Thinc meeting location:Prague, Czech Republic date:29-30 January 2009
Lens epithelium-derived growth factor (LEDGF/p75) is a transcriptional co-activator high-jacked by the integrase (IN) of HIV-1 to tether the integration complex to the chromatin. Interference with the IN-LEDGF/p75 interaction adversely affects HIV-1 replication.
The molecular movement of eGFP-tagged LEDGF/p75 was tracked in live HeLa cells by means of continuous fluorescence photobleaching (CP) and fluorescence correlation spectroscopy (FCS). CP measurements revealed a concentration-dependent fraction of molecules that was more sensitive to photobleaching, consistent with a high affinity for chromatin. Importantly, the specific point mutations K56D and R74D in the PWWP-domain of LEDGF/p75 eliminated this photobleaching-sensitive fraction, suggesting a role for this domain in high affinity chromatin interaction. FCS measurements showed that LEDGF/p75 is continuously interacting with chromatin. Both PWWP mutants still displayed a very slow dynamical behaviour, albeit in a faster timescale than the wild type protein. Next, we co-expressed mRFP-tagged IN to verify its intracellular interaction with LEDGF/p75 by fluorescence cross-correlation spectroscopy (FCCS) and to study the LEDGF/p75-mediated chromatin anchoring of IN. In wild type HeLa cells the observed CP pattern for IN was consistent with IN being anchored to the chromatin. In cells with a stable shRNA-mediated knock-down of endogenous LEDGF/p75, IN did not show any significant bleaching. Finally, when we back-complemented these cells with eGFP-LEDGF/p75, mRFP-IN again showed an increased bleaching. These data prove that LEDGF/p75 is the chromatin anchor for IN. CP and fluorescence recovery after photobleaching (FRAP) experiments indicate that the affinity of the IN-LEDGF/p75 complex for chromatin is exceptionally high.
In conclusion, the following model can be proposed. LEDGF/p75 is almost permanently in contact with chromatin moving around by a dynamic sliding or hopping mechanism. On occasion, it encounters a high affinity binding site and stops. Soon after, it dissociates again from this site and continues its movement. In the presence of IN, however, a stably chromatin-anchored protein complex is formed. Although a more quantitative analysis of the chromatin-anchoring is part of ongoing research, we believe that this work is already useful for understanding the exact role of LEDGF/p75 during HIV integration.