Nadine Wagenknecht

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B3 Interference of the viral effector proteins pp71 and IE1 with ND10-mediated intrinsic immunity against human cytomegalovirus infections

Principal investigator
Thomas Stamminger

Mentor
Alexander Steinkasserer

PhD exam: 23.03.2015

Contribution of ND10 to the regulation of HCMV latency in dendritic cell precursors

The nuclear domain 10 (ND10) has been discovered as a nuclear substructure that is targeted by a variety of viruses belonging to different viral families. ND10 domains are dynamic, spherical and macromolecular structures that are defined by the presence of the major components PML, hDaxx and Sp100 which accumulate in distinct foci within the interchromosomal space of the nucleus. Infection with human cytomegalovirus, a member of the beta-herpesvirus subfamily, results in the association of viral genomes and replication factors with ND10. Studies performed by several groups demonstrated that all three major ND10 proteins function as cellular restriction factors that are capable of repressing HCMV replication by inhibiting the initiation of viral immediate-early (IE) gene expression. This intrinsic defense mechanism against HCMV is antagonized by viral regulatory proteins. It was shown that the tegument protein pp71 is required for a proteasome-dependent degradation of hDaxx and that IE1 overcomes the PML-mediated repression by inducing the disruption of this subnuclear structure. It was also suggested that a failure of the tegument protein pp71 to antagonize hDaxx mediated repression may contribute to the establishment of viral latency.
The aim of my project is to investigate the role of individual ND10 proteins for the establishment and maintenance of HCMV latency in dendritic cell (DC) precursors which are regarded as the natural site of latent HCMV. Interestingly, differentiation of these cells to a mature DC phenotype is linked to reactivation of infectious virus. Thus, I will analyse the localization pattern as well as the expression profile of the major ND10 constituents in immature dendritic cells compared to mature dendritic cells by indirect immunofluorescence and western blot analysis, respectively. Furthermore, I will try to knockdown PML, hDaxx and Sp100 in dendritic cell precursors by the transduction of these cells with lentiviral shRNA vectors containing an additional EGFP cassette. The knockdown of individual ND10 proteins will be verified by confocal microscopy followed by infection of the transduced cells with clinical HCMV isolates like TB40E, in order to determine the relevance of these ND10 factors for the establishment of HCMV latency.

 

Figure: Indirect immunofluorescence analysis for the detection of ND10 proteins in DCs. PML, hDaxx and Sp100 form ND10-like accumulations in DCs.

 

Publications

Scherer, M., Reuter, N., Wagenknecht, N., Otto, V., Sticht, H. and Stamminger, T.(2013). SUMO pathway-mediated enhancement of human cytomegalovirus replication correlates with a recruitment of SUMO-1/3 proteins to viral replication compartments. J Gen Virol 94, 1373-1884.

Zielke, B., Wagenknecht, N., Pfeifer, C., Zielke, K., Thomas, M. and Stamminger, T. (2012). Transfer of the UAP56-interaction motif of human cytomegalovirus pUL69 to its murine cytomegalovirus homolog converts the protein into a functional mRNA-export factor that can substitute for pUL69 during viral infection. J Virol 86(13),7448-7453.

Kaiser, N., Lischka, P., Wagenknecht, N., Stamminger, T. (2009). Inhibition of human cytomegalovirus replication via peptide aptamers directed against the nonconventional nuclear localization signal of the essential viral replication factor pUL84. J Virol 83, 11902-11913.

 

Presentations

 

July 2014 6th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
“Infection of primary human monocytes with an endotheliotropic human cytomegalovirus strain results in a subpopulation of cells that initiate the lytic replication cycle”
Talk
     
July 2013 5th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
HCMV replication in monocytes and dendritic cells - role of the antiviral protein complex ND10
Talk
     
July 2013 38th Annual International Herpesvirus Workshop, Grand Rapids, Michigan, USA
Differentiation of monocytes to mature dendritic cells is associated with a profound structural alteration of the antiviral protein complex ND10
Poster
     
July 2013 European Seminars in Virology, Bertinoro, Italy
Differentiation of monocytes is associated with a profound structural alteration of the cellular antiviral protein complex ND10
Talk
     
June 2013 Methods in Molecular Virology, Erlangen
HCMV replication in monocytes and dendritic cells - role of the antiviral protein complex ND10
Talk
     
July 2012 4th Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Contribution of ND10 to the regulation of HCMV latency in dendritic cell precursors
Poster
     
June 2012 International Symposium: Fourty Years of Virology at the University of Erlangen-Nürnberg, Erlangen, Germany
Differentiation of monocytes to immature and mature dendritic cells is associated with a profound structural alteration of the antiviral protein complex ND10
Poster
     
March 2012 22nd Annual Meeting of the Society for Virology, Essen, Germany
Differentiation of monocytes to immature and mature dendritic cells is associated with a profound structural alteration of the antiviral protein complex ND10
Poster
     
October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
Analysis of the role of the major ND10 components PML, hDaxx and Sp100 for the regulation of HCMV latency in the dendritic cell precursors
Poster
     
July 2011 3rd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Contribution of ND10 to the regulation of HCMV latency in dendritic cell precursors
Talk and Poster
     
May 2011 13th International CMV/BetaHerpesvirus Workshop, Nueremberg, Germany
Analysis of the role of the major ND10 components PML, hDaxx and Sp100 for epigenetic regulation of the HCMV replication
Poster
     
April 2011 2nd Annual Spring Meeting of the SFB 796, Erlangen, Germany
Rolle von ND10 Proteinen für die Cytomegalovirus-Replikation in Monozyten und dendritischen Zellen
Talk
     

 

Awards

Best Talk Award

second place

6th Annual Retreat, Erlangen School of Molecular Communication Kloster Banz, Bad Staffelstein, Germany, July 2014
“Infection of primary human monocytes with an endotheliotropic human cytomegalovirus strain results in a subpopulation of cells that initiate the lytic replication cycle”