A global neutralization resistance phenotype of human immunodeficiency virus type 1 is determined by distinct mechanisms mediating enhanced infectivity and conformational change of the envelope complex

Abstract

We have described previously genetic characterization of neutralization-resistant, high-infectivity, and neutralization-sensitive, low-infectivity mutants of human immunodeficiency virus type 1 (HIV-1) MN envelope. The distinct phenotypes of these clones are attributable to six mutations affecting functional interactions between the gp120 C4-V5 regions and the gp41 leucine zipper. In the present study we examined mechanisms responsible for the phenotypic differences between these envelopes using neutralization and immunofluorescence assays (IFA). Most monoclonal antibodies (MAbs) tested against gp120 epitopes (V3, CD4 binding site, and CD4-induced) were 20 to 100 times more efficient at neutralizing pseudovirus expressing sensitive rather than resistant envelope. By IFA cells expressing neutralization sensitive envelope bound MAbs to gp120 epitopes more, but gp41 epitopes less, than neutralization-resistant envelope. This binding difference appeared to reflect conformational change, since it did not correlate with the level of protein expression or gp120-gp41 dissociation. This conformational change was mostly attributable to one mutation, L544P, which contributes to neutralization resistance but not to infectivity enhancement. The V420I mutation, which contributes a major effect to both high infectivity and neutralization resistance, had no apparent effect on conformation. Notably, a conformation-dependent V3 neutralization epitope remained sensitive to neutralization and accessible to binding by MAbs on neutralization-resistant HIV-1 envelope. Sensitivity to sCD4 did not distinguish the clones, suggesting that the phenotypes may be related to post-CD4-binding effects. The results demonstrate that neutralization resistance can be determined by distinguishable effects of mutations, which cause changes in envelope conformation and/or function(s) related to infectivity. A conformation-dependent V3 epitope may be an important target for neutralization of resistant strains of HIV-1.

FIG. 1

Chimera J constructed by exchanging fragments of previously constructed env genes (see Materials and Methods) (16, 17). (A) Schematic diagram of the HIV-1 env gene. Locations are indicated for the coding sequences for the cleavage site between gp120 and gp41, restriction enzyme recognition sequences, and landmarks in mature envelope proteins (V1 to V5, variable regions of gp120; F, fusion domain; LZ, leucine zipper-like alpha-helical domain; AH, membrane proximal alpha-helical domain; TM, transmembrane domain; C, cytoplasmic domain). (B) Mutant env genes. The V5 gene encodes the neutralization-sensitive envelope characteristic of the laboratory adapted MN virus. The E6 gene encodes the MN variant selected in vitro with the neutralization-resistant, high-infectivity phenotype. Chimera J comprises the N-terminal region from E6 which includes the coding sequences for the V1-V2 region, the junction region between gp120 and gp41 from the E6 clone (which encodes the neutralization resistance phenotype), and other regions from V5. (C) Specific mutations which are responsible for the E6 neutralization resistance phenotype.

FIG. 2

Comparative binding of the anti-V3 MAb, 257D, and the anti-gp41 MAb, 50-69, to cells expressing envelope glycoproteins of MN strain variant clones V5 and E6. Transfected 293T cells were prepared as described in Materials and Methods and analyzed for immunofluorescence by using flow cytometry. The percent fluorescence-positive cells (% Cells +) and median cell fluorescence (MCF) were calculated after background subtraction by using WinList software (2). Histograms are shown for total cell populations (unfilled areas) and positive cell populations (filled areas).

FIG. 3

Comparative binding of MAb to mutant MN strain envelopes. The E6 clone is neutralization resistant. The six mutations which encode this phenotype were introduced singly or in combinations into the neutralization sensitive V5 clone. Chimera J incorporates the neutralization resistance mutations and the V1-V2 regions from E6 into the V5 sequence (see Fig. 1). Immunofluorescence analyses were performed by using flow cytometry, as described in Materials and Methods. Results are expressed as ratios: median cell fluorescence (MCF) obtained in testing the indicated clones/MCF for V5 tested in parallel. Each point indicates the result of an individual experiment. The results shown for the antibody 48d were obtained after preincubation of the transfected cells with sCD4 at 10 μg/ml at 4°C for 30 min.