Lens epithelium-derived growth factor/p75 prevents proteasomal degradation of HIV-1 integrase.
The transcriptional coactivator lens epithelium-derived growth factor (LEDGF)/p75 acts as a chromatin tethering factor for human immunodeficiency virus type 1 (HIV-1) integrase protein, determining its nuclear localization and its tight association with nuclear DNA. Here we identify a second function for the LEDGF/p75-integrase interaction. We observed that stable introduction of HIV-1 integrase (IN) transcription units into cells made stringently LEDGF/p75-deficient by RNAi resulted in much lower steady state levels of IN protein than introduction into LEDGF/p75 wild type cells. The same LEDGF/p75-dependent disparity was observed for feline immunodeficiency virus IN. However, IN 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 IN protein was found to be much shorter in LEDGF/p75-deficient cells. Proteasome inhibition fully countered this extreme instability, increasing IN protein levels to those seen in LEDGF/p75 wild type cells and implicating proteasomal destruction as the main cause of IN instability. Consistent with these data, increased ubiquitinated HIV-1 IN 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 IN is exclusively cytoplasmic in LEDGF/p75-deficient cells, but mainly nuclear in LEDGF/p75 wild type cells, and that cytoplasmic HIV-1 IN has a shorter half-life than nuclear HIV-1 IN. However, using LEDGF proteins defective for nuclear localization and IN interaction, we further determined that protection of HIV-1 IN from the proteasome requires neither chromatin tethering nor nuclear residence. Protection requires only interaction with LEDGF/p75, and it is independent of the subcellular localization of the IN-LEDGF complex.
Molecular Medicine Program and Department of Immunology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
Manuel
M
Llano
Sharon
S
Delgado
Maria
M
Vanegas
Eric M
EM
Poeschla
D015153:Blotting, Western; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D002843:Chromatin / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D019427:HIV Integrase / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D019426:Integrases / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins / Q000378:metabolism / Q000502:physiology*; D016086:Lentivirus / Q000235:genetics; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D061988:Proteasome Inhibitors; D011499:Protein Processing, Post-Translational; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism
D002843:Chromatin; D018076:DNA, Complementary; D036341:Intercellular Signaling Peptides and Proteins; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D012333:RNA, Messenger; D025801:Ubiquitin; C119380:lens epithelium-derived growth factor; D004247:DNA; D019427:HIV Integrase; D019426:Integrases; D046988:Proteasome Endopeptidase Complex