Interference of the viral effector proteins pp71 and IE1 with ND10-mediated intrinsic immunity against human cytomegalovirus infections
Infection with DNA viruses commonly results in the association of viral genomes with a cellular sub-nuclear structure known as nuclear domain 10 (ND10) or PML bodies. ND10 represent discrete interchromosomal accumulations of multiple cellular proteins including hDaxx, Sp100 or SUMO-1, that require the PML protein for their formation. During the previous funding period we could show that specific ND10 proteins mediate an intrinsic immune response against infections with DNA viruses via the silencing of viral gene expression and are thus part of an antiviral defense mechanism of the cell. In particular, we could show that Sp100 exerts a dual antiviral role during the immediate-early and late phases of human cytomegalovirus replication. Moreover, we demonstrated in a close collaboration with project B1 that Sp100 also strongly represses the gammaherpesvirus Herpesvirus saimiri and we characterized in collaboration with project B2 the subcellular localization of ND10 factors in primary human monocytes. However, this intrinsic cellular defense mechanism is antagonized by viral regulatory proteins: we could show that the HCMV IE1 protein not only induces a deSUMOylation of PML but also directly affects the SUMOylation of Sp100 thus leading to a disruption of ND10. In Herpesvirus saimiri a tegument protein with homology to the cellular enzyme formylglycinamide ribotide amidotransferase (FGARAT) was found to selectively degrade the Sp100 protein in a proteasome dependent manner, while PML remained intact. Finally, in close collaboration with project A3 we succeeded in the purification, crystallization and structural determination of the primate cytomegalovirus IE1 protein at 2.3 A. In the second funding period of the CRC796 we will continue our efforts to elucidate the silencing mechanism of ND10. Since we detected that gene expression of Herpesvirus saimiri is fully restricted by PML even under conditions of high multiplicity infection, this will be an ideal system to analyze chromatin modifications instituted by PML using ChIP technology (in collaboration with B1). The determination of the structure of the hydrophobic core of primate cytomegalovirus IE1 is a milestone towards understanding the function of this ND10 antagonistic protein. Since this part of IE1 binds with high affinity to PML, we will try to co-express and co-crystallize both proteins (collaboration with A3). Furthermore, mutations will be predicted by project A2 that should interfere with IE1 multimerization and PML interaction and these mutations will be introduced into recombinant viruses for functional analyses. We already generated mutants of IE1 SUMO-interaction motifs (SIMs) and observed defects in ND10 disruption, while PML colocalization and interaction were fully preserved. These mutants should be extremely useful to define the cellular factors involved in ND10 dispersal which will be done by MS analyses of purified protein complexes.
Schematic representation of the current model of HCMV-ND10 interplay during infection. (1) After entry of cytomegalovirus into host cells, viral nucleoprotein complexes associate with ND10 proteins. This induces a silencing of viral gene expression. The tegument protein pp71 acts as an antagonist of ND10-mediated silencing via the proteasomal degradation of hDaxx. (2) Viral gene expression initiates, leading to the synthesis of the IE1 protein. (3) The IE1 protein acts as an additional antagonist of ND10 functions. This is mediated via a dispersal of ND10 structures and correlates with the induction of efficient lytic replication.
Project relevant publications
- Zheng, Y., Stamminger, T., Hearing, P. (2016). E2F/Rb family proteins mediate interferon induced repression of adenovirus immediate early transcription to promote persistent viral infection. PLoS Path, 12(1):e1005415.
- Graf, L., Feichtinger, S., Naing, Z., Hutterer, C., Milbradt, J., Webel, R., Wagner, S., Scott, G.M., Hamilton, S.T., Rawlinson, W.D., Stamminger, T., Thomas, M., Marschall, M. (2016). New insight into the phosphorylation-regulated intranuclear localization of human cytomegalovirus pUL69 mediated by cyclin-dependent kinases (CDKs) and viral CDK ortholog pUL97. J. Gen. Virol., 97(1): 144-51.
- Kahle, T., Volkmann, B., Eissmann, K., Schmitt, S., Merkel, L., Wittmann, S., Reuter, N., Stamminger, T., Gramberg, T. (2015). TRIM19/PML restricts HIV infection in a cell type-dependent manner. Viruses 8(1): pii: E2. doi: 10.3390/v8010002.
