Revisiting promyelocytic leukemia protein targeting by human cytomegalovirus immediate-early protein 1

This work was supported by a grant (MR/P022146/1) from the Medical Research Council (https://mrc.ukri.org) to MMN, a grant (T16/28) from Tenovus Scotland (https://tenovus-scotland.org.uk) to CP, a European Union Erasmus+ grant (https://www.erasmusplus.org.uk) to BW and the Wellcome Trust Institutional Strategic Support Fund (https://wellcome.ac.uk) to CP and MMN.Promyelocytic leukemia (PML) bodies are nuclear organelles implicated in intrinsic and innate antiviral defense. The eponymous PML proteins, central to the self-organization of PML bodies, and other restriction factors found in these organelles are common targets of viral antagonism. The 72-kDa immediate-early protein 1 (IE1) is the principal antagonist of PML bodies encoded by the human cytomegalovirus (hCMV). IE1 is believed to disrupt PML bodies by inhibiting PML SUMOylation, while PML was proposed to act as an E3 ligase for IE1 SUMOylation. PML targeting by IE1 is considered to be crucial for hCMV replication at low multiplicities of infection, in part via counteracting antiviral gene induction linked to the cellular interferon (IFN) response. However, current concepts of IE1-PML interaction are largely derived from mutant IE1 proteins known or predicted to be metabolically unstable and globally misfolded. We performed systematic clustered charge-to-alanine scanning mutagenesis and identified a stable IE1 mutant protein (IE1cc172-176) with wild-type characteristics except for neither interacting with PML proteins nor inhibiting PML SUMOylation. Consequently, IE1cc172-176 does not associate with PML bodies and is selectively impaired for disrupting these organelles. Surprisingly, functional analysis of IE1cc172-176 revealed that the protein is hypermodified by mixed SUMO chains and that IE1 SUMOylation depends on nucleosome rather than PML binding. Furthermore, a mutant hCMV expressing IE1cc172-176 was only slightly attenuated compared to an IE1-null virus even at low multiplicities of infection. Finally, hCMV-induced expression of cytokine and IFN-stimulated genes turned out to be reduced rather than increased in the presence of IE1cc172-176 relative to wild-type IE1. Our findings challenge present views on the relationship of IE1 with PML and the role of PML in hCMV replication. This study also provides initial evidence for the idea that disruption of PML bodies upon viral infection is linked to activation rather than inhibition of innate immunity.Publisher PDFPeer reviewe

Human cytomegalovirus immediate-early 1 protein rewires upstream STAT3 to downstream STAT1 signaling switching an IL6-type to an IFNγ-like response

MN and CP were supported by the Wellcome Trust (www.wellcome.ac.uk) Institutional Strategic Support Fund and CP was supported by the Deutsche Forschungsgemeinschaft (PA 815/2-1; www.dfg.de).The human cytomegalovirus (hCMV) major immediate-early 1 protein (IE1) is best known for activating transcription to facilitate viral replication. Here we present transcriptome data indicating that IE1 is as significant a repressor as it is an activator of host gene expression. Human cells induced to express IE1 exhibit global repression of IL6- and oncostatin M-responsive STAT3 target genes. This repression is followed by STAT1 phosphorylation and activation of STAT1 target genes normally induced by IFNγ. The observed repression and subsequent activation are both mediated through the same region (amino acids 410 to 445) in the C-terminal domain of IE1, and this region serves as a binding site for STAT3. Depletion of STAT3 phenocopies the STAT1-dependent IFNγ-like response to IE1. In contrast, depletion of the IL6 receptor (IL6ST) or the STAT kinase JAK1 prevents this response. Accordingly, treatment with IL6 leads to prolonged STAT1 instead of STAT3 activation in wild-type IE1 expressing cells, but not in cells expressing a mutant protein (IE1dl410-420) deficient for STAT3 binding. A very similar STAT1-directed response to IL6 is also present in cells infected with a wild-type or revertant hCMV, but not an IE1dl410-420 mutant virus, and this response results in restricted viral replication. We conclude that IE1 is sufficient and necessary to rewire upstream IL6-type to downstream IFNγ-like signaling, two pathways linked to opposing actions, resulting in repressed STAT3- and activated STAT1-responsive genes. These findings relate transcriptional repressor and activator functions of IE1 and suggest unexpected outcomes relevant to viral pathogenesis in response to cytokines or growth factors that signal through the IL6ST-JAK1-STAT3 axis in hCMV-infected cells. Our results also reveal that IE1, a protein considered to be a key activator of the hCMV productive cycle, has an unanticipated role in tempering viral replication.Publisher PDFPeer reviewe

