The cells were incubated overnight with the supernatants containing pseudovirus. form a salt bridge with a negatively charged residue, Glu137 (E137), in gp41 CHR. Substitution CEP-37440 of R46 with the hydrophobic residue Ala (R46A) or the negatively charged residue Glu (R46E) resulted in disruption of the hydrogen bond network, breakage of the salt bridge and reduction of 6-HBs stability, leading to impairment of viral fusion and decreased inhibition of N36, an NHR peptide. Similarly, CHR peptide C34 with substitution of E137 for Ala (E137A) or Arg (E137R) also exhibited reduced inhibitory activity against HIV-1 infection and HIV-1-mediated cell-to-cell fusion. These results suggest that the positively charged residue R46 and its hydrogen bond network, together with the salt bridge between R46 and E137, are important for viral fusion and entry and may therefore serve as a target for designing novel HIV fusion/entry inhibitors. Introduction MMP7 The fusion of human immunodeficiency virus 1 (HIV-1) and its target cell is mediated by the envelope glycoprotein consisting of surface subunit gp120 and transmembrane subunit gp41 which are associated with non-covalent interactions [1]. To initiate infection, the gp120 binds to its receptor CD4 on the surface of the target cell and then to coreceptors (CCR5 or CXCR4), events which trigger a cascade of conformational changes of gp41, facilitating the fusion between membranes of HIV and its target cell [2], [3], [4]. The HIV-1 gp41 consists of three major functional domains, CEP-37440 including CEP-37440 the fusion peptide (FP), the N-terminal heptad repeat (NHR), and the C-terminal heptad repeat (CHR). The peptides derived from the NHR and CHR, e.g., N36 and C34, exhibited potent anti-HIV-1 activity ( Figure 1A ) [5], [6]. Previous studies have revealed that the conformation of gp120/gp41 complex finally changes from native state to a CEP-37440 hairpin state through a pre-hairpin fusion intermediate (PFI) [7]C[9]. In the fusion state, the residues at the and positions of one NHR domain interact with those at the and positions of another NHR domain to form N-helix trimer, while the residues at the and positions of one NHR domain interact with those at the and positions of the CHR domain to form a six-helix bundle (6-HB) core ( Figure 1B and C ), in which three N-terminal heptad repeats (NHR) form an interior, parallel coiled-coil trimer with three C-terminal heptad repeats (CHR) inserting into its highly conserved, hydrophobic cavity on the surface [5] ( Figure 1D ). Open in a separate window Figure 1 Schematic representations of the HIV-1 gp41 molecule, the core structure and the NHR/CHR interactions.(A) The functional domains in the gp41 molecule and the sequences of the NHR peptide N36 and the CHR peptide C34, as well as their mutants. (B) Interactions between the amino acid residues in the gp41 NHR and CHR. The black dashed lines between the NHR and CHR domains indicate the interaction between the residues located at the and the positions in the NHR and CHR, respectively. The red and pink solid lines represent the ionic interactions between R46 and E137, as well as K63 and D121, respectively, while the blue dotted line denotes the hydrogen bond between R46 and N43. Pocket-forming domain (PFD) in the NHR and pocket-binding domain (PBD) in CHR, as well as lipid-binding domain (LBD) in the MPER, are highlighted in green, violet and orange, respectively. (C) X-ray crystal structure of the HIV-1 gp41 6-HB core formed by N36 and C34 (adapted from [5]). NHR is colored in green, and CHR is colored in blue. (D) Model of the gp41 6-HB showing the locations of R46 and N43 in the N-helix wheel and E137 in the C-helix wheel. The residues located at the positions (yellow) in one of the N-helices interact.