Effect of mutations in the human immunodeficiency virus type 1 protease on cleavage of the gp41 cytoplasmic tail

Abstract

We previously reported that human immunodeficiency virus type 1 (HIV-1) develops resistance to the cholesterol-binding compound amphotericin B methyl ester (AME) by acquiring mutations (P203L and S205L) in the cytoplasmic tail of the transmembrane envelope glycoprotein gp41 that create cleavage sites for the viral protease (PR). In the present study, we observed that a PR inhibitor-resistant (PIR) HIV-1 mutant is unable to efficiently cleave the gp41 cytoplasmic tail in P203L and S205L virions, resulting in loss of AME resistance. To define the pathway to AME resistance in the context of the PIR PR, we selected for resistance with an HIV-1 isolate expressing the mutant enzyme. We identified a new gp41 mutation, R236L, that results in cleavage of the gp41 tail by the PIR PR. These results highlight the central role of gp41 cleavage as the primary mechanism of AME resistance.

FIG. 1.

PIR mutations abrogate the ability of the P203L and S205L gp41 mutations to confer AME resistance. (A) 293T cells were transfected with WT pNL4-3 (WT) (1) or AME-resistant Env mutants (P203L and S205L) (27, 28) encoding WT or PIR PR. The PIR substitutions (L10R/M46I/L63P/V82T/I84V) (10) were introduced into the pNL4-3/P203L and pNL4-3/S205L mutants by subcloning the 3,737-bp ApaI-EcoRI fragment containing the PIR changes as described previously (2). Virus-containing supernatants were harvested and virions were concentrated by ultracentrifugation (14). Viral lysates were subjected to immunoblotting with the 2F5 monoclonal anti-gp41 antibody (11). Molecular mass markers are shown on the left. NTF, gp41 N-terminal fragment. (B) TZM-bl cells were infected with virus stocks produced by transfecting 293T cells with molecular clones encoding WT or AME-resistant Env mutants (P203L, S205L) and WT or PIR PR. Infections were carried out for 2 h in the absence or presence of 1, 5, or 10 μM AME. Infected cells were washed and cultured in the absence of AME; luciferase activity was measured 2 days postinfection. The infectivity of the PIR mutant was similar to that of the WT. Data shown are means ± standard errors (SEs) (n = 4).

FIG. 2.

The R236L mutation confers resistance to AME in the context of WT or PIR PR. (A) Identification of the R236L mutation. The env region was PCR amplified from genomic DNA purified at the peak of viral replication from WT- and PIR-infected Jurkat cultures treated with AME, and the PCR product was sequenced as described previously (27). The organization of HIV-1 Env is indicated, with an arrowhead showing the cellular protease cleavage site between gp120 and gp41. Locations of the R236L mutation and the previously described AME-resistant mutants (P203L, S205L) selected in the context of WT PR (27, 28) are shown. MSD, membrane-spanning domain. (B) TZM-bl cells were infected with virions bearing WT or R236L Env and WT or PIR PR. The R236L mutant was constructed by QuikChange site-directed mutagenesis (Stratagene), and the PIR mutations were introduced into the pNL4-3/R236L clone as described in the legend to Fig. 1. Infections were carried out in the absence or presence of 1, 5, or 10 μM AME as described in the legend to Fig. 1. Data shown are means ± SEs of the results from three independent experiments performed in duplicate. The relative infectivities of the NL4-3/PIR, NL4-3/R236L, and NL4-3/PIR/R236L mutants, compared to that of the WT, were 97%, 87%, and 72%, respectively. (C) Five million Jurkat cells were transfected with 5 μg of the indicated molecular clones (5 μg) by using the DEAE-dextran transfection procedure and were cultured in the absence or continual presence (+) of 5 μM AME. Cultures were split 1:3 every 2 days, and supernatants were reserved for RT assay at each time point.

FIG. 3.

Cleavage of the R236L gp41 CT by WT and PIR PR. (A) 293T cells were transfected with WT or the R236L mutant molecular clones encoding WT or PIR PR. Cell and viral lysates were prepared as described previously (28) and subjected to immunoblotting with the 2F5 anti-gp41 monoclonal antibody. To evaluate the size of the truncated gp41 from the R236L Env mutant, gp41 CT truncation mutants CTdel-144 (144) and CTdel-104 (104) (13, 16, 20) were included for comparison. (B) 293T cells were transfected with WT or AME-resistant mutants (P203L, S205L, R236L, PIR/R236L), and viral lysates were subjected to immunoblotting with the Fitzgerald polyclonal anti-gp41 antibody or the anti-gp41 monoclonal antibody NEA-9303 (19) as described previously (28). Positions of gp160, gp41, and the gp41 N-terminal fragment (NTF) and C-terminal fragment (CTF) are shown. On the right side of the Fitzgerald blot is an overexposed lane of the R236L mutant (R236L o.e.) that shows the ≈13-kDa CTF for this mutant. Molecular mass markers are indicated.

FIG. 4.

Identification of the PR cleavage site in R236L gp41. (A) Separation of viral proteins by HPLC. Virions were prepared in 293T cells by transfection with pNL4-3 derivatives encoding WT (WT) or R236L Env (R236L) mutants with WT or PIR PR (PIR/R236L). Virions were concentrated by ultracentrifugation, and viral proteins were separated by HPLC as described previously (28); the A₂₈₀ chromatograms are shown. Major viral protein peaks identified by immunoblot or protein sequence analysis are labeled. Peaks representing matrix (MA), capsid (CA), full-length gp41, gp41 NTF, and gp41 CTF are shown. The NTF and CTF derived from R236L and PIR/R236L virions were subjected to Edman degradation, and the N-terminal sequences were analyzed by using an automated 477 protein sequencer (Applied Biosystems, Foster City, CA). (B) Cleavage site in the R236L gp41. The organization of HIV-1 Env is as shown in Fig. 2A, and the position of the residue 236 mutation is indicated in bold. The PR-mediated cleavage site between gp41 residues 236 and 237 is indicated by an arrow.

FIG. 5.

The P204L and R237L mutations in the AD8 strain of HIV-1 confer resistance to AME. (A) 293T cells were transfected with WT pAD8 or the P204L and R237L AD8 mutants constructed by QuikChange site-directed mutagenesis (Stratagene). One day posttransfection, viral lysates were prepared as described in the legends to Fig. 1 and 3 and were subjected to immunoblotting with the 2F5 anti-gp41 monoclonal antibody. In this experiment, NL4-3 and its AME-resistant derivatives (P203L and R236L) were included as controls. (B) TZM-bl cells were infected with virus stocks derived from WT NL4-3 or AD8 or the indicated mutants. Infections were carried out in the absence or presence of 1, 5, or 10 μM AME as described in the legend to Fig. 1. Data shown are means ± SEs of the results from three independent experiments performed in duplicate.