SFB796 - Sub project C3

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Regulation of nuclear egress of human cytomegalovirus through a multifunctional viral-cellular protein complex

 

Project summary

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The replication of herpesviruses, including human cytomegalovirus (HCMV), is based on a nuclear phase followed by viral egress through the nuclear envelope. For nuclear egress, the transient destabilization of the nuclear envelope, notably the nuclear lamina, is an event with central importance. The destabilization of this rigid proteinaceous network is achieved by the function of lamina-associated effector proteins (HCMV egress proteins) which particularly recruit protein kinases to phosphorylate nuclear lamins. This process is reminiscent of a similar sequence of events occurring during cellular mitosis. In contrast to mitosis, however, nuclear destabilization during HCMV replication does not involve the entire nuclear envelope but is restricted to locally defined areas and this reprogramming seems to be achieved by a small number of viral effectors. Thus, the virus-specific nuclear destabilization is based on the formation and activity of a viral-cellular nuclear egress complex (NEC). The composition of the NEC is pivotal for the access of viral capsids to the inner nuclear membrane. The main goals fort he second funding period of this project are (i) isolation of the native NEC from HCMV-infected cells and analysis of NEC dynamics, (ii) elucidation of the functional nature of NEC components, in particular the viral kinase pUL97, (iii) definition of protein domains and structural elements responsible for multiple protein interactions of NEC components, and (iv) development of novel strategies of therapeutic intervention by targeting small inhibitory molecules to essential positions of the NEC. Collectively, these analyses will help to explain the reprogramming activity of the NEC that fills a key position in virus-cell interaction.




Model scheme describing the nuclear egress complex in cytomegalovirus-infected cells



Morphological alterations of the nuclear lamina in cytomegalovirus-infected human fibroblasts (confocal laser-scanning microscopy used for the detection of fluorescence signals: blue, Dapi; red, lamin A/C; green, autofluorescent HCMV particles)

 

Project relevant publications

  • Sonntag, E., Hamilton, S.T., Bahsi, H., Wagner, S., Jonjic, S., Rawlinson, W.D., Marschall, M. & Milbradt, J.   (2016).   Cytomegalovirus pUL50 is the multi-interacting determinant of the core nuclear egress complex (NEC) that recruits cellular accessory NEC components.   J. Gen. Virol.. [in press, Epub ahead of print, doi: 10.1099/jgv.0.000495 ].

  • Lamm, C., Schmitt, K., Link, K., Milbradt, J., Marschall, M. & Sonnewald, U.   (2016).   Human cytomegalovirus nuclear egress proteins ectopically expressed in a hetereologous environment are targeted to the nuclear envelope of plant cells.   Viruses, 8: 73, doi 10.3390.

  • 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: 144-151.

  • 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.   (2015b).   The broad-spectrum antiinfective drug artesunate interferes with the canonical nuclear factor kappa B (NF-κB) pathway by targeting RelA/p65.   Antiviral Res., 124: 101-109.

  • Walzer, S.A., Egerer-Sieber, C., Sticht, H., Sevvana, M., Hohl, H., Milbradt, M., Muller, Y.A. & Marschall, M.   (2015).   Crystal structure of the human cytomegalovirus pUL50-pUL53 core nuclear egress complex provides insight into a unique assembly scaffold for virus-host protein interactions.   J. Biol. Chem., 290: 27452-27458.

  • Reiter, C., Fröhlich, T., Zeino, M., Marschall, M., Bahsi, H., Leidenberger, M., Friedrich, O., Kappes, B., Hampel, F., Efferth, T. & Tsogoeva, S.B.   (2015).   New efficient artemisinin derived agents against human leukemia cells, human cytomegalovirus and Plasmodium falciparum: 2nd generation 1,2,4-trioxane-ferrocene hybrids.   Eur. J. Med. Chem. 97: 164-172.

  • Steingruber, M., Socher, E., Hutterer, C., Webel, R., Bergbrede, T. Lenac, T., Sticht, H. & Marschall, M.   (2015).   The interaction between cyclin B1 and cytomegalovirus protein kinase pUL97 is determined by an active kinase domain.   Viruses 7: 4582-4601.

  • Hutterer, C., Eickhoff, J., Milbradt, J., Korn, K., Zeitträger, I., Bahsi, H., Wagner, S., Zischinsky, G., Wolf, A., Degenhart, C., Unger, A., Baumann, M., Klebl, B. & Marschall, M.   (2015a).   A novel CDK7 inhibitor of the pyrazolo-triazine class exerts broad-spectrum antiviral activity at nanomolar concentrations.   Antimicrob. Agents Chemother. 59: 2062-2071.

  • Hamilton, S.T., Milbradt, J., Marschall, M. & Rawlinson, W.D.   (2014).   Human cytomegalovirus replication is strictly inhibited by siRNAs targeting UL54, UL97 or UL122 gene transcripts suggesting a high potential for novel siRNA-based antiviral strategies.   PLoS One 9: e97231.

  • Held, C., Webel, R., Palmisano, R., Hutterer, C., Marschall, M. & Wittenberg, T.   (2014).   Using multi channel level sets for the establishment of a measurement for the nuclear localization of pUL97 in GFP::β-gal fusion constructs.   J. Virol. Methods 199: 61-67.

  • Dell'Oste, V., Gatti, D., Gugliesi, F., De Andrea, M., Bawadekar, M., Lo Cigno, I., Vallino, M., Marschall, M., Gariglio, M. & Landolfo, S.   (2014).   Human cytomegalovirus escapes the antiviral activity of the restriction factor IFI16 by incorporating it into maturing virions.   J. Virol. 88: 9670-9682.

