Rike Webel

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

Principal investigator
Manfred Marschall

Mentor
Heinrich Sticht

PhD exam: 04.04.2014

Functional analysis of the protein kinase pUL97 of human cytomegalovirus regarding regulatory protein interactions

The replication of 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 lamina, a rigid proteinaceous network of the nuclear envelope, is required. This step is achieved by the function of lamina-associated proteins which particularly recruit protein kinases (PKs) such as the viral PK pUL97 to phosphorylate nuclear lamins. The viral PK pUL97 is a multifunctional determinant of the efficiency of viral replication and phosphorylates viral as well as cellular substrate proteins. As recently identified, pUL97 is expressed in two isoforms with molecular masses of approximately 90 and 100 kDa. ORF UL97 comprises an unusual coding strategy in that five in-frame ATG start codons are contained within the N-terminal 157 amino acids. Site directed mutagenesis, transient expression of point and deletion mutants and proteomic analyses accumulated evidence that the formation of the large and small isoforms results from alternative initiation of translation with start points at amino acids 1 and 74, respectively. In vitro kinase assays demonstrated that catalytic activity, in terms of autophosphorylation and histone substrate phosphorylation, was indistinguishable for the two isoforms. An analysis of intracellular distribution of pUL97 by confocal laser-scanning microscopy demonstrated that both isoforms have a pronounced nuclear localization. Surprisingly, mapping experiments performed to identify the nuclear localization signal (NLS) of pUL97 strongly suggest that the mechanism of nuclear transport is distinct for the two isoforms. While the extreme N-terminus (large isoform) comprises a highly efficient, bipartite NLS (amino acids 6 to 35), a second sequence apparently conferring a less efficient mode of nuclear translocation was identified downstream of amino acid 74 (small and large isoforms). Combined, the findings argue for a complex mechanism of nuclear translocation of pUL97 which might be linked with fine-regulatory differences between the two isoforms. The functional importance of the two pUL97 isoforms, during viral nuclear egress and other replicative processes, will be further analyzed.

 

 

Figure: Schematical description of the cytomegaloviral protein kinase pUL97. This scheme depicts the functionally important regions of pUL97, as summarizing the data of studies published by several research groups: the nuclear localization signals (NLS), the kinase domain, the interaction domains for the cellular protein p32, the tumor suppressor protein Rb (three binding motifs), the viral DNA polymerase processivity factor pUL44, the viral nuclear mRNA export factor pUL69, the nucleoside analog ganciclovir, the pUL97 self-interaction domain and the unknown interaction site for nuclear lamins.

 

Publications

Webel, R., Solbak, S. M., Held, C., Milbradt, J., Gross, A., Eichler, J., Wittenberg, T., Jardin, C., Sticht, H., Fossen, T. and 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 alpha binding. J Gen Viorol 93, 1756-1768.

Held, C., Wenzel, J., Webel, R., Marschall, M., Lang, R., Palmisano, R. and Wittenberg, T. (2011). Using Multimodal Information for the Segmentation of Fluorescent Micrographs with Application to Virology and Microbiology. Conf Proc IEEE Eng Med Biol Soc, pp. 6487-6490.

Webel, R., Milbradt, J., Auerochs, S., Schregel, V., Held, C., Nobauer, K., Razzazi-Fazeli, E., Jardin, C., Wittenberg, T., Sticht, H. and 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.

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, 13979-13989.

 

Presentations

July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Cytomegalovirus protein kinase pUL97: formation of three isoforms and their relevance for efficient viral replication
Talk
     
July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Characterization of the HCMV protein kinase pUL97 highlighting differences in the isoform-specific nuclear import
Talk
     
July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Specification of the HCMV pUL97 isoforms and their differences in nuclear import
Poster
     
June 2012 International Symposium: Fourty Years of Virology at the University of Erlangen-Nürnberg, Erlangen, Germany
Specification of the HCMV pUL97 isoforms and their differences in nuclear import
Poster
     
March 2012 22nd Annual Meeting of the Society for Virology, Essen, Germany
Two NLS sequences within the cytomegaloviral protein kinase pUL97 regulate the nuclear import of both isoforms through the classical importin alpha/beta pathway
Talk
     
October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
Characterization of the nuclear import mechanism of two isoforms of the HCMV protein kinase pUL97
Poster
     
July 2011 3rd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Nuclear transport regulation of two isoforms of the cytomegalovirus protein kinase pUL97
Talk and Poster
     
May 2011 13th International CMV/BetaHerpesvirus Workshop, Nueremberg, Germany
Identification of two NLS sequences within the HCMV protein kinase pUL97 differentially regulating the nuclear translocation of two isoforms
Poster
     
March 2011 21st Annual Meeting of the Society for Virology, Freiburg, Germany ,
The HCMV protein kinase pUL97 contains two NLS sequences responsible for regulation of nuclear translocation of two isoforms
Poster
     
November 2010 Methods in Molecular Virology, Institute of Virology, Erlangen, Germany
The two pUL97 isoforms of HCMV are differentially regulated in their nuclear translocation
Talk
     
July 2010 35th International Herpesvirus Workshop (IHW 2010), Salt Lake City, USA
The UL97 gene of human cytomegalovirus encodes two isoforms showing regulatory similarities and differences
Poster
     
May 2009 Methods in Molecular Virology, Institute of Virology, Erlangen, Germany
Functional analysis of isoforms and deletion mutants of the HCMV protein kinase pUL97
Talk