N-terminal substitutions in HIV-1 gp41 reduce the expression of non-trimeric envelope glycoproteins on the virus

Abstract

The native, functional HIV-1 envelope glycoprotein (Env) complex is a trimer of two non-covalently associated subunits: the gp120 surface glycoprotein and the gp41 transmembrane glycoprotein. However, various non-functional forms of Env are present on virus particles and HIV-1-infected cells, some of which probably arise as the native complex decays. The aberrant forms include gp120-gp41 monomers and oligomers, as well as gp41 subunits from which gp120 has dissociated. The presence of non-functional Env creates binding sites for antibodies that do not recognize native Env complexes and that are, therefore, non-neutralizing. Non-native Env forms (monomers, dimers, tetramers and aggregates) can also arise when soluble gp140 proteins, lacking the cytoplasmic and transmembrane domains of gp41, are expressed for vaccine studies. We recently identified five amino acids in the gp41 N-terminal region (I535, Q543, S553, K567 and R588) that promote gp140 trimerization. We have now studied their influence on the function and antigenic properties of JR-FL Env expressed on the surfaces of pseudoviruses and Env-transfected cells. The 5 substitutions in gp41 reduce the expression of non-trimeric gp160s, without affecting trimer levels. Pseudovirions bearing the mutant Env are fully infectious with similar kinetics of Env-mediated fusion. Various non-neutralizing antibodies bind less strongly to the Env mutant, but neutralizing antibody binding is unaffected. Hence the gp41 substitutions do not adversely affect Env structure, supporting their use for making new Env-based vaccines. The mutant Env might also help in studies intended to correlate antibody binding to virus neutralization. Of note is that the 5 residues are much more frequent, individually or collectively, in viruses from subtypes other than B.

Fig.1

Effect of gp41 N-terminus substitutions on Env incorporation into pseudovirions. The JR-FL WT and gp41 NT 1–5 mutant viruses were produced by transfection of HEK 293T cells and pelleted from clarified supernatants. The gp120, gp41 and p24 proteins were resolved by SDS-PAGE and analyzed by Western blotting with the appropriate antibodies.

Fig.2

Effect of gp41 N-terminal changes on the Env forms present on pseudovirions. (A) Virions, normalized for p24 content and expressing either the JR-FL WT or gp41 NT 1–5 mutant Envs, were solubilized then analyzed under native conditions on a 4–12% Bis-Tris NuPAGE gel and Western blotted with the anti-gp120 MAb ARP3119. Env tetramers and dimers are highlighted with black arrows, trimers with a grey arrow. (B) The histogram shows the relative proportions of the different Env forms present on the WT (black bars) and mutant (grey bars) pseudovirions. The densitometric data represents the Mean ± Standard Deviation of values from four independent experiments.

Fig.3

Effect of gp41 substitutions on soluble CD4- and temperature-induced gp120 shedding from pseudovirions. (A) Pseudovirions expressing the JR-FL WT or gp41 NT 1–5 mutant Envs were incubated for 2h with the sCD4 concentrations indicated, at either 4°C or 37°C. (B) The pseudovirions were incubated for 2h at the temperatures indicated, in the absence of sCD4. In both experiments, the H×B2 Env-pseudotyped virus served as a reference standard. The amount of virion-bound Env is expressed relative to that present on each virus in the absence of sCD4 at 4°C (= 100%).

Fig.3

Effect of gp41 substitutions on soluble CD4- and temperature-induced gp120 shedding from pseudovirions. (A) Pseudovirions expressing the JR-FL WT or gp41 NT 1–5 mutant Envs were incubated for 2h with the sCD4 concentrations indicated, at either 4°C or 37°C. (B) The pseudovirions were incubated for 2h at the temperatures indicated, in the absence of sCD4. In both experiments, the H×B2 Env-pseudotyped virus served as a reference standard. The amount of virion-bound Env is expressed relative to that present on each virus in the absence of sCD4 at 4°C (= 100%).

Fig.4

Effect of gp41 N-terminal substitutions on Env-pseudotyped virus infectivity. Pseudovirions, containing normalized amounts of p24 antigen and bearing the WT or mutant forms of JR-FL Env, were serially diluted and used to infect U87.CD4.CCR5 cells. Infectivity was quantified by measuring luciferase activity 4 days post infection.

Fig.5

Effect of gp41 N-terminal substitutions on Env-mediated cell-cell fusion. The kinetics of cell-cell fusion mediated by the WT (black squares) and mutant (grey triangles) forms of JR-FL Env were determined in a β-lactamase reporter assay using HeLa-CD4/CCR5 cells expressing either relatively high (JC53 cells; panels A and B) or low levels of both levels of CD4 and CCR5 (RC49 cells; panels C and D). The extent of fusion is expressed as the percentage of the maximal fusion mediated by each Env (panels A and C), or of the maximal fusion mediated by the WT Env (panels B and D). The data represent the mean ± standard error of three independent experiments. The various kinetic parameters are described in Table 2.

Fig.6

Effect of gp41 N-terminal substitutions on the binding of MAbs to pseudovirions. Equal amounts (judged by p24 antigen content) of virions expressing either the WT (black bars) or mutant (white bars) forms of JR-FL Env were tested in a virus capture assay. The amount of p24 antigen captured by each of the indicted MAbs is recorded.

Fig.7

Cell-surface expression of wild-type and gp41 mutant Env glycoproteins and their reactivity with CD4-IgG2 and MAbs. (A) Cell surface expressed Envs were biotinylated, avidin-precipitated and detected using MAb ARP3119. Cell surface expressed CD47 served as a loading control (lower panel). (B) The WT and gp41 mutant Env glycoproteins were stained with 10 µg/ml of biotinylated MAbs, followed by streptavidin-PE. Background fluorescence due to the secondary antibody was determined using isotype controls and subtracted from experimental values. The MFI values are shown as Mean ± Standard Deviation from a representative experiment performed in triplicates.