Lens epithelium-derived growth factor (LEDGF)/p75 is a cellular cofactor for HIV-1 DNA integration. It is well established that the simultaneous binding of LEDGF/p75 to chromatin and to HIV-1 integrase is required for its cofactor activity. However, the exact molecular mechanism of LEDGF/p75 in HIV-1 integration is not yet completely understood. Our hypothesis is that evolutionarily conserved regions in LEDGF/p75 exposed to solvent and harboring posttranslational modifications may be involved in its HIV-1 cofactor activity. Therefore, a panel of LEDGF/p75 deletion mutants targeting these protein regions were evaluated for their HIV-1 cofactor activity, chromatin binding, integrase interaction, and integrase-to-chromatin-tethering activity by using different cellular and biochemical approaches. The deletion of amino acids 267 to 281 reduced the cofactor activity of LEDGF/p75 to levels observed for chromatin-binding-defective mutants. This region contains a serine cluster (residues 271, 273, and 275) recurrently found to be phosphorylated in both human and mouse cells. Importantly, the conversion of these Ser residues to Ala was sufficient to impair the ability of LEDGF/p75 to mediate HIV-1 DNA integration, although these mutations did not alter chromatin binding, integrase binding, or the integrase-to-chromatin-tethering capability of LEDGF/p75. These results clearly indicated that serine residues 271, 273, and 275 influence the HIV-1 cofactor activity of integrase-to-chromatin-tetheringcompetent LEDGF/p75.

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Lens epithelium-derived growth factor (LEDGF) proteins, p75 and p52, are transcriptional co-activators that connect sequence-specific activators to the basal transcription machinery. We have found that these proteins are post translationally modified by the Small Ubiquitin-like Modifier (SUMO)-1 and SUMO-3. Three SUMOylation sites, K75, K250 and K254, were mapped in the shared N-terminal region of these molecules, while a fourth site, K364, was identified in the C-terminal part exclusive of LEDGF/p75. The N-terminal SUMO targets are located in evolutionarily conserved charge-rich regions that lack resemblance to the described consensus SUMOylation motif, whereas the C-terminal SUMO target is solvent-exposed and situated in a typical consensus motif. SUMOylation did not affect the cellular localization of LEDGF proteins and was not necessary for their chromatin-binding ability, nor did it affect this activity. However, lysine to arginine mutations of the identified SUMO acceptor sites drastically inhibited LEDGF SUMOylation, extended the half-life of LEDGF/p75 and significantly increased its transcriptional activity on the Heat shock protein 27 (Hsp27) promoter, indicating a negative effect of SUMOylation on the transcriptional activity of LEDGF/p75. Considering that SUMOylation is known to negatively affect the transcriptional activity of all transcription factors known to transactivate Hsp27 expression, these findings support the paradigm establishing SUMOylation as a global neutralizer of cellular processes upregulated upon cellular stress.

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