMOylieren. Dies führt zu einer Zerstreuung von ND10-assoziierten Proteinen, aber nicht von PML selbst, auf einen mindestens zweistufigen Prozess der ND10-Auflösung schließen lässt – vermittelt durch verschiedene Domänen des Proteins. Zuletzt war es möglich anhand der Kristallstruktur von IE1 mutierte Proteine zu generieren und eine erste Charakterisierung vorzunehmen. Die Struktur stellt daher ein hilfreiches Mittel zur Aufklärung des Mechanismus dar, über den IE1 die ND10-vermittelte Immunität überwindet.