Seaton Kelly E, Smith Charles D
Department of Pharmacology, Penn State College of Medicine, Hershey, PA, USA.
Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, SC, USA.
J Gen Virol. 2008 Jan;89(Pt 1):288-296. doi: 10.1099/vir.0.83412-0.
Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by N-myristoyltransferase (NMT) is a key process in retroviral replication and virulence, yet remains incompletely characterized. Therefore, the roles of the two isozymes, NMT1 and NMT2, in myristoylating Gag and Nef were examined using biochemical and molecular approaches. Fluorescently labelled peptides corresponding to the N terminus of HIV-1 Gag or Nef were myristoylated by recombinant human NMT1 and NMT2. Kinetic analyses indicated that NMT1 and NMT2 had 30- and 130-fold lower K(m )values for Nef than Gag, respectively. Values for K(cat) indicated that, once Gag or Nef binds to the enzyme, myristoylation by NMT1 and NMT2 proceeds at comparable rates. Furthermore, the catalytic efficiencies for the processing of Gag by NMT1 and NMT2 were equivalent. In contrast, NMT2 had approximately 5-fold higher catalytic efficiency for the myristoylation of Nef than NMT1. Competition experiments confirmed that the Nef peptide acts as a competitive inhibitor for the myristoylation of Gag. Experiments using full-length recombinant Nef protein also indicated a lower K(m) for Nef myristoylation by NMT2 than NMT1. Small interfering RNAs were used to selectively deplete NMT1 and/or NMT2 from HEK293T cells expressing a recombinant Nef-sgGFP fusion protein. Depletion of NMT1 had minimal effect on the intracellular distribution of Nef-sgGFP, whereas depletion of NMT2 altered distribution to a diffuse, widespread pattern, mimicking that of a myristoylation-deficient mutant of Nef-sgGFP. Together, these findings indicate that Nef is preferentially myristoylated by NMT2, suggesting that selective inhibition of NMT2 may provide a novel means of blocking HIV virulence.
N-肉豆蔻酰基转移酶（NMT）对人类免疫缺陷病毒1型（HIV-1）蛋白Gag和Nef进行肉豆蔻酰化是逆转录病毒复制和毒力的关键过程，但仍未完全明确其特征。因此，使用生化和分子方法研究了两种同工酶NMT1和NMT2在对Gag和Nef进行肉豆蔻酰化中的作用。与HIV-1 Gag或Nef N端对应的荧光标记肽被重组人NMT1和NMT2进行肉豆蔻酰化。动力学分析表明，NMT1和NMT2对Nef的K(m)值分别比对Gag低30倍和130倍。K(cat)值表明，一旦Gag或Nef与酶结合，NMT1和NMT2进行肉豆蔻酰化的速率相当。此外，NMT1和NMT2处理Gag的催化效率相当。相比之下，NMT2对Nef进行肉豆蔻酰化的催化效率比NMT1高约5倍。竞争实验证实，Nef肽是Gag肉豆蔻酰化的竞争性抑制剂。使用全长重组Nef蛋白的实验还表明，NMT2对Nef进行肉豆蔻酰化的K(m)比NMT1低。小干扰RNA被用于从表达重组Nef-sgGFP融合蛋白的HEK293T细胞中选择性耗尽NMT1和/或NMT2。耗尽NMT1对Nef-sgGFP的细胞内分布影响最小，而耗尽NMT2则将分布改变为弥漫、广泛的模式，类似于Nef-sgGFP肉豆蔻酰化缺陷突变体的模式。总之，这些发现表明Nef优先被NMT2肉豆蔻酰化，这表明选择性抑制NMT2可能提供一种阻断HIV毒力的新方法。
