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 e, g and the a, d 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 a, d positions (yellow) in one of the N-helices interact with those at the d, a positions (yellow) in another N-helix, respectively, resulting in formation of the NHR-trimer. The residues located at the e, g positions (red) in one of the N-helices associate with those at the a, d positions in one of the C-helices, respectively, leading to the formation of 6-HB.
Figure 2.
Analysis of putative interactions of R46 in the gp41 NHR with the residues in the 6-HB formed between C34 and N36 or their mutants.
(A) The 6-HB formed between C34 and N36; (B) The 6-HB formed between C34 and N36 R46A; (C) The 6-HB formed between C34 and R46E; (D) The 6-HB formed between C34 E137A and N36; and (E) The 6-HB formed between C34 E137R and N36. The putative interactions via hydrogen bond or salt bridge were predicted by using the MOE program. NHRs are colored in blue; CHR is colored in green.
Table 1.
Using the MOE program to predict hydrogen bonds and salt bridges in 6-HB formed by C34 and N36, or N36’s mutants, or by N36 and C34, or C34’s mutants.
Figure 3.
CD spectrographic analysis of the complexes formed between N36 and C34 or their mutants.
(A) The secondary structures of N36 and its mutants; (B) The secondary structures of the complexes formed by C34 and N36 or N36’s mutants; (C) The secondary structures of the complexes formed by N36 and C34 or C34’s mutants; and (D) The stability of complexes formed by C34 with N36 and its mutants, as measured by thermal denaturation analysis.
Figure 4.
Interaction of C34 with N36 and its mutants to form 6-HB, as determined by FN-PAGE.
C34 was labeled with FAM (carboxyfluorescein, Molecular Probes, Eugene, OR), which could be viewed under UV light.
Figure 5.
Effect of R46 mutations in gp41 NHR region on viral infectivity and Env expression.
(A) Infectivity of HIV-1 pseudoviruses carrying wild-type N36 and its mutants. Luciferase assay was used to measure the single-cycle infection of pseudovirus on TZM-bl cells. (B) Expression of gp120 and gp160 on pseudoviruses bearing wild-type N36 sequence and its mutants as determined by Western blot.
Table 2.
Inhibition of the peptides N36, C34 and their mutants on HIV-1IIIB-mediated cell-cell fusion and HIV-1 IIIB infection.