Identification of functional inhibitors of HIV-1 Rev in astrocytes
Final Report Abstract
HIV-1 Rev stimulates the production of viral gene products, thereby being essential for HIV-1 replication. Rev activity is much weaker in astrocytes, which suppress HIV replication, than in cells capable of high-level virus production (e.g. HeLa cells). We hypothesize that host-cell dependent differences may exist in the interactions of Rev with cellular factors. However, interaction of Rev with cellular factors has not been investigated for astrocytes so far. Therefore the goal of this work was to identify Rev-interacting factors in astrocytes and to investigate their influence on HIV-l replication. We established an assay for capture of Rev-interacting proteins entailing chromatography of cell lysates through columns with immobilized recombinant Rev-bait proteins. This approach generated experimental evidence that Rev can interact with a group of multifunctional cellular RNA binding proteins. This group consist of at least five hnRNPs (hnRNP-A 1,-Q, -K, -R and -U) which alt feature an RGG box. Interaction required amino acids 9-14 of Rev, indicating that this sequence represents a new functional motif in Rev for interaction with cellular RNA binding proteins. Mutational analysis of the RGG-box of hnRNP-Al revealed that this domain is essential for interaction with Rev. Differences in the interactions of Rev with hnRNP-Al in astrocytic and HeLa cell lysates suggest the existence of cellular mechanisms for regulation of interactions of Rev with hnRNP-A 1. In conclusion, we identified an acidic motif at the Rev N-terminus that functions in recognition of Rev by multiple hnRNPs. This novel function of Rev may facilitate linkage of post-transcriptional processes of HIV late-phase gene expression with cellular machineries and may contribute to cellular regulafion of Rev-dependent gene expression.
Publications
- Analysis of the influence of subcellular localization of the HIV Rev protein on Rev-dependent gene expression by multi-fluorescence live-cell imaging. Experimental Celt Research, 2006,312(4):443-456
Wolff H, Hadian K, Ziegler M, Weierich C, Kramer-Hämmerle S, Kleinschmidt A, Erfle V and Brack-Werner R
- Live-celt assay for simultaneous monitoring of expression and interaction of proteins. Biotechniques 2006, 41 (6): 688,690,692
Wolff H, Hartl A, Hadian K, Ziegler M and Brack-Werner R
