Jens Milbradt

Suche


C3 Regulation of the cytomegalovirus nuclear egress through a viral-cellular multiprotein complex

Principal investigator
Manfred Marschall

Mentor
Thomas Stamminger

PhD exam: 18.10.2010

Functional characterization of a multiple protein complex which regulates the nuclear capsid egress of the human cytomegalovirus

The nucleo-cytoplasmic export of viral capsids is a crucial step in herpesviral replication. As recently demonstrated for human cytomegalovirus (HCMV), a nuclear egress complex (NEC) is composed by viral (Fig. 1) and cellular components. Importantly, the NEC recruits protein kinases to the nuclear envelope to induce a destabilization of the egress-limiting nuclear lamina by phosphorylation of nuclear lamins. The HCMV-encoded protein kinase pUL97 as well as the cellular protein kinase C (PKC) are involved in this process. As shown by in vitro kinase assays, pUL97 and PKC were capable of phosphorylating nuclear lamins. Various analyses of protein-protein interactions strongly suggested that the kinases are recruited to the NEC (consisting of at least pUL50, pUL53, p32, PKC, pUL97 and lamin B receptor) as the active components. During the PhD thesis, focus will be set on verifying the composition of the postulated NEC, characterizing involved protein interactions and revealing the functional relevance for HCMV nuclear egress. Verification of the complex composition will be accomplished by isolation of the native NEC from HCMV-infected cells following mass spectrometric analysis. Furthermore, characterization of protein interactions by mapping the interaction domains will provide better insight in the regulation of the NEC formation. Finally, confocal immunofluorescence analyses of transfected as well as HCMV-infected cells will be performed to reveal the ability of viral and cellular protein kinases to induce morpholocial alterations of the nuclear lamina, necessary for herpesviral nuclear capsid egress. Thus, a detailed examination of the NEC will not only contribute to a better understanding of cytomegaloviral nuclear egress, but also be essential for new approaches considering the NEC as a novel target for antiviral therapy.

Figure: Involvement of viral-encoded proteins pUL50 and pUL53 in the nuclear capsid egress of HCMV. (a) Schematic overview of herpesviral capsids leaving the nucleus of infected cells. Modified figure after Sanchez and Spector, 2002. (b) Predicted C-terminal transmembrane domain localizes pUL50 at the nuclear rim of transfected HeLa cells (panels a, b). pUL53 is recruited by pUL50 from a diffuse nuclear distribution (panels c, d) to a perfect colocalization with pUL50 at the nuclear rim upon coexression (panels e-h). (c) Interaction of pUL50 and pUL53. An HA-tagged version of pUL50, lacking its transmembrane domain, was transiently coexpressed in 293T cells with FLAG (F-) tagged pUL53 or pUL97, respectively. Western blot analysis demonstrates the ability of an HA-specific antibody, targeting pUL50(1-358)-HA, to coprecipitate pUL53, but not pUL97 (upper panel). This indicates a direct interaction of pUL50 with pUL53. Preimmune serum was used as a negative control to monitor specificity. Expression controls demonstrate the protein expression of transfected plasmids (lower panel).

 

Publications

Milbradt, J., Webel, R., Auerochs, S., Sticht, H. and Marschall, M. (2010). Novel mode of phosphorylation-triggered reorganisation of the nuclear lamina during nuclear egress of human cytomegalovirus. J Biol Chem. 285(18), 13979-13989.

Milbradt, J., Auerochs, S., Korn, K. and Marschall, M. (2009). Sensitivity of human herpesvirus 6 and other human herpesviruses to the broad-spectrum antiinfective drug artesunate. J Clin Virol 46, 24-28.

Thomas, M., Rechter, S., Milbradt, J., Auerochs, S., Müller, R., Stamminger, T. and Marschall, M. (2009). 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., 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.

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

 

Presentations

September 2010 2nd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Phosphorylation-triggered reorganization of the nuclear lamina during nuclear egress of human cytomegalovirus”
Talk
     
April 2010 4th European Congress of Virology, Cernobbio, Lake Como, Italy
Cytomegaloviral nuclear casid egress through lamina-depleted areas induced by viral and cellular kinase activity”
Poster and Talk
     
September 2009 First Annual Retreat, Erlangen School of Molecular Communication, Schloss Atzelsberg, Atzelsberg, Germany
Combined protein kinase activities of cytomegaloviral pUL97 and cellular PKC create lamina-free areas as potential sites for viral nuclear egress”
Talk
     
September 2009 Leica Symposium, Erlangen, Germany
Visualization of cytomegaloviral nuclear capsid egress by confocal imaging”
Talk
     
July 2009 34th International Herpesvirus Workshop (IHW 2009), Ithaca, USA
“Combined protein kinase activities of cytomegaloviral pUL97 and cellular PKC create lamina-free areas as potential sites for viral nuclear egress”
Poster
     
March 2009 19th Annual Meeting of the Society for Virology, Leipzig, Germany
“Activity of cytomegaloviral protein kinase pUL97 creates lamina-free areas as potential sites of nuclear egress”
Poster and Talk
     
June 2008 6th International Conference on HHV-6 & 7, Baltimore, USA
“Sensitivity of HHV-6 and other human herpesviruses towards the pluripotent drug artesunate”
Talk
     
March 2008 18th Annual Meeting of the Society for Virology, Heidelberg, Germany
“The nuclear egress complex of human cytomegalovirus: a network of lamina-associated proteins and protein kinases”
Talk
     
May 2008 11th International CMV and Beta Herpesvirus Workshop, Toulouse, France
“The cytomegaloviral UL50 and UL53 proteins interact with each other and recruit PKC to the nuclear lamina"
Poster
     
September 2007 3rd European Congress of Virology, Nuremberg, Germany
“Association of the cytomegaloviral proteins UL50 and UL53 with the nuclear envelope and specific recruitment of PKC”
Poster