- Scherer, M., Otto, V., Stump, J., Klingl, S., Müller, R., Reuter, N., Muller, Y., Sticht, H., Stamminger, T. (2015). Characterization of recombinant human cytomegaloviruses encoding IE1 mutants L174P and 1-382 reveals that viral targeting of PML bodies perturbs both intrinsic and innate immune responses. J. Virol. 90(3):1190-205.
- Hutterer, C., Niemann, I., Milbradt, J., Fröhlich, T., Reiter, C., Kadioglu, O., Bahsi, H., Zeitträger, I., Wagner, S., Einsiedel, J., Gmeiner, P., Vogel, N., Wandinger, S., Godl, K. Stamminger, T., Efferth, T., Tsogoeva, S.B., Marschall, M. (2015). The broad-spectrum antiinfective drug artesunate interferes with the canonical nuclear factor kappa B (NF-κB) pathway by targeting RelA/p65. Antiviral Res. 124: 101-9.
- Thomas, M., Sonntag, E., Müller, R., Schmidt, S., Zielke, B., Fossen, T., Stamminger, T. (2015). pUL69 of human cytomegalovirus recruits the cellular protein arginine methyltransferase 6 via a domain that is crucial for mRNA export and efficient virus replication. J. Virol. 89: 9601-15.
- Klingl, S., Scherer, M., Stamminger, T., Muller, Y.A. (2015). Controlled crystal dehydration triggers a space-group switch and shapes the tertiary structure of cytomegalovirus immediate-early 1 (IE1) protein. Acta Crystallogr D Biol Crystallogr 71: 1493-504.
- Wagenknecht, N., Reuter, N., Scherer, M., Reichel, A., Müller, R., Stamminger, T. (2015). Contribution of the major ND10 proteins PML, hDaxx and Sp100 to the regulation of human cytomegalovirus latency and lytic replication in the monocytic cell line THP-1. Viruses 7(6): 2884-907.
- Vink, El, Zheng, Y., Yeasmin, R., Stamminger, T., Krug, L.T., Hearing, P. (2015). Impact of adenovirus E4-ORF3 oligomerization and protein localization on cellular gene expression. Viruses 7(5): 2428-49.
- Scherer, M., Klingl, S., Sevvana, M., Otto, V., Schilling, E.-A., Stump, J.D., Müller, R., Reuter, N., Sticht, H., Muller, Y.A., Stamminger, T. (2014). Crystal structure of cytomegalovirus IE1 protein reveals targeting of TRIM family member PML via coiled-coil interactions. PLoS Pathog. 19(11): e1004512.
- Niemann, I., Reichel, A., Stamminger, T. (2014). Intracellular trafficking of the human cytomegalovirus-encoded 7-trans-membrane protein homologs pUL27 and pUL78 during viral infection: a comparative analysis. Viruses Feb 10;6(2):661-82. doi: 0.3390/v6020661.
- Full, F., Jungnickl, D., Reuter, N., Bogner, E., Brulois, K., Scholz, B., Stürzl, M., Myoung, J., Jung, J.U., Stamminger, T., Ensser, A. (2014). Kaposi's sarcoma associated herpesvirus tegument protein ORF75 is essential for viral lytic replication and plays a critical role in the antagonization of ND10-instituted instrinsic immunity. PLoS Pathog. 2014 Jan; 10(1): e1003863.
- Hutterer, C., Wandinger, S.K., Wagner, S., Müller, R., Stamminger, T., Zeitträger, I., Godl, K., Baumgartner, R., Strobl, S., Marschall, M. (2013). Profiling of the kinome of cytomegalovirus-infected cells reveals the functional importance of host kinases Aurora A, ABL and AMPK. Antiviral Res. 99: 139-148.
- Hesse, J., Reyda, S., Tenzer, S., Besold, K., Reuter, N., Krauter, S., Büscher, N., Stamminger, T., Plachter, B. (2013). Human cytomegalovirus pp71 stimulates major histocompatibility complex class I presentation of IE1-derived peptides at immediate early times of infection. J. Virol. 87: 5229-38.
- Scherer, M., Reuter, N., Wagenknecht, N., Otto, V., Sticht, H., Stamminger, T. (2013). Small ubiquitin-related modifier (SUMO) pathway-mediated enhancement of human cytomegalovirus replication correlates with a recruitment of SUMO-1/3 proteins to viral replication compartments. J. Gen. Virol. 94: 1373-84.
- Ulbricht, T., Alzrigat, M., Horch, A., Reuter, N., von Mikecz, A., Steimle, V., Schmitt, E., Krämer, O.H., Stamminger, T., Hemmerich, P. (2012). PML promotes MHC class II gene expression by stabilizing the class II transactivator. J. Cell Biol. 199, 49-63.
