Llano Manuel, Delgado Sharon, Vanegas Maria, Poeschla Eric M
Molecular Medicine Program and Department of Immunology, Mayo Clinic College of Medicine, 200 First Street SW, Rochester, MN 55905, USA.
J Biol Chem. 2004 Dec 31;279(53):55570-7. doi: 10.1074/jbc.M408508200. Epub 2004 Oct 8.
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.
转录共激活因子晶状体上皮衍生生长因子（LEDGF）/p75作为1型人类免疫缺陷病毒（HIV-1）整合酶蛋白的染色质拴系因子，决定其核定位以及与核DNA的紧密结合。在此，我们确定了LEDGF/p75-整合酶相互作用的第二种功能。我们观察到，通过RNA干扰使细胞严格缺乏LEDGF/p75后，将HIV-1整合酶（IN）转录单位稳定导入细胞，与导入LEDGF/p75野生型细胞相比，IN蛋白的稳态水平要低得多。对于猫免疫缺陷病毒IN，也观察到了同样依赖LEDGF/p75的差异。然而，无论有无LEDGF/p75，IN mRNA水平都是相当的。当发现HIV-1 IN蛋白在缺乏LEDGF/p75的细胞中的半衰期短得多时，证实了一种翻译后机制。蛋白酶体抑制完全抵消了这种极端的不稳定性，使IN蛋白水平增加到在LEDGF/p75野生型细胞中所见的水平，这表明蛋白酶体破坏是IN不稳定性的主要原因。与这些数据一致，在LEDGF/p75敲低的细胞中发现泛素化的HIV-1 IN增加。此外将LEDGF/p75恢复到敲低的克隆中可挽救HIV-1 IN的稳定性。亚细胞分级分离显示，HIV-1 IN在缺乏LEDGF/p75的细胞中仅存在于细胞质中，但在LEDGF/p75野生型细胞中主要存在于细胞核中，并且细胞质中的HIV-1 IN半衰期比细胞核中的HIV-1 IN短。然而，使用对核定位和IN相互作用有缺陷的LEDGF蛋白，我们进一步确定，保护HIV-1 IN免受蛋白酶体降解既不需要染色质拴系也不需要核定位。这种保护仅需要与LEDGF/p75相互作用，并且独立于IN-LEDGF复合物的亚细胞定位。
