Mirjam Steingruber

Suche


C3 Molecular mode of interaction between the cytomegalovirus CDK ortholog pUL97 and cellular cyclins

Principal investigator
Manfred Marschall

Mentor
Heinrich Sticht

Molecular mode of interaction between the cytomegalovirus CDK ortholog pUL97 and cellular cyclins

Replication of human cytomegalovirus (HCMV) is characterized by a tight virus-host cell interaction. In particular, cyclin-dependent protein kinases (CDKs) are functionally integrated into viral gene expression and protein modification. The HCMV-encoded protein kinase pUL97 acts as a CDK ortholog showing structural and functional similarities. Recently, we reported a strong interaction of pUL97 with cyclin B1. The finding is surprising and important regarding that this is the only example of a herpesviral kinase-cyclin interaction identified so far that may illustrate a cross-talk between cyclins representing regulatory effectors of the cell cycle and viral replication. Importantly, we were able to demonstrate that the HCMV-encoded protein kinase pUL97 does not exclusively bind to one cyclin but undergoes interactions with several types of cyclins, which is reminiscent to the variable combinations of CDK-cyclin interaction. As a striking feature, the interaction between pUL97 and cyclin B1 is strictly dependent on pUL97 activity, i.e. our data demonstrate a block of cyclin B1 interaction by treatment with pUL97 inhibitors. Thus, the mechanism of pUL97-cyclin interaction is highly interesting and will be studied by a combination of intense bioinformatics and biochemical analyses, in particular focusing a putative impact of the interaction on the phosphorylation-dependent regulatory activities of pUL97. Especially, it will be challenging to ascertain the importance of pUL97-cyclin interaction in the context of viral replication.

 

Figure: Interaction between HCMV pUL97 and endogenous cyclin B1. (A) CoIP analysis showing an activity-dependent interaction between pUL97 and cyclin B1. pUL97-Flag and the catalytically inactive mutant pUL97(K355M)-Flag were transiently expressed in 293T cells. Controls included a separately expressed RFP (transfection control) as well as CoIP specificity controls such as an Fc fragment (Fc), pre-immune serum (PS) and a control sample without antibody (no). Optionally, one day before harvesting the cells MBV was added to the medium (1 µM or 3 µM). At two days post-transfection, cells were lysed and endogenous cyclin B1 was immunoprecipitated using a polyclonal antibody. Coimmunoprecipitates and expression control samples were subjected to Western blot analysis using the antibodies indicated. The precipitation control staining indicated the presence of constant levels of cyclin B1 in the precipitates and the expression control stainings illustrated reliable expression of all relevant proteins. β-actin staining was used as a loading control. CoIP, coimmunoprecipitation; IP antibody, immunoprecipitation antibody; WB, Western blot. (B and C) Intracellular colocalization between cyclin B1 and pUL97 determined by confocal imaging. HFFs were infected with HCMV AD169 (MOI approx. 0.5, B: panels 5-9, C: panels 1-12) or remained uninfected (B: mock, panels 1-4; fixed 48 hours post-infection, hpi). At 72 hpi (B) or 96 hpi (C), cells were subjected to indirect immunofluorescence analysis using cyclin B1- and pUL97-specific antibodies. Cell nuclei were counterstained with DAPI. Scale bars, 20 µm.

 

Publications

Hutterer, C., Hamilton, S., Steingruber, M., Zeitträger, I., Bahsi, H., Thuma, N., Naing, Z., Örfi, Z., Örfi, L., Socher, E., Sticht, H., Rawlinson, W., Chou, S., Haupt, V. J. and Marschall, M. (2016). The chemical class of quinazoline compounds provides a core structure for the design and optimization of anticytomegaloviral kinase inhibitors. Antiviral Res 134, 130-143

Steingruber, M., Socher, E., Hutterer, C., Webel, R., Bergbrede, T., Lenac, T., Sticht, H. and Marschall, M. (2015). The Interaction between Cyclin B1 and Cytomegalovirus Protein Kinase pUL97 is Determined by an Active Kinase Domain. Viruses 7(8), 4582-4601;

Steingruber, M., Kraut, A., Socher, E., Sticht, H., Reichel, A., Stamminger, T., Amin, B., Couté, Y., Hutterer, C. and Marschall, M. (2016). Proteomics-Based Exploration of the Cyclin Association of 2 the Cyclin-Dependent Kinase Ortholog pUL97 and further 3 Cytomegalovirus Proteins. Viruses.18 (8), 219.

 

Presentations 

October 2016

Virology Research Seminar, UNSW & Prince of Wales Hospital, Sydney, Australia; “Viral mimicry - human cytomegalovirus CDK orthologue pUL97”

Talk
     
May 2016 Methods in Molecular Virology, Institute of Virology, Erlangen, Germany
”New insights into the interaction between the viral CDK ortholog pUL97
and cellular cyclins”
Talk
     
April 2016 26th Annual Meeting of the Society for Virology, Münster, Germany
”First demonstration of a kinase activity-dependent interaction between the herpesviral CDK ortholog pUL97 and human cyclin B1”
Poster
     
October 2015 2nd International SFB 796 Conference: Mechanisms of microbial host cell manipulation: From plants to humans, Erlangen, Germany
”The importance of an active kinase domain of cytomegalovirus protein kinase pUL97 for the interaction with endogenous cyclin B1”
Poster
     
March 2015 25th Annual Meeting of the Society for Virology, Bochum, Germany
“Molecular mode of interaction between the cytomegalovirus protein kinase pUL97 and human cyclins”
Poster
     
January 2015 Methods in Molecular Virology, Institute of Virology, Erlangen, Germany
“Analysis of the interaction between HCMV protein kinase pUL97 and cyclins”
Talk