Background:  It has been shown that Nef increases the viral load in the infected host by enhancing viral infectivity and particle release. Whether Nef-mediated signalling in T cells directly targets viral transcription is not clear.

Methods:  A yeast 2-hybrid system was used to screen for a novel Nef-interacting protein. The shuttling activity of the identified Eed protein was confirmed by microinjection and heterokaryon assays. Co-immunoprecipitation experiments, immunofluorescence techniques, and confocal microscopy was used to validate the Nef- and integrin-dependent recruitment of Eed to the cytoplasm. The association of Eed with the LTR and the Nef- and integrin-dependent removal was verified by CHIP assay. Transcriptional activation after transient transfection was measured using luciferase reporter gene assays.

Results:  Our previous results suggested that proteins binding to the Nef N-terminus are required for viral replication. Employing a yeast 2-hybrid screen using the N-terminal a helix of Nef we identified the Polycomb group protein Eed as a Nef-interacting protein. This finding was rather unusual as Eed, a member of a chromatin-silencing complex, was so far known to be strictly nuclear. However, we demonstrated that Eed can shuttle between nucleus and cytoplasm. Moreover, we found that this redistribution could be induced upon Nef expression. Also, the activation of integrins, which had been shown to interact with Eed via their cytoplasmic tails, induced Eed to relocate to the cytoplasm. The repressive Eed factor associates with the HIV LTR  and is removed upon Nef expression as well as integrin activation. Furthermore we  showed that this removal leads to an increase in Tat-dependent transcription.

Conclusions:  The Nef-dependent removal of the inhibitory Eed factor from the LTR may constitute a de-repression signal that allows efficient Tat-dependent transcription to promote viral replication. To induce this effect Nef obviously mimicks a physiological integrin receptor signal.

Keywords: Nef; viral replication; Eed