The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor-Mediated Type I IFN Transcription

The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life-long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK-binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses

Charakterisierung der HCMV Proteine UL35 und UL35a als neue Typ I Interferon Antagonisten

Human cytomegalovirus (HCMV) is an opportunistic, human specific pathogen which is distributed worldwide with seroprevalences between 45 and 100% depending on the geographical location and socioeconomic status. While primary infection of immunocompetent individuals is mostly asymptomatic, transplant recipients and AIDS patients with a suppressed immune system often develop life-threatening disseminating disease. Moreover, HCMV can infect the fetus in utero leading to severe congenital birth defects. Once infected, HCMV is present life-long and persists in a dormant state with sporadic reactivation events. HCMV infection is detected by germline-encoded pattern recognition receptors (PRRs) leading to the production of type I interferons (IFN) and proinflammatory cytokines. Through the long co-evolution with its host, HCMV developed efficient mechanisms to counteract the host’s immune response to ensure the establishment of a life-long infection. In this study, two HCMV-encoded proteins were characterized, the tegument protein UL35, and the shorter, C-terminal identical protein UL35a, for their potential, to interfere with the PRR-mediated signaling. Luciferase reporter assays showed that UL35 inhibited PRR-mediated activation of IFNβ between the key kinase TANK-binding kinase 1 (TBK1) and the transcription factor interferon regulatory factor 3 (IRF3). In contrast, UL35a inhibited downstream of IRF3 showing that both proteins interfere with the cellular type I IFN response at different levels. In addition, the antagonistic potential of UL35 and UL35a was analyzed in HCMV-permissive human primary fibroblasts. Finally, infection experiments with a recombinant UL35-deficiant HCMV were performed and confirmed the immunomodulatory function of UL35 during infection. Additionally, a UL35-specific mouse monoclonal antibody was generated to study the expression kinetics and subcellular localization of HCMV-derived UL35 protein. Further, signaling processes upon PRR-activation were characterized and showed that UL35 interacts with TBK1 and interferes with its activation. In summary, this work identified the HCMV tegument protein UL35 as a novel type I IFN antagonist that targets multiple PRRs by inhibiting the activation of the key kinase TBK1.Das humane Cytomegalievirus (HCMV) ist ein opportunistischer Krankheitserreger für Menschen und betrifft 45 bis 100% der Bevölkerung weltweit, abhängig von sozialem Status und geografischer Lage. Die primäre Infektion verläuft in der Regel asymptomatisch bei gesunden Individuen. Bei immunsupprimierten Personen wie AIDS Patienten oder Organempfängern kann die Infektion jedoch lebensbedrohlich verlaufen. Darüber hinaus kann sich eine HCMV Infektion auch von der Schwangeren auf den Fötus übertragen und zu bleibenden Gesundheitsschäden führen. HCMV Infektionen bleiben lebenslang bestehen und können sporadisch wiederausbrechen. Keimbahn-kodierte Mustererkennungsrezeptoren (PRRs) erkennen eine HCMV Infektion und lösen die Produktion von Typ I Interferonen und proinflammatorischen Cytokinen aus. Im Zuge der langen Co-Evolution von HCMV mit seinem Wirt, entwickelte HCMV effiziente Strategien die der Immunreaktion des Wirtes entgegenwirken und eine lebenslange Infektion ermöglichen. In dieser Arbeit wurden zwei HCMV-Proteine, das Tegumentprotein UL35 sowie das kürzere, C-terminal identische UL35a, auf ihr Potential getestet, der PRR-induzierten Interferonantwort entgegenzuwirken. Mit Hilfe von Luciferase Reporterexperimenten konnte gezeigt werden, dass UL35 den Interferon Signalweg zwischen der Kinase TANK-binding kinase 1 (TBK1) und dem Transkriptionsfaktor interferon regulatory factor 3 (IRF3) inhibiert. Im Vergleich zu UL35 inhibierte UL35a die Interferon Antwort unterhalb von IRF3 und nutzt daher einen anderen Mechanismus. Anschließende Experimente mit HCMV-permissiven humanen Fibroblasten bestätigten die Erkenntnisse der Reporterexperimente und zeigten die antagonistische Wirkung von UL35 und UL35a auf die Interferon Transkription. Weiters zeigte die Infektion mit einer rekombinanten HCMV Mutante, welche kein UL35 exprimiert, eine erhöhte Interferonantwort im Vergleich zu HCMV Wildtyp. Zur Charakterisierung der Expressionskinetik und der intrazellulären Lokalisation von viralem UL35 Protein, wurde ein monoklonaler Maus Antikörper hergestellt. Schlussendlich enthüllten detaillierte Analysen der PRR-induzierten Signalkaskade, TBK1 als Interaktionspartner von UL35 und es konnte gezeigt werden, dass die Kinasefunktion von TBK1 in Gegenwart von UL35 gehemmt wird

The Cytomegalovirus Tegument Protein UL35 Antagonizes Pattern Recognition Receptor- Mediated Type I IFN Transcription

Abstract: The rapid activation of pattern recognition receptor (PRR)-mediated type I interferon (IFN) signaling is crucial for the host response to infection. In turn, human cytomegalovirus (HCMV) must evade this potent response to establish life- long infection. Here, we reveal that the HCMV tegument protein UL35 antagonizes the activation of type I IFN transcription downstream of the DNA and RNA sensors cGAS and RIG-I, respectively. We show that ectopic expression of UL35 diminishes the type I IFN response, while infection with a recombinant HCMV lacking UL35 induces an elevated type I IFN response compared to wildtype HCMV. With a series of luciferase reporter assays and the analysis of signaling kinetics upon HCMV infection, we observed that UL35 downmodulates PRR signaling at the level of the key signaling factor TANK- binding kinase 1 (TBK1). Finally, we demonstrate that UL35 and TBK1 co-immunoprecipitate when co-expressed in HEK293T cells. In addition, we show that a previously reported cellular binding partner of UL35, O-GlcNAc transferase (OGT), post-translationally GlcNAcylates UL35, but that this modification is not required for the antagonizing effect of UL35 on PRR signaling. In summary, we have identified UL35 as the first HCMV protein to antagonize the type I IFN response at the level of TBK1, thereby enriching our understanding of how this important herpesvirus escapes host immune responses