N-Myristoyltransferase isozymes exhibit differential specificity for human immunodeficiency virus type 1 Gag and Nef

Abstract

Myristoylation of the human immunodeficiency virus type 1 (HIV-1) proteins Gag and Nef by Nmyristoyltransferase (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. INTRODUCTIONAIDS is a growing problem worldwide, with UNAIDS estimating that 40 million individuals are currently infected by human immunodeficiency virus type 1 (HIV-1). No cure for AIDS exists, and new therapies are needed to combat the growing problem of HIV-1 drug resistance. Two important HIV-1 proteins, Gag and Nef, contain Nterminal myristic acid, which is added to the proteins during viral processing (Guy et al., 1987;Veronese et al., 1988). This protein lipidation occurs via transfer of a myristate moiety from myristoyl-CoA to the N terminus of the proteins through the action of the N-myristoyltransferase isozymes (NMT1 and NMT2). This involves recognition of an 8 aa target sequence at the N terminus of HIV-1 Gag and Nef proteins by NMT activity in the host cell.Gag is a structural protein that binds tightly to the plasma membrane upon myristoylation (Bryant & Ratner, 1990;Zhou et al., 1994). Plasma membrane binding of Gag initiates recruitment of Gag molecules, RNA and various viral proteins, ultimately supporting the budding of newly formed viral particles (Bouamr et al., 2003;Gottlinger et al., 1989). Importantly, it has been demonstrated that nonmyristoylated Gag mutants fail to assemble into active viral particles and are unable to bud from the cell surface (Bryant & Ratner, ...

Suppression of Human Immunodeficiency Virus Type-1 Production by Coexpression of Catalytic-Region-Deleted N-Myristoyltransferase Mutants

“…16,17) In other words, these results indicate that the production of HIV-1 WT depends on both NMT1 and NMT2, whereas the production of HIV-1 nefG2A depends on mainly NMT1. Since the competition of NMT1DC and NMT2DC with endogenous NMT isozymes for the targeting to the ribosome showed isozyme specificity (Fig.…”

“…Additionally, it has been suggested that each NMT isozyme has a specific role in vivo. [14][15][16][17] We therefore hypothesized that the lower identity of the N-terminal region between NMT1 and NMT2 is associated with the specific role of each isozyme at the ribosome. It was then examined whether the expressions of NMT1DC and NMT2DC can affect the ribosomal localization of endogenous NMT1 and NMT2.…”

“…14,15) Additionally, siRNA using studies suggested that NMT1 and NMT2 are respectively associated with the membrane targeting of Pr55 gag and Nef. 16,17) In a previous study of the NMT1 isozyme, it was reported that the N-terminal region is not required for the catalytic activity 11) but is associated with ribosomal targeting. 18) However, it remains to be elucidated whether the N-terminal region differs in property in between NMT1 and NMT2.…”

Acide myristique : nouvelles fonctions de régulation et de signalisation

“…Il est connu depuis plusieurs années que le domaine matriciel amino-terminal de Pr55 gag est myristoylé [45]. Des études récentes suggèrent que la protéine virale myristoylable Gag a une affinité différente pour les deux isoformes NMT1 et NMT2 présentes dans la cellule infectée mais ces résultats sont parfois discordants [46,47]. Cette myristoylation, associée à la présence d'un domaine polybasique, permet l'ancrage à la membrane du précurseur polyprotéique Pr55 gag , étape nécessaire au clivage par les protéases virales qui libère alors une protéine MA myristoylée mature ( Figure 5).…”

Protein myristoylation in health and disease

“…One recent report suggests that a drug may be able to exploit the distinct substrate profiles of human NMT isozymes [116]. The authors reported that both isozymes prefer Nef over Gag, with NMT2 showing increased affinity in vitro and in the cell.…”