Kartierung immunmodulatorischer Funktionen im Zytomegalievirus IE1-Protein

Das humane Zytomegalievirus (hCMV), der größte Vertreter der human-pathogenen Herpesviren, verursacht lebensbedrohliche Krankheitsbilder bei Menschen mit geschwächtem Immunsystem, wie AIDS- oder Transplantationspatienten. Zudem kann es bei einer diaplazentaren Übertragung des Virus während der Schwangerschaft zu einer schweren Schädigung des ungeborenen Kindes kommen. Dagegen zeigen Immungesunde im Rahmen einer hCMV-Infektion nur sehr selten Anzeichen einer ernsthaften Erkrankung. Die sogenannten immediate early- (IE-) Proteine sind die ersten viralen Genprodukte, die nach der Infektion in den Wirtszellen gebildet werden. Dazu gehört das IE1-Protein, das unter anderem als Transkriptionsaktivator für eine Vielzahl viraler Gene fungiert. Darüber hinaus ist dieses Protein in der Lage, die Genexpression der Wirtszelle zu beeinflussen. So schützt es hCMV unter anderem vor dem Zugriff des angeborenen Immunsystems, indem es in Typ I-Interferon- (IFN-) vermittelte Signalwege eingreift und über deren Blockierung die Transkription antiviral wirksamer Faktoren verhindert. Zusätzlich interagiert IE1 mit Proteinkomplexen des Zellkerns, den sogenannten PML-Körpern, für die ebenfalls eine Bedeutung bei der angeborenen Immunabwehr von Infektionen beschrieben ist. IE1 kann an PML-Körper binden und diese kurze Zeit später zerstören, sodass das virale Protein auch hier immunevasiv wirkt. Neben diesen hemmenden Eigenschaften besitzt IE1 jedoch auch die Fähigkeit zur Aktivierung von Genen der Wirtszelle. So ist es in der Lage, die Expression einer Reihe von zellulären Zytokinen und anderen Proteinen zu stimulieren, die normalerweise durch IFN-γ (Typ II-IFN) aktiviert werden. Für die IE1-vermittelte Aktivierung dieser Gene ist die Anwesenheit von Typ II-IFN jedoch nicht notwendig. Allerdings ist eine Phosphorylierung des signal transducer and activator of transcription 1 (STAT1) an Tyrosin 701 erforderlich, die zu dessen nukleärer Translokation und sequenzspezifischer DNA-Bindung und damit zur Aktivierung der Transkription führt. Um den Organismus nicht durch eine überschießende Reaktion des Immunsystems zu schädigen, unterliegen IFN-induzierte Prozesse einer negativen Rückkoppelung, an der unter anderem Proteine der suppressor of cytokine signaling- (SOCS-) Familie beteiligt sind. Über eine Repression der SOCS3-Transkription greift das IE1-Protein auch in diesen Mechanismus ein und verhindert damit offenbar eine effiziente Herunterregulierung IFN-induzierter Vorgänge. Im Rahmen dieser Arbeit wurden verschiedene IE1-Mutanten hergestellt, die in einem induzierbaren Zellsystem außerhalb der komplexen Vorgänge einer Virusinfektion auf ihre Funktionalität bezüglich der beschriebenen immunmodulatorischen Abläufe untersucht wurden. Dabei stellte sich heraus, dass verschiedene carboxy-terminale Abschnitte im