Davis D A, Newcomb F M, Moskovitz J, Wingfield P T, Stahl S J, Kaufman J, Fales H M, Levine R L, Yarchoan R
HIV and AIDS Malignancy Branch, National Cancer Institute, 9000 Rockville Pike, National Institutes of Health, Bethesda, MD 20892-1906, USA.
Biochem J. 2000 Mar 1;346 Pt 2(Pt 2):305-11.
Human immunodeficiency viruses encode a homodimeric protease that is essential for the production of infectious virus. Previous studies have shown that HIV-1 protease is susceptible to oxidative inactivation at the dimer interface at Cys-95, a process that can be reversed both chemically and enzymically. Here we demonstrate a related yet distinct mechanism of reversible inactivation of the HIV-2 protease. Exposure of the HIV-2 protease to H(2)O(2) resulted in conversion of the two methionine residues (Met-76 and Met-95) to methionine sulphoxide as determined by amino acid analysis and mass spectrometry. This oxidation completely inactivated protease activity. However, the activity could be restored (up to 40%) after exposure of the oxidized protease to methionine sulphoxide reductase. This treatment resulted in the reduction of methionine sulphoxide 95 but not methionine sulphoxide 76 to methionine, as determined by peptide mapping/mass spectrometry. We also found that exposure of immature HIV-2 particles to H(2)O(2) led to the inhibition of polyprotein processing in maturing virus particles comparable to that demonstrated for HIV-1 particles. Thus oxidative inactivation of the HIV protease in vitro and in maturing viral particles is not restricted to the type 1 proteases. These studies indicate that two distinct retroviral proteases are susceptible to inactivation after a very minor modification at residue 95 of the dimer interface and suggest that the dimer interface might be a viable target for the development of novel protease inhibitors.
人类免疫缺陷病毒编码一种同二聚体蛋白酶，它对于感染性病毒的产生至关重要。先前的研究表明，HIV-1蛋白酶在二聚体界面的半胱氨酸-95处易受氧化失活作用，这一过程在化学和酶学方面均可逆转。在此，我们展示了HIV-2蛋白酶可逆失活的一种相关但又不同的机制。通过氨基酸分析和质谱测定发现，将HIV-2蛋白酶暴露于过氧化氢会导致两个甲硫氨酸残基（甲硫氨酸-76和甲硫氨酸-95）转化为甲硫氨酸亚砜。这种氧化作用完全使蛋白酶活性失活。然而，将氧化后的蛋白酶暴露于甲硫氨酸亚砜还原酶后，其活性能够恢复（高达40%）。通过肽图分析/质谱测定可知，这种处理使得甲硫氨酸亚砜95还原为甲硫氨酸，但甲硫氨酸亚砜76并未还原。我们还发现，将未成熟的HIV-2颗粒暴露于过氧化氢会导致成熟病毒颗粒中的多蛋白加工受到抑制，这与HIV-1颗粒的情况类似。因此，HIV蛋白酶在体外和成熟病毒颗粒中的氧化失活并不局限于1型蛋白酶。这些研究表明，两种不同的逆转录病毒蛋白酶在二聚体界面的95位残基发生非常微小的修饰后就易受失活影响，这表明二聚体界面可能是开发新型蛋白酶抑制剂的一个可行靶点。
