Cathrin Schmeiser

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

Principal investigator
Manfred Marschall

Mentor
Thomas Stamminger

PhD exam 13.12.2013

Studies on the functional role of cellular protein kinases during nuclear egress of human cytomegalovirus

The export of cytomegaloviral capsids from the nucleus to the cytoplasm is limited by the natural barrier of the nuclear envelope. In particular, the protein network of the nuclear lamina constitutes an obstacle, which can be overcome via the virus-induced nuclear egress complex (NEC). The nuclear lamina is not static, but underlies dynamic changes during the different phases of the cell cycle. Those changes are mediated by site-specific phosphorylation which is accomplished by various cellular kinases like cyclin-dependent kinases (CDKs) and protein kinase C (PKC). Usually CDK1 is responsible for the disassembly of the lamina in mitosis while PKC plays a similar role during apoptosis. During the replication of human cytomegalovirus (HCMV), PKC and the viral kinase pUL97 are known functional constituents of the NEC and lead to the disassembly of the nuclear lamina by phosphorylating the lamins. Notably, conserved herpesviral protein kinases such as pUL97 recognize very similar Ser-specific phosphorylation motifs within the substrate as cellular CDKs. For this reason, it has been repeatedly assumed that CDK1 (or likewise the closely related CDK2) may also be involved in the disassembly of the lamina during the nuclear egress of HCMV.
The Ph.D. thesis will specifically address the question whether CDK1, CDK2 or other cellular protein kinases contribute to the nuclear egress of HCMV by dissolving the nuclear lamina via phosphorylation of the lamins. A first screening for potential interaction between known NEC proteins and CDKs as well as cyclins will be accomplished by yeast two hybrid studies. In the next step, recombinant cytomegaloviruses will be created by BACmid recombination for the production of epitope-tagged NEC components pUL50 and pUL53. In addition to their use in coimmunoprecipitation analyses with CDKs and cyclins, they can be particularly helpful for the isolation of the NEC from cells infected with the recombinant HCMVs and a subsequently performed mass spectrometry analysis. Another approach to illustrate possible influences of CDK activity on NEC formation and the nuclear egress of HCMV will be the application of CDK- or other kinase inhibitors in analyzing the morphology of HCMV-infected cells. During the course of infection, the NEC components accumulate in a characteristic ring pattern around the nuclear lamina, which is visible by fluorescence labeling in confocal laser scanning microscopy. If CDK activity influences the NEC formation, its inhibition by classical or novel CDK inhibitors may change this pattern. Finally, the potential phosphorylation of NEC proteins by CDKs will be studied in an in vitro kinase assay. These experimental settings may confirm the postulated involvement of CDKs in HCMV nuclear egress.

 

 

Figure: Involvement of the viral proteins pUL50 and pUL53 as well as cellular protein kinases in the nuclear egress of HCMV capsids. (A) Schematic overview of confirmed and speculated involvement of viral and cellular proteins and protein kinases in the formation of the NEC. (B) MRC-5 cells were infected with recombinant HCMVs expressing GFP as a reporter as well as HA-tagged pUL50 or FLAG-tagged pUL53. In uninfected cells (Mock), pUL50- or pUL53-specific antibodies showed no or only marginal background signal (panels a-h), whereas in HCMV-infected cells, the typical localization of pUL50 and pUL53 along the nuclear rim became visible. This could be shown with pUL50- or pUL53-specific antibodies (panels m-p and u-x) as well as with tag-specific antibodies (panels i-l and q-t).

 

Publications

Schmeiser, C., Borst, E., Sticht, H., Marschall, M. and 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, in press.

 

Presentations

 

July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Mechanistic and functional aspects of the nuclear import of the two cytomegalovirus nuclear egress core proteins
Talk
     
July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Cytomegalovirus egress proteins pUL50 and pUL53 translocate to the nuclear rim in an interaction-mediated fashion and associate with cellular proteins
Poster
     
June 2012 International Symposium: Fourty Years of Virology at the University of Erlangen-Nürnberg, Erlangen, Germany
Functional analysis of pUL50 and pUL53 by use of recombinant cytomegaloviruses: pUL50-pUL53 interaction, nuclear translocation and association with cellular proteins
Poster
     
October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
Functional analysis of pUL50 and pUL53 by use of recombinant cytomegaloviruses: pUL50-pUL53 interaction, nuclear translocation and association with cellular proteins
Poster
     
December 2011 Method-Seminar, Institute of Virology, Erlangen, Germany
Study of the HCMV nuclear egress proteins pUL50 and pUL53 - regulation of nuclear localization and functional protein interactions
Talk
     
July 2011 3rd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Functional analysis of pUL50 and pUL53 by use of recombinant cytomegaloviruses
Talk
     
July 2011 3nd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Functional analysis of pUL50 and pUL53 by use of recombinant cytomegalo-viruses: pUL50-pUL53 interaction, nuclear translocation and association with cellular proteins
Poster
     
March 2011 21. Annual Meeting of Society for Virology, Freiburg, Germany
Generation of recombinant cytomegaloviruses with tagged egress proteins pUL50 and pUL53 for the analysis of cellular protein kinases involved in viral nuclear egress
Poster
     

 

Awards

Poster Award
3rd Annual Retreat of the Erlangen School of Molecular Communication
Kloster Banz, Bad Staffelstein, Germany, July 2011
Functional analysis of pUL50 and pUL53 by use of recombinant cytomegaloviruses.