IE1-Protein, und zwar sowohl die acidic domain 2 (AD2) (Aminosäure 421 bis 445) als auch die benachbarten Sequenzen im Bereich der AD1 und Serin/Prolin- (S/P-) reichen Region (Aminosäure 373 bis 420), an der Hochregulierung Typ II-IFN-stimulierter Gene sowie der Suppression von SOCS3 beteiligt sind. Während jedoch für die Induktion Typ II-IFN-stimulierter Gene beiden Domänen eine essenzielle Bedeutung zukommt, ist für die Unterdrückung der SOCS3-Expression im Wesentlichen der Abschnitt AD1-S/P wichtig, wohingegen AD2 nur eine unterstützende Rolle übernimmt. Ein vergleichbares Bild lieferte die Blockierung des Typ I-IFN-vermittelten Signalweges, die ebenfalls hauptsächlich über AD1-S/P und weniger stark über AD2 vermittelt wird, wie sich bereits in früher publizierten Arbeiten gezeigt hatte. Damit erscheint eine komplette Kopplung der drei untersuchten IE1-Funktionen (Inhibition der Typ I-IFN-Antwort, Aktivierung einer Typ II-IFN-ähnlichen Antwort, Inhibition der SOCS3-Expression) unwahrscheinlich, wenngleich es möglich ist, dass die jeweiligen Abschnitte sich in ihrer Funktion unterstützen. Im Rahmen der Hochregulierung IFN-γ-stimulierter Gene konnte zudem eine direkte oder indirekte Interaktion zwischen STAT1 und IE1 nachgewiesen werden, die ebenfalls über AD1-S/P und AD2 vermittelt wird und ein wesentlicher Bestandteil dieses Prozesses zu sein scheint. Neben der Wechselwirkung mit STAT1 ist möglicherweise auch eine Assoziation mit PML-Körpern, nicht jedoch deren Auflösung, an der Vermittlung der Typ II-IFN-ähnlichen Antwort durch IE1 beteiligt. Im zweiten Teil der Arbeit wurde mittels IE1-defizienter Virusmutanten die Bedeutung dieses viralen Proteins für das klinische hCMV-Isolat TB40/E im Hinblick auf seine Replikation unter verschiedenen Infektionsbedingungen sowie die Resistenz gegenüber Typ I- und Typ II-IFN analysiert. Dabei stellte sich heraus, dass IE1 vor allem bei niedriger Infektionsmultiplizität für TB40/E überaus wichtig ist und eine Abwesenheit dieses Proteins unter diesen Bedingungen zu keiner nennenswerten Virusreplikation führt. Für den Schutz vor IFN-vermittelten antiviralen Effekten ist IE1 im Kontext von TB40/E jedoch weitaus weniger bedeutsam als erwartet, sodass davon auszugehen ist, dass neben diesem Protein noch andere virale Faktoren für die effiziente Hemmung IFN-abhängiger Prozesse erforderlich sind. Insgesamt zeigen die Ergebnisse dieser Arbeit, dass es sich bei der differentiellen Modulation verschiedener angeborener Immunprozesse durch das hCMV IE1-Protein um Funktionen handelt, die auf überlappenden Bereichen im carboxy-terminalen Abschnitt des viralen Proteins kodiert, aber wahrscheinlich nicht vollständig gekoppelt sind. Diese immunmodulatorischen Aktivitäten des IE1-Proteins spielen bei der Infektion in vivo vermutlich eine größere Rolle als in vitro und könnten künftig für verbesserte antivirale Therapiestrategien genutzt werden

