Background:  The transcriptional co-activator LEDGF/p75 acts as a chromatin-tethering factor for HIV-1 integrase protein, determining its nuclear localization and its tight association with nuclear DNA. We observed that stable introduction of HIV-1 integrase transcription units into cells made stringently LEDGF/p75-deficient by RNAi resulted in much lower steady state levels of integrase protein than introduction into LEDGF/p75 wild type cells.

Methods:  We determined by immunoblotting the levels of lentiviral integrases in wild type and LEDGF/p75 deficient cells and in cells treated with proteasome inhibitors or co-transfected with different LEDGF/p75 mutants. We also evaluated full-length IN mRNA levels by RT-PCR.

Results:  The initial observation that the expression of HIV integrase in LEDGF/p75-deficient cells resulted in much lower steady state levels of integrase protein than in wild type cells was verified by immunoblotting in different clones. The same LEDGF/p75-dependent disparity was observed for FIV integrase. However, integrase mRNA levels were equivalent in the presence and absence of LEDGF/p75. A post-translational mechanism was confirmed when the half-life of HIV-1 integrase protein was found to be much shorter in LEDGF/p75-deficient cells. Proteasome inhibition fully countered this extreme instability, increasing integrase protein levels to those seen in LEDGF/p75-wild type cells and implicating proteasomal destruction as the main cause of integrase instability. Consistent with these data, increased ubiquitinated HIV-1 integrase was found in the LEDGF/p75 knock-down cells. Moreover, restoration of LEDGF/p75 to knocked-down clones rescued HIV-1 IN stability. Subcellular fractionation showed that HIV-1 integrase is exclusively cytoplasmic in LEDGF/p75-deficient cells, but mainly nuclear in LEDGF/p75-wild type cells and that cytoplasmic HIV-1 integrase has a shorter half-life than nuclear HIV-1 integrase. However, using LEDGF proteins defective for nuclear localization and integrase interaction, we further determined that protection of HIV-1 integrase from the proteasome requires neither chromatin tethering nor nuclear residence. Protection requires only interaction with LEDGF/p75.

Conclusions:  Here we identify a second function for the LEDGF/p75-integrase interaction. LEDGF/p75 protects HIV integrase from proteasome degradation. Protection requires integrase-LEDGF/p75 interaction and it is independent of the subcellular localization of this protein complex. We postulated that LEDGF/p75 prevents the recognition of HIV integrase by an E3 ligase.

Keywords: HIV integrase; LEDGF/p75; Proteasome