Human immunodeficiency virus type 1 variants resistant to first- and second-version fusion inhibitors and cytopathic in ex vivo human lymphoid tissue

Abstract

Human immunodeficiency virus type 1 (HIV-1) fusion inhibitors blocking viral entry by binding the gp41 heptad repeat 1 (HR1) region offer great promise for antiretroviral therapy, and the first of these inhibitors, T20 (Fuzeon; enfuvirtide), is successfully used in the clinic. It has been reported previously that changes in the 3-amino-acid GIV motif at positions 36 to 38 of gp41 HR1 mediate resistance to T20 but usually not to second-version fusion inhibitors, such as T1249, which target an overlapping but distinct region in HR1 including a conserved hydrophobic pocket (HP). Based on the common lack of cross-resistance and the difficulty of selecting T1249-resistant HIV-1 variants, it has been suggested that the determinants of resistance to first- and second-version fusion inhibitors may be different. To further assess HIV-1 resistance to fusion inhibitors and to analyze where changes in HR1 are tolerated, we randomized 16 codons in the HR1 region, including those making contact with HR2 codons and/or encoding residues in the GIV motif and the HP. We found that changes only at positions 37I, 38V, and 40Q near the N terminus of HR1 were tolerated. The propagation of randomly gp41-mutated HIV-1 variants in the presence of T1249 allowed the effective selection of highly resistant forms, all containing changes in the IV residues. Overall, the extent of T1249 resistance was inversely correlated to viral fitness and cytopathicity. Notably, one HIV-1 mutant showing approximately 10-fold-reduced susceptibility to T1249 inhibition replicated with wild type-like kinetics and caused substantial CD4+-T-cell depletion in ex vivo-infected human lymphoid tissue in the presence and absence of an inhibitor. Taken together, our results show that the GIV motif also plays a key role in resistance to second-version fusion inhibitors and suggest that some resistant HIV-1 variants may be pathogenic in vivo.

FIG. 1.

Random mutagenesis of the HIV-1 gp41 HR1 region. (A) Helical wheel diagram of the six-helix bundle (modified from reference with permission). The left panel shows the positions of heptad repeat residues in a cross-section of the helices in the gp41 core. The residues at the g and e positions in the N helices (N) and the a and d positions in the C helices (C) that are important for the interaction between the coiled-coil domain and the antiparallel external helices are highlighted in yellow and green, respectively. The right panel shows the amino acid residues at positions g and e of the N helix subjected to random mutagenesis. (B) Levels of infectivity of gp41-mutated HIV-1 NL4-3 variants containing randomized codons corresponding to the HR1 region. The amino acid sequences of the N-helical region and the positions of the randomized residues (denoted by X's) are indicated. Viral infectivity was measured by infecting TZM-bl cells with virus stocks containing normalized amounts (1 ng) of the p24 core antigen, and the data represent average values ± standard deviations derived from triplicate infections. The numbers to the right of the bars indicate the reductions (n-fold) in infectivity compared to that of the NL4-3 wt virus. Similar results were obtained in an independent experiment. RLU/s, relative light units per second. (C) Sequence analysis of the NL4-3 HR1 IV/XX-VQ/XX mutant mix. Proviral DNA encompassing the mutated positions was subjected to direct sequence analysis. The nucleotide and deduced amino acid sequences of the mutated region are shown. Similar results were obtained for the remaining nine HR1 and HP mutants except that complex nucleotide mixtures were observed at different positions.

FIG. 2.