Ingenuity analysis¹ of human genes repressed by IE1 and activated by STAT3, IL6 or OSM.

<p>Ingenuity analysis<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1005748#t001fn001" target="_blank">¹</a> of human genes repressed by IE1 and activated by STAT3, IL6 or OSM.</p

Residues within IE1 region 410–445 are required for phosphorylation of STAT1 and up-regulation of IFNγ-responsive genes.

<p>(A) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Whole cell protein extracts were prepared and analyzed by immunoblotting for IE1 (HA tag), pSTAT1 (Y701), pSTAT1 (S727), total STAT1 and GAPDH. (B) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT1 target genes CXCL10 and CXCL11. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).</p

siRNAs used in this study.

<p>siRNAs used in this study.</p

Systematic deletion analysis of C-terminal IE1 residues 373–491.

<p>(A) Schematic overview of amino acids 373–491 in the tested wild-type and mutant IE1 proteins. Positions of the low-complexity motifs (acidic domains AD1-3 and serine/proline-rich region S/P), the SUMOylation site (K450) and the chromatin tethering domain (CTD) are shown. (B) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Whole cell protein extracts were prepared and analyzed by immunoblotting for IE1 (HA tag) and GAPDH. (C) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Whole cell extracts prepared in the presence of N-ethylmaleimide were used for immunoprecipitation with anti-HA-agarose, and samples were analyzed by immunoblotting for IE1 (HA tag) and SUMO1.</p

Residues within IE1 region 410–445 are required for targeting of STAT3 and down-regulation of STAT3-responsive genes.

<p>(A) TetR cells without (w/o) or with inducible expression of the indicated HA-IE1 proteins were treated with dox for 48 h. During the final 24 h of dox treatment, cells were kept in medium with 0.5% FBS. Subcellular localization of endogenous STAT3α in IE1 expressing cells was analyzed by indirect immunofluorescence microscopy. Samples were simultaneously reacted with a rabbit monoclonal antibody to STAT3α and a mouse monoclonal antibody to HA-tagged IE1, followed by incubation with a rabbit-specific Alexa Fluor 594 conjugate and a mouse-specific Alexa Fluor 488 conjugate. Host cell nuclei were visualized by 4',6-diamidino-2-phenylindole (DAPI) staining. Additionally, merge images of STAT3α, IE1 and DAPI signals are presented. (B) The percentage of cells with i) predominantly nuclear STAT3α staining (N>C), ii) equally strong nuclear and cytoplasmic STAT3α staining (N = C) and iii) predominantly cytoplasmic STAT3α staining (C>N) was determined for 100 randomly selected cells per sample described in (A). (C) TetR cells without or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent (w/o) or IL6 plus IL6R (IL6/Rα) for 30 min. Cytoplasmic and nuclear extracts were prepared and analyzed by immunoblotting for histone H2B, STAT2, STAT3α and IE1. (D) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h. Whole cell extracts were prepared and used for immunoprecipitations (IPs) with anti-HA-agarose. Samples of lysates and immunoprecipitates were analyzed by immunoblotting for IE1 and STAT3α. (E) TetR cells without (w/o) or with inducible expression of HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with IL6 plus IL6R for 30 min. Samples were subjected to ChIP with rabbit polyclonal antibodies to STAT3 or normal rabbit IgG and primers specific for sequences in the SOCS3 promoter or coding region. The percentage of output versus input DNA is presented as the difference between STAT3 and normal IgG ChIPs. Means and standard deviations of two biological and two technical replicates are shown. (F) TetR cells without (w/o) or with inducible expression of the indicated HA-tagged wild-type or mutant IE1 proteins were treated with dox for 72 h. Relative mRNA expression levels were determined by RT-qPCR with primers specific for the STAT3 target genes CXCL12 and SOCS3. Results were normalized to TUBB, and means and standard deviations of two biological and two technical replicates are shown in comparison to IE1-negative TetR cells (set to 1).</p

IE1 switches an IL6-type to an IFNγ-like response.

<p>(A) TetR (w/o) or TetR-IE1 (IE1) cells were treated with dox for 72 h and with IL6 plus IL6R (IL6/Rα) for the indicated times. Whole cell protein extracts were prepared and analyzed by immunoblotting for IE1, total STAT1, pSTAT1 (Y701), total STAT3α, pSTAT3 (Y705) and GAPDH. (B) TetR (w/o) or TetR-IE1 cells expressing HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent, IFNα, IFNγ or IL6 plus IL6R (IL6/Rα) for 24 h. Whole cell protein extracts were analyzed by immunoblotting for IE1, total STAT1, pSTAT1 (Y701) and GAPDH. (C) TetR (w/o) or TetR-IE1 cells expressing HA-tagged wild-type IE1 or IE1dl410-420 were treated with dox for 72 h and with solvent, IFNα, IFNγ or IL6 plus IL6R (IL6/Rα) for 24 h. Relative mRNA levels were determined by RT-qPCR for the type I IFN/STAT2 target genes OAS1 and EIF2AK2 (protein kinase R) (left panels), the type II IFN/STAT1 target genes CXCL10 and CXCL11 (middle panels) and the IL6/STAT3 target genes CXCL12 and SOCS3 (right panels). Results were normalized to TUBB, and means and standard deviations of biological triplicates are shown in comparison to solvent-treated TetR cells (set to 1).</p