Amino acid 36 in the human immunodeficiency virus type 1 gp41 ectodomain controls fusogenic activity: implications for the molecular mechanism of viral escape from a fusion inhibitor

Abstract

We have previously described a human immunodeficiency virus type 1 (HIV-1) proviral clone, pL2, derived from defective viral particles with higher fusogenicity than the prototypic NL4-3 virus. In this study, we attempted to determine the region that confers the enhanced fusion activity by creating envelope recombinants between pL2 and pNL4-3, as well as point mutants based on pNL4-3. The results indicate that amino acid 36 of gp41 is key for the fusogenic activity and infectivity enhancement and that glycine 36 (36G) of gp41 in pL2 is conserved in nearly all HIV-1 isolates except for pNL4-3. The mutation 36G-->D in a primary-isolate-derived Env decreased syncytium-forming activity and infectivity. The assays for cell-cell fusion and viral binding suggested that the enhanced fusion mediated by the 36D-->G mutation is not due to increased binding efficiency but is directly due to actual enhancement of viral fusion activity. Interestingly, this amino acid position is exactly equivalent to that at which the mutation of HIV-1 isolates that have escaped from a fusion inhibitor, enfuvirtide (T-20), has been frequently observed. The correlation between these previous findings and our findings was suggested by structural analysis. Our finding, therefore, has implications for a molecular basis of the viral escape from this drug.

FIG. 1.

Construction of gp120 and gp41 chimeric full-length proviral DNAs and their syncytium-forming activities. An HIV-1 envelope cassette vector, pNL-envCT (WT), harbors MroI, BlpI, and NotI restriction enzyme sites immediately upstream of the gp120, gp41, and nef genes of pNL4-3, respectively. The envelope gp160, gp120, and gp41 genes of pLAI and pL2 were replaced with the corresponding regions of pNL-envCT. MOLT-4, M8166, CEMx174, or H9 cells (5 × 10⁴) were incubated with 200 ng of p24 antigen of each virus. After 48 h, cells were fixed and observed by microscopy. Arrows indicate the formation of syncytia.

FIG. 2.

Construction of gp41 recombinants and point mutants and their syncytium-forming activities and infectivities. (A) Chimeric viruses carrying three different regions of gp41 derived from pLAI or pL2 were constructed by utilizing the restriction enzyme sites BlpI, HindIII, BamHI and NotI. (B) Amino acid substitutions (21C→A, 22T→R, 22T→A, 36D→G, or 91L→F) were introduced within the BlpI-HindIII region of gp41 by site-directed mutagenesis. Asterisks show mutant constructs harboring a mutation commonly seen in both pLAI and pL2. The syncytium-formation assay (A and B) was performed as described for Fig. 1. Arrows indicate the formation of syncytia. (C) To determine infectivity, 5 × 10⁵ MAGIC5A cells were infected with 10 ng of NL-Luc-envCT (WT) or NL-Luc-D36G (D36G), harboring the Luc gene in place of nef. After 48 h, cells were lysed and subjected to Luc assay. Averages from three independent experiments with standard deviations are indicated. RLU, relative light units.

FIG. 3.

Effect of the amino acid change at position 36 in gp41 in a primary-isolate-derived Env. (A) A syncytium formation assay was performed with MOLT-4 cells inoculated with NL-1549 (1549-WT) viruses carrying a primary-isolate (QH1549)-derived Env or with NL-1549-G36D (1549-G36D) viruses harboring the amino acid change 36G→D in 1549 gp41. Arrows indicate the formation of syncytia. (B) Infectivities of NL-Luc-1549 (1549-WT) and NL-Luc-1549-G36D (1549-G36D) viruses were measured by Luc assay as described for Fig. 2C. Averages from three independent experiments with standard deviations are indicated. RLU, relative light units.

FIG. 4.

Comparisons of cell-cell fusion (A) and viral binding (B) between WT and D36G mutant Envs. (A) 293T cells as the effector cells were transfected with pNLnΔBs (WT) or pNLnΔBs-D36G (D36G) and Tat expression plasmids, while MAGIC5A cells as the target cells were transfected with pLTR-hLucP+. Forty-eight hours later, both cell types were washed, trypsinized, and cocultured. After 5 h, cells were lysed and subjected to Luc assay. Averages from three independent experiments with standard deviations are indicated. RLU, relative light units. (B) MAGIC5A cells (1.5 × 10⁶) were incubated with medium containing 100 ng of NL-Δenv, NL-envCT (WT), or NL-gp41-D36G (36G) viruses at 4°C. After 3 h, cells were extensively washed with ice-cold PBS three times and lysed with lysis buffer. Cell lysates were analyzed for p24 levels by ELISA. The background obtained with NL-Δenv viruses was subtracted from sample values. Averages from three independent experiments with standard deviations are indicated.

FIG. 5.

Molecular modeling of HIV-1 gp41 ectodomain trimer. The 3-D model of an HIV-1 gp41 ectodomain trimer was constructed by the homology modeling technique using the MOE. HIV-1 gp41 ectodomain trimers of D36G (upper left) and NL4-3 (upper right) are displayed. Target portions of each trimer around amino acid position 36 are shown with side chains of position 36 in the N-peptide region and related amino acids within the C-peptide region. Arrows indicate the side chains of N and C helices of D36G (lower left) and NL4-3 gp41 (lower right).

FIG. 6.

A model of the HIV-1 Env-mediated fusion mechanism and a putative difference in hairpin formation between D36G and NL4-3 Envs. This model is adapted from that of Koshiba and Chan (22). Binding of gp120 to CD4 and a coreceptor (not shown) induces a conformational change in gp120 that allows exposure of gp41 fusion peptide (red) and its penetration into the target cellular membrane and allows the formation of the prehairpin intermediate (upper panel). As shown in the lower left panel, hairpin (six-helix bundle) formation occurs between trimeric N-peptide (gray) and C-peptide (blue) regions. Enlargements of putative six-helix bundles of D36G and NL4-3 gp41 ectodomains are displayed in the lower right panel.