Identification of functional inhibitors of HIV-1 Rev in astrocytes

• Applicant: Professorin Dr. Ruth Brack-Werner
• Subject Area: Virology
• Term: from 2004 to 2009
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 5419952

The HIV Rev protein stimulates expression of HIV RNAs by enhancing stability, export, and translation of HIV target RNAs. Rev is crucial for replication of HIV. Rev accumulates in the nucleus and shuttles between nuclear and cytoplasmatic campartments by using active transport pathways. We have demonstrated that inefficient production of HIV in human astrocytes is associated with cytoplasmatic accumulation and retarded nuclear import kinetics of the Rev protein. Cell fusion experiments indicate that the inhibiton of nuclear import in astrocytes is caused by dominant negative factors. The goal of this collaborative project is to isolate and characterize Rev-interacting factors from astrocytes and to analyze their effects on nuclear import of Rev in an attempt to identify cellular inhibitors of Rev. We will adapt previously established nuclear import assay systems to monitor the inhibitory activity of cytoplasmatic extracts obtained from astrocytes on nuclear import of Rev. Rev-interacting factors will be isolated from cytoplasmatic extracts of astrocytes by affinity chromatography with immobilized Rev. In parallel we plan to isolate Rev-associated protein complexes from the cytoplasm of intact astrocytes, using tandem affinity purification (TAP) methodoloy. Isolated factors will be characterized with regard to their capacity to bind Rev, their influence on nuclear import and localization of Rev and their specific occurence in astrocytes or HeLa cells. Proteins will be identified by mass spectrometry. This work will contribute to understanding mechanisms of cellular control of HIV replication and may lead to novel anti-viral therapies.

• DFG Programme: Research Grants