SFB796  - Sub project A1
(sub project concluded on Dec 31 2012)

Suche


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

 

Project summary

The multi-functional viral protein R (Vpr), which represents a highly conserved regulatory protein of HIV-1, has been shown to interact with several host cell factors and a number of biological functions have been attributed to its presence in various cellular and extra cellular compartments. Vpr can be envisioned as a typical effector molecule since it deploys several functions in the virus replication cycle. Even more, due to its presence as a virus-free soluble molecule in the peripheral blood, and due to its capability to transduce cell membranes, Vpr can affect a variety of target cells, even those that can not be infected with HIV. Although dispensable for growth of HIV-1 in dividing cultured T-cells, Vpr appears to play an important role in virus replication in vivo. The most intensively investigated biological functions of Vpr are those affecting the (i) translocation of the pre-integration complex (PIC) of the incoming virus from the cytoplasm to the nucleus, the (ii) induction of a cell cycle arrest in the G2 phase, and the (iii) apoptosis in infected T-cells. Our recent structural investigation of the conformational heterogeneity of the proline residues in the N-terminus of Vpr suggested a functional interaction between Vpr and a host peptidyl-prolyl cis/trans isomerase (PPIase) that might regulate the interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. The subsequently identified physical interaction between Vpr and the major host PPIase, cyclophilin A (CypA) involves the N-terminal region of Vpr and includes an essential role of proline in position 35 (Pro-35). From these preliminary studies it was concluded that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA appears to be important for the de novo synthesis of Vpr and the Vpr-mediated cell cycle arrest. We will further investigate whether Pro-35, potentially in concert with CypA or other host cell chaperones, regulates the conformation of the hydrophobic core of the molecule, whose integrity is required for encapsidation of Vpr, and thus is necessary for a productive infection of macrophages. Since Vpr is present in several intra- and extracellular compartments we will identify further Vpr interacting host factors. Those potential binding partners include components of the ubiquitin proteasome system (UPS) and certain caspases.

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.

 

Project relevant publications

  • 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, 9572-9566.
  • Bruns, K., Fossen, T., Wray, V., Henklein, P., Tessmer, U. and Schubert, U. (2003). Structural characterization of the HIV-1 Vpr N terminus: evidence of cis/trans-proline isomerism. J Biol Chem 278, 43188-431201.
  • Sherman, M.P., Schubert, U., Williams, S.A., de Noronha, C.M., Kreisberg, J.F., Henklein, P. and Greene, W.C. (2002). HIV-1 Vpr displays natural protein-transducing properties: implications for viral pathogenesis. Virology 302,95-105.
  • Henklein, P., Bruns, K., Sherman, M.P., Tessmer, U., Licha, K., Kopp, J., de Noronha, C.M., Greene, W.C., Wray, V. and Schubert, U. (2000). Functional and structural characterization of synthetic HIV-1 Vpr that transduces cells, localizes to the nucleus, and induces G2 cell cycle arrest. J Biol Chem 275, 32016-32026.