Stefan Sörgel

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A1 Interaction of the HIV-1 regulatory virus protein R (Vpr) with host cell factors

Principal investigator
Ulrich Schubert

Mentor
Manfred Marschall

PhD exam: 28.03.2012

Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2 arrest and virus replication

The HIV-1 accessory protein Vpr induces G2 arrest, apoptosis and facilitates the transport of the HIV-1 preintegration-complex into the nucleus of non-dividing cells. Vpr, which localizes in the nucleus, in mitochondria and in the cytoplasm, is known to interact with members of the nuclear pore complex, especially nucleoporin, and alters its permeability during induction of apoptosis. It was reported by others that Vpr accumulation at the nuclear lamina is dependent on the integrity of the N-terminal helix-α1 in Vpr, which is important for its capacity to induce G2 arrest. Thus, we set out to investigate the role of the nuclear transport of Vpr and the permeability of the nuclear pore for the function of Vpr. Treatment of HeLa cells with the caspase 3 inhibitor Z-DEVD caused accumulation of Vpr at the nuclear lamina. As caspases regulate the pore morphology during induction of apoptosis, reduced permeability may result from an altered pore complex assembly caused by the caspase 3 inhibition. Consistently, HIV-1 infected Jurkat cells showed an increased induction of G2 arrest when treated with Z-DEVD. Mutation of the proline 35 in the N-terminal part of Vpr, which is important for the formation of helix-α1, abrogated Z-DEVD mediated enhancement of G2 arrest. Replication analysis of HIV-1 in PBMCs revealed that Z-DEVD treatment elevates virus replication. These results indicate that the shuttling of Vpr between nucleus and cytoplasm is dependent on the permeability of the nuclear pores. Furthermore, caspase 3 activity influences the localization of Vpr and the induction of G2 arrest by Vpr, which ultimately affects the Vpr dependent HIV-1 replication in primary cells.

Figure: Influence of Caspase 3 inhibition on induction of Vpr mediated G2 arrest. Jurkat T-cells infected with HIV-1NL4-3, HIV-1NL4-3VprP35A or HIV-1NL4-3DVpr were treated with 40µM Z-DEVD every 3rd day. At peak of infection (day 12 p.i.), productively infected cells were identified by immunostaining with FITC conjugated anti-CA antibodies. Infected cells were then assayed for DNA content by staining with To-Pro 3 and analyzed by FACS, indicating that accumulation of Vpr at the nuclear envelope is essential for induction of G2-arrest. Vpr induced G2-arrest is elevated in HIV-1NL4-3 infected cells after Z-DEVD treatment. The HIV-1 VprP35A mutant induced G2-arrest at the same level as the untreated wt strain. However, the induction of G2-arrest was not elevated by inhibition of Caspase 3. The enhancing effect of Z-DEVD on induction of G2-arrest depends on the presence of Vpr, in as much as the Vpr deletion mutant did not induce G2-arrest whether Caspase 3 was inhibited or not.

 

Publications

Votteler, J., Iavnilovitch, E., Fingrut, O., Shemesh, V., Taglicht, D., Erez, O., Sörgel, S., Walther, T., Bannert, N., Schubert, U. and Reiss, Y. (2009). Exploring the functional interaction between POSH and ALIX and the relevance to HIV-1 release. BMC Biochem 10, 12.

Votteler, J., Studtrucker, N., Sörgel, S., Münch, J., Rücker, E., Kirchhoff, F., Schick, B., Henklein, P., Fossen, T., Bruns, K., Sharma, A., Wray, V. and Schubert, U. (2007). Proline 35 of human immunodeficiency virus type 1 (HIV-1) Vpr regulates the integrity of the N-terminal helix and the incorporation of Vpr into virus particles and supports the replication of R5-tropic HIV-1 in human lymphoid tissue ex vivo. J Virol 81, 9572-9576.

 

Presentations

October 2011 First International SFB 796 Conference: Mechanisms of viral host cell manipulations: from plants to humans, Bamberg, Germany
Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2 arrest
Talk and Poster
     
July 2011 3rd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2 arrest
Talk and Poster
     
September 2010 2nd Annual Retreat, Erlangen School of Molecular Communication, Kloster Banz, Bad Staffelstein, Germany
Perinuclear localisation and cellular interaction partners of the HIV-1 regulatory protein Vpr
Talk
     
September 2009 First Annual Retreat, Erlangen School of Molecular Communication, Schloss Atzelsberg, Atzelsberg, Germany
Perinuclear localisation and cellular interaction partners of the HIV-1 regulatory protein Vpr
Talk
     
May 2009 Cold Spring Harbor Laboratory Meeting “Retroviruses”, Cold Spring Harbor, U.S.A.
Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2 arrest
Poster
     
March 2009 19th Annual Meeting of the Society for Virology. Leipzig, Germany
Perinuclear localization of the HIV-1 regulatory protein Vpr is important for induction of G2 arrest and virus replication
Poster
     
March 2008 18th Annual Meeting of the Society for Virology. Heidelberg, Germany
POSH and ALIX cooperate to facilitate efficient HIV-1 budding
Poster
     
September 2007 3rd European Congress of Virology. Nuremberg, Germany
Proline 35 of HIV-1 Vpr regulates incorporation in virus particles and is required for replication in macrophages.
Poster