Interaction with CD4 and Antibodies to CD4-Induced
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病菌学杂志 2005年第11期
Departments of Neurology
Medicine
Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
We previously showed that the envelope glycoprotein from an in vitro microglia-adapted human immunodeficiency virus type 1 isolate (HIV-1Bori-15) is able to use lower levels of CD4 for infection and demonstrates greater exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody than the envelope of its parental, peripheral isolate (HIV-1Bori). We investigated whether these phenotypic changes were related to a different interaction of their soluble monomeric gp120 proteins with CD4 or 17b. Equilibrium binding analyses showed no difference between Bori and Bori-15 gp120s. However, kinetic analysis of surface plasmon resonance-based, real-time binding experiments showed that while both proteins have similar association rates, Bori-15 gp120 has a statistically significant, 3-fold-lower dissociation rate from immobilized CD4 than Bori and a statistically significant, 14-fold-lower dissociation rate from 17b than Bori in the absence of soluble CD4. In addition, using the sensitivity to inhibition by anti-CD4 antibodies as a surrogate for CD4:trimeric envelope interaction, we found that Bori-15 envelope-pseudotyped viruses were significantly less sensitive than Bori pseudotypes, with four- to sixfold-higher 50% inhibitory concentration values for the three anti-CD4 antibodies tested. These differences, though small, suggest that adaptation to microglia correlates with the generation of a gp120 that forms a more stable interaction with CD4. Nonetheless, the observation of limited binding changes leaves open the possibility that HIV-1 adaptation to microglia and HIV-associated dementia may be related not only to diminished CD4 dependence but also to changes in other molecular factors involved in the infection process.
INTRODUCTION
Central nervous system (CNS) invasion by human immunodeficiency virus type 1 (HIV-1) often occurs during primary infection, but HIV-associated dementia (HAD) is mostly a late feature in patients who have developed AIDS. Although highly active antiretroviral therapy has dramatically decreased the incidence of HAD, the prevalence of minor cognitive and/or motor disorders is increasing and may continue to pose a significant problem as HIV-positive individuals survive longer (15, 45, 51, 83). HIV encephalitis, the pathological correlate of HAD, is defined by the presence of multinucleated giant cells or syncytia, thought to be the result of fusion among infected and uninfected microglia and brain macrophages (6, 14, 70).
The viral mediators of cell-to-cell fusion are the trimeric spikes formed by noncovalently associated surface protein gp120 and transmembrane protein gp41 present on the surface of HIV-1 virions. The heavily glycosylated gp120 (40, 42) has a core defined by five conserved regions (C1 to C5) and variable loop-like structures (V1/V2, V3, V4, and V5) with high flexibility (36, 48, 64, 90). The gp41 protein contains the fusion peptide (4, 21). Entry into cells requires sequential specific binding of gp120 to CD4 and a chemokine receptor, most commonly CCR5 or CXCR4 (12, 16, 52, 80, 88). Binding to CD4 triggers a conformational change in gp120, primarily involving V1/V2 and V3, which results in the exposure of conserved regions previously folded into the core structure (66, 77-79, 88, 91, 92). These CD4-induced (CD4i) regions include discontinuous epitopes recognized by the human neutralizing monoclonal antibodies (MAbs) 17b and 48d, known to interfere with chemokine receptor binding (36, 77-79, 90-92). Thus, the CD4i conformational change is thought to expose a high-affinity coreceptor binding site that collocates with these epitopes. Additionally, fusion kinetics and entry are determined to some extent by the affinity of the interaction between gp120 and the chemokine receptor (63).
Microglial cells and perivascular macrophages support productive viral infection within the brain. Similarly to macrophages from other tissues (39, 41), they express low levels of CD4 (13, 29, 57, 85), as well as CCR5 and CXCR4 (44). Since viruses isolated from the brain are macrophage tropic and use mainly CCR5 (1, 26, 71), it is likely that viral tropism for microglia and macrophages is determined by similar mechanisms (3, 49, 59). Genetic analyses have shown compartmentalization of HIV-1 sequences in the CNS (19, 33, 53, 61, 86), leading to the hypothesis that there is independent viral evolution and potential adaptation to the brain microenvironment.
We previously reported that in vitro adaptation to microglia of the primary peripheral isolate HIV-1Bori generated a virus (HIV-1Bori-15) with an increased ability to replicate in microglia/macrophages and a robust syncytium-forming phenotype, with only four amino acid differences in the V1/V2 region of gp120 being responsible for the phenotypic changes (72, 76). In addition, in the context of trimeric spikes, the envelope glycoprotein of the microglia-adapted virus showed (i) an increased ability to use low levels of CD4 for infection and increased sensitivity to neutralization with soluble CD4 (sCD4) and (ii) greater exposure of the CD4i 17b epitope, with enhanced sensitivity to neutralization by the human 17b MAb (43), suggesting a partially triggered conformation and potential differences in the affinity of the interaction with receptors.
Thus, we tested in this study whether phenotypic differences between the parental and microglia-adapted virus correlated with differences in the affinity for CD4 and the Abs to CD4i epitopes (as a surrogate for CCR5). We produced soluble, monomeric gp120 molecules from HIV-1Bori and HIV-1Bori-15; using surface plasmon resonance (SPR)-based optical biosensor technology, we noted that the gp120 of the microglia-adapted Bori-15 has association rates similar to the parental Bori gp120 but dissociation rates from CD4 and 17b MAb that are statistically significantly lower, resulting in moderately higher affinities. In addition, as a surrogate for CD4-trimeric envelope affinity, we analyzed the infection by envelope-pseudotyped viruses in the presence of three anti-CD4 MAbs, finding that Bori-15 has statistically significant four- to sixfold-higher 50% inhibitory concