PMC

The V2 domain of HIV-1 gp120 contains sulfated tyrosines. (A) Sequence alignment of the CCR5 N-terminal domain and the conserved central region of the V2 domain of HIV-1 gp120 (consensus sequence for subtype B). Two colinear conserved tyrosine residues present in CCR5 and gp120 V2 are highlighted in green. (B) Detection of sulfated tyrosines by Western blot in gp120 purified from HeLa cells expressing WT HIV-1 BaL gp160 or a partially V2-deleted mutant (Δ164–190) lacking Tyr173 and Tyr177 by vaccinia technology. gp120 was immunoprecipitated from the cell surface and analyzed by Western blot using a specific mAb for sulfated tyrosines (1C-A2); a murine anti-gp120 mAb (b13) was tested in parallel as a loading control (gp120). (C) Detection of sulfated tyrosines by autoradiography in metabolically labeled HEK293 cells expressing HIV-1 BaL WT or Δ164–190 gp160 by vaccinia technology. The cells were labeled with free [³⁵S]sulfate or [³⁵S]cysteine/[³⁵S]methionine by overnight culture in sulfate-free or cysteine/methionine-free medium, respectively. The films were exposed for 48 h for [³⁵S]sulfate labeling and for 18 h for [³⁵S]cysteine/[³⁵S]methionine labeling. (D) Detection of sulfated tyrosines by Western blot in HEK293 cells transfected with a plasmid encoding WT HIV-1 BaL gp160 or the phenylalanine-substituted mutants BaL Y173F, BaL Y177F, and BaL Y173F/Y177F. Western blot analysis was performed as in B.

Competition of a tyrosine-sulfated V2 mimetic peptide with mAb 412d binding to gp120. (A) Effect of a tyrosine-sulfated V2-loop mimetic peptide (pV2α-Tys; amino acids 168–185) on mAb 412d binding to gp120 as assessed by surface plasmon resonance. A tyrosine-sulfated peptide derived from the CCR5 N terminus (pCCR5-Tys; amino acids 1–22) and unsulfated peptides from V2 (pV2α) and CCR5 (pCCR5) were tested in parallel as controls. MAb 412d was immobilized on the sensor surface and tested for binding to gp120 (BaL) pretreated with two-domain sCD4 in the presence or absence of the peptides used at 166 μM. A control antibody (F105; no sCD4 pretreatment) was tested in parallel as a control. The data are from a representative experiment of three that were performed with similar results. (B) Effect of tyrosine-sulfated and unsulfated V2-loop and CCR5 N terminus mimetic peptides on HIV-1 virion capture by mAb 412d. Infectious viral stocks from HIV-1 BaL were pretreated with sCD4 (5 μg/mL) in the presence or absence of the indicated peptides (each at 100 μM) and then were mixed with immunomagnetic beads prearmed with mAb 412d. A control antibody (2G12; no sCD4 pretreatment) was tested in parallel as a further specificity control. The data presented are mean values (± SD) from three independent experiments. Asterisks denote significant differences from the virion capture in the peptide-untreated control (P < 0.01 by unpaired Student t test).

Effect of V2 tyrosine sulfation on gp120 (BaL) epitope exposure and neutralization. (A) Modulation of V2 tyrosine sulfation alters gp120 (BaL) epitope accessibility. Flow cytometry was performed on HeLa cells expressing full-length, cleavable HIV-1 BaL gp160 on their surface. V2 tyrosine sulfation was enhanced by overexpression of the sulfotransferase TPST2 or inhibited by treatment with the sulfotransferase inhibitor NaClO₃ (30 mM). A panel of human mAbs directed to the indicated domains of gp120 and gp41 or four-domain soluble CD4 (sCD4) (each at 5 μg/mL) was used. BS, binding site; CoR, coreceptor; OD, outer domain. The sequence of the tyrosine-sulfated segment of V2 (amino acids 170–181) of HIV-1 BaL is QKEYALFYELDI. The data represent mean fluorescence intensity values after subtraction of background fluorescence levels. The signals obtained with the reference antibody 2F5 were used to verify that cell-surface envelope expression levels were comparable in the three cultures. The data presented are from a representative experiment of three that were performed with similar results. (B) Modulation of V2 tyrosine sulfation alters HIV-1 BaL neutralization sensitivity. Neutralization experiments were performed using an HIV-1 envelope-mediated fusion assay with the same panel of anti-gp120/gp41 antibodies and sCD4 as in A, which were tested at the indicated concentrations. The fusion assay was performed using vaccinia technology as described () with HeLa cells expressing full-length cleavable HIV-1 BaL gp160 as effector cells and NIH 3T3 cells expressing human CD4 and CCR5 as target cells. Tyrosine sulfation was enhanced and inhibited as described in A. The fusion values were normalized to the value obtained with untreated controls for each culture condition. The mean absolute levels of envelope-mediated fusion for the experiment shown, after background subtraction, were the following: untreated, 1.136 optical density units; NaClO₃-treated, 1.192 optical density units; TPST2 overexpression, 0.891 optical density units. The data presented are mean values (± SE) of duplicate wells from a representative experiment of three that were performed with similar results.

Efficiency of V2 tyrosine sulfation in recombinant gp120 produced in continuous cell lines and virion-associated gp120 produced in primary CD4⁺ T cells. (A) Detection of sulfated tyrosines in recombinant gp120 produced in CHO cells. The effects of overexpression of the sulfotransferase TPST2 or treatment with the sulfotransferase inhibitor NaClO₃ (30 mM) were tested in parallel. Gp120 was expressed by transfection, purified from the cell culture supernatants by lectin affinity, and quantified. Identical amounts of serially diluted gp120s were loaded onto the gel, as indicated over each lane. The presence of sulfated tyrosines was evaluated by Western blot with the specific mAb 1C-A2; loading controls were revealed with a murine anti-gp120 mAb (b24). To visualize low levels of tyrosine sulfation, high amounts of gp120 were used (fivefold dilutions starting from 1,250 ng per lane). (B) Direct side-by-side comparison of tyrosine sulfation levels in a reference sulfated mAb (412d) versus virion-associated gp120 (BaL) produced by infected primary human CD4⁺ T cells (V-gp120) and recombinant gp120 (BaL) produced in HEK293 cells (R-gp120). Identical amounts of serially diluted mAb 412d and purified gp120s were loaded onto the gels under nondenaturing conditions, as indicated over each lane. For V-gp120, the protein was immunoprecipitated directly from the infectious viral stock (BaL), quantified, and analyzed by Western blot as described in A. The loading control for mAb 412d was revealed using a murine mAb specific for human IgG. (C) Detection of sulfated tyrosines in virion-associated gp120 purified from different HIV-1 isolates grown in primary human CD4⁺ T cells. Infectious viral stocks from six primary and laboratory isolates with different coreceptor-use phenotypes were used: Three were CCR5-tropic (BaL, JR-FL, ADA), two were dual-tropic (92US077, 92HT599), and one was CXCR4-tropic (IIIB). The sequences of the tyrosine-sulfated segments of V2 (amino acids 170–181) from the tested isolates are as follows: JR-FL: QKEYALFYKLDV; BaL: QKEYALFYELDI; 92US077: QKEDAFFYKSDV; ADA: KKDYALFYRLDV; 92HT599: QKEYALFSKLDV; IIIB: QKEYAFFYKLDI. Although BaL contains an unusual acidic residue (Glu) at position 178, which may favor tyrosine sulfation at position 177, all the other isolates contained a basic residue (Lys or Arg) at position 178. The gp120 proteins were immunoprecipitated directly from the viral stocks, quantified, and analyzed by Western blot as in A.