Selection of T1249-resistant HIV-1 variants. (A) Levels of infectivity of the indicated HIV-1 NL4-3 stocks (see Fig. 1B for details). Viral infectivity was measured by infecting TZM-bl cells with virus stocks containing normalized amounts (1 ng) of the p24 core antigen, and data represent average values ± standard deviations derived from triplicate infections. HR1 wo IV-VQ, HR1 pool without the IV/XX-VQ/XX viral stock. (B) Changes in the HR1 region reduce the susceptibility of HIV-1 to fusion inhibitors. TZM-bl indicator cells were infected with wt NL4-3, the HR1 pool, or the IV/XX-VQ/XX viral stock in the presence of the indicated concentrations of T1249 or T20. Shown are average values derived from triplicate infections with virus stocks containing 20 ng of the p24 antigen. (C) Changes in the gp41 HR1 region allow efficient viral replication in the presence of T1249. CEMx174 5.25M7 cells were infected in triplicate with the indicated virus stocks in the presence of 50 nM T1249. At day 15, the concentration of the inhibitor was increased to 100 nM. The arrow indicates the point at which the gp41 region was amplified by RT-PCR from the cell-free culture supernatants. The replication curves indicate average levels of p24 derived from triplicate infections. (D) The selected T1249-resistant HIV-1 mutants contain alterations mainly or exclusively in the IV residues. Shown is the result of direct sequence analysis of the RT-PCR product obtained from the cell-free supernatant of CEMx174 5.25M7 cells infected with the HR1 pool mentioned in the legend to panel B. Codons that were subjected to random PCR mutagenesis are underlined in the nucleotide sequence.

FIG. 3.

Expression of wt and gp41 mutant Env proteins. Cell-free supernatants from transfected 293T cells were centrifuged to pellet viral particles, and the pellets were probed with antibodies against gp120, gp41, and p24. Similar results were obtained in an independent experiment.

FIG. 4.

Replication of T1249-resistant HIV-1 mutants. (A) Cumulative production of p24 by infected PBMC or PM1 cells. Values were measured at 3, 6, 9, 12, and 15 days after infection. Shown are representative levels of p24 production expressed as percentages of those measured in cultures infected with the wild-type virus. Similar results were obtained in independent experiments. uninf., uninfected. (B) Correlation between the replicative capacities and the levels of infectivity and T1249 resistance of gp41-mutated HIV-1 NL4-3 variants. Shown are the correlations between levels of p24 production in PBMC and PM1 cells (left panel), virus infectivity for TZM-bl cells and virus production by PBMC (left middle panel), virus infectivity and the IC₅₀ of T1249 (right middle panel), and viral replication in PBMC and the IC₅₀ (right panel). The values obtained for the L33V and L33S mutants are indicated by gray squares.

FIG. 5.

Replication and cytopathicity of gp41-mutated HIV-1 variants in lymphoid tissue ex vivo. (A) Representative replication kinetics of wt NL4-3 and gp41-mutated variants. uninf., unifected. (B) Average virus production. Matched tissues from seven donors were inoculated with the wt virus or with gp41-mutated variants as indicated, and for each condition, the cumulative production of p24 over 15 days was measured. Presented are means ± standard errors of the means of these measurements expressed as percentages of p24 production in cultures infected with the wt virus. (C) Correlation between virus production by infected PBMC and that by HLT ex vivo. (D) CD4⁺-T-cell depletion in HLT infected ex vivo with gp41-mutated HIV-1 variants. To evaluate CD4⁺-T-cell depletion, cells were mechanically isolated from matched control and infected tissues (18 pooled blocks for each variant) on day 15 postinfection, stained for CD3, CD4, CD8, and p24, and analyzed with flow cytometry as described previously (21, 22). Both T-cell populations in the tissue blocks (Blocks) and those that migrated in the gel foam (Foam) on which the tissues were incubated were analyzed. Presented are average depletion values ± standard errors of the means for tissues from seven donors. (E) Correlation between CD4⁺-T-cell depletion and p24 production in ex vivo-infected HLT cultures.

FIG. 6.

T1249-resistant HIV-1 causes CD4⁺-T-cell depletion in the presence of an inhibitor. (A) Cumulative p24 production over 15 days of HLT infection with the HIV-1 NL4-3 wt or the IV/VT mutant in the absence of an inhibitor (−) or in the presence of 50 nM T1249. Cumulative virus production was monitored at 3, 6, 9, 12, and 15 days postinfection. Results shown in panels A and B are average values ± standard errors of the means for tissues from three independent donors. (B) CD4⁺-T-cell depletion in the tissue blocks (Blocks) and among the cells that migrated in the gel foam (Foam) at the end of culture at 15 days postinfection. Tissue culture, infections, and fluorescence-activated cell sorter analysis were performed as described previously (21, 22).