- Frankenberg, N., Lischka, P., Pepperl-Klindworth, S., Stamminger, T., Plachter, B. (2012). Nucleocytoplasmic shuttling and CRM1-dependent MHC class I peptide presentation of human cytomegalovirus pp65. Med. Microbiol. Immunol., 567-79.
- Sörgel, S., Fraedrich, K., Votteler, J., Thomas, M., Stamminger, T., Schubert, U. (2012). Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of a G2-arrest. Virology 432, 444-51.
- Zielke, B., Wagenknecht, N., Pfeifer, C., Zielke, K., Thomas, M., Stamminger, T. (2012). Transfer of the UAP56 interaction motif of human cytomegalovirus pUL69 to ist murine cytomegalovirus homolog converts the protein into a functional mRNA export factor that can substitute for pUL69 during viral infection. J. Virol. 86, 7448-53.
- Full, F.*, Reuter, N.*, Zielke, K., Stamminger, T.*, Ensser, A.* (2012). Herpesvirus saimiri antagonizes nuclear domain 10-instituted intrinsic immunity via an ORF3-mediated selective degradation of cellular protein Sp100. J. Virol. 86, 3541-53. (*these authors contributed equally)
- Zydek, M., Uecker, R., Tavalai, N., Stamminger, T., Hagemeier, C., Wiebusch, L. (2011). General blockade of human cytomegalovirus immediate-early mRNA expression in the S/G2 phase by a nuclear, Daxx- and PML-independent mechanism. J. Gen. Virol. 92, 2757-69.
- Tavalai, N., Adler, M., Scherer, M., Riedl, Y., Stamminger, T. (2011). Evidence for a dual antiviral role of the major nuclear domain 10 component Sp100 during the immediate-early and late phases of the human cytomegalovirus replication cycle. J. Virol. 85, 9447-58.
- Adler, M., Tavalai, N., Müller, R., Stamminger, T. (2011). Human cytomegalovirus immediate-early gene expression is restricted by the nuclear domain 10 component Sp100. J. Gen. Virol. 92, 1532-8.
- Zakaryan, H., Stamminger, T. (2011). Nuclear remodelling during viral infections. Cell. Microbiol. 13, 806-13.
- Tavalai, N., Stamminger, T. (2011). Intrinsic cellular defense mechanisms targeting human cytomegalovirus. Virus Res. 157, 128-33.
- Jiang, M., Entezami, P., Gamez, M., Stamminger, T., Imperiale, M.J. (2011). Functional reorganization of promyelocytic leukemia nuclear bodies during BK virus infection. MBio. doi: 10.1128/mBio.00281-11.
- Tavalai, N., and Stamminger, T. (2009). Interplay between herpesvirus infection and host defense by PML nuclear bodies. Viruses 1(3): 1240-1265; doi: 10.3390/v1031240.
- Berndt, A., Hofmann-Winkler, H., Tavalai, N., Hahn, G., and Stamminger, T. (2009). Importance of covalent and noncovalent SUMO interactions with the major human cytomegalovirus transactivator IE2p86 for viral infection. J. Virol., 83, 12881-12894.
- Tavalai, N. and Stamminger, T. (2008). New insights into the role of the subnuclear structure ND10 for viral infection. Biochim Biophys Acta, 1783(11), 2207-2221.
- Tavalai, N., Papior, P., Rechter, S., and Stamminger, T. (2008). Nuclear domain 10 components promyelocytic leukemia protein and hDaxx independently contribute to an intrinsic antiviral defense against human cytomegalovirus infection. J Virol 82, 126-137.
- Tavalai, N., Kraiger, M., Kaiser, N., and Stamminger, T. (2008). Insertion of an EYFP-pp71 (UL82) coding sequence into the human cytomegalovirus genome results in a recombinant virus with enhanced viral growth. J Virol 82, 10553-10555.
- Sourvinos, G., Tavalai, N., Berndt, A., Spandidos, D. A., and Stamminger, T. (2007). Recruitment of human cytomegalovirus immediate-early 2 protein onto parental viral genomes in association with ND10 in live-infected cells. J Virol 81, 10123-10136.
- Tavalai, N., Papior, P., Rechter, S., Leis, M., Stamminger, T. (2006). Evidence for a role of the cellular ND10 protein PML in mediating intrinsic immunity against human cytomegalovirus infections. J Virol 80, 8006-8018