  • Milbradt, J., Kraut, A., Hutterer, C., Sonntag, E., Schmeiser, C., Ferro, M., Wagner, S., Lenac, T., Claus, C., Pinkert, S., Hamilton, S.T., Rawlinson, W.D., Sticht, H., Couté, Y. & Marschall, M.   (2014).   Proteomic analysis of the multimeric nuclear egress complex of human cytomegalovirus.   Mol. Cell. Proteomics, 13: 2132-2146.

  • Webel, R., Hakki, M., Prichard, M., Rawlinson, W.D., Marschall, M. & Chou, S.   (2014).   Differential properties of three isoforms of cytomegalovirus protein kinase pUL97 affect viral replication and maribavir susceptibility.   J. Virol. 88: 4776-4785.

  • Graf, L., Webel, R., Wagner, S., Hamilton, S.T., Rawlinson, W.D., Sticht, H. & Marschall, M.   (2013).   The cyclin-dependent kinase ortholog pUL97 of human cytomegalovirus interacts with cyclins.   Viruses 5: 3213-3230.

  • 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.

  • Schmeiser, C., Borst, E., Sticht, H., Marschall, M. & Milbradt, J.   (2013).   The cytomegalovirus egress proteins pUL50 and pUL53 are translocated to the nuclear envelope through two distinct modes of nuclear import.   J. Gen. Virol., 94: 2056-2069.

  • Marschall, M., Stamminger, T., Urban, A., Wildum, S., Ruebsamen-Schaeff, H., Zimmermann, H. & Lischka, P.   (2012).   In vitro evaluation of the activities of the novel anti-cytomegalovirus compound AIC246 (letermovir) against herpesviruses and other human pathogenic viruses.   Antimicrob. Agents Chemother. 56: 1135-1137.

  • Milbradt, M., Auerochs, S., Sevvana, M., Muller, Y.A. Sticht, H. & Marschall, M.   (2012).   Specific residues of a conserved domain in the N-terminus of human cytomegalovirus pUL50 protein determine its intranuclear interaction with pUL53.   J. Biol. Chem. 287: 24004-24016.

  • Webel, R., Solbak, S.M.Ø., Held, C., Milbradt, J., Groß, A., Eichler, J., Wittenberg, T., Jardin, C., Sticht, H., Fossen, T. & Marschall, M.   (2012).   The nuclear import of isoforms of the cytomegalovirus kinase pUL97 is mediated by differential activity of NLS1 and NLS2 both acting through classical importin-α binding.   J. Gen. Virol. 93: 1756-1768.

  • Auerochs, S., Korn, K. & Marschall, M.   (2011).   A reporter system for Epstein-Barr virus (EBV) lytic replication: anti-EBV activity of the broad anti-herpesviral drug artesunate.   J. Virol. Methods 173: 334-339.

  • Webel, R., Milbradt, J., Auerochs, S., Schregel, V., Held, C., Nöbauer, K., Razzazi-Fazeli, E., Jardin, C., Wittenberg, T., Sticht, H. & Marschall, M.   (2011).   Two isoforms of the protein kinase pUL97 of human cytomegalovirus are differentially regulated in their nuclear translocation.   J. Gen. Virol. 92: 638-649.

  • Becke, S., Fabre-Mersseman, V., Aue, S., Auerochs, S. Sedmak, T., Wolfrum, U., Strand, D., Marschall, M., Plachter, B. & Reyda, S.   (2010).   Modification of the major tegument protein pp65 of 1 human cytomegalovirus inhibits viral growth and leads to the enhancement of a protein complex with pUL69 and pUL97 in infected cells.   J. Gen. Virol. 91: 2531-2541.

  • Gershburg, S., Murphy, L., Marschall, M. & Gershburg, E.   (2010).   Key motifs in EBV (Epstein-Barr virus)-encoded protein kinase for phosphorylation activity and nuclear localization.   Biochem. J. 431: 227-235.

  • Milbradt, J., Webel, R., Auerochs, S., Sticht, H. & Marschall, M.   (2010).   Novel mode of phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus.   J. Biol. Chem. 285: 13979-13989.

  • Milbradt, M., Auerochs, S., Sticht, H. and Marschall, M.   (2009).   Cytomegaloviral proteins that associate with the nuclear lamina: components of a postulated nuclear egress complex.   J Gen Virol 90, 579-590.

  • Thomas, M., Rechter, S., Milbradt, J., Auerochs, S., Müller, R., Stamminger, T. and Marschall, M.   (2009).   The cytomegaloviral protein kinase pUL97 interacts with the nuclear mRNA export factor pUL69 to modulate its intranuclear localization and activity.   J Gen Virol 90, 567-578.

  • Milbradt, J., Auerochs, S. and Marschall, M.   (2007).   The cytomegaloviral proteins pUL50 and pUL53 are associated with the nuclear lamina and interact with cellular protein kinase C.   J Gen Virol 88, 2642-2650.

  • Romaker, D., Schregel, V., Maurer, K., Auerochs, S., Marzi, A., Sticht, H. and Marschall, M.   (2006).   Analysis of the structure-activity relationship of four UL97 subfamily herpesviral protein kinases reveals partial but not full functional conservation.   J Med Chem 49, 7044-7053.

  • Marschall, M., Marzi, A., aus dem Siepen, P., Jochmann, R., Kalmer, M., Auerochs, S., Lischka, P., Leis, M. and Stamminger, T.   (2005).   Cellular p32 recruits cytomegalovirus kinase pUL97 to redistribute the nuclear lamina. J Biol Chem 280, 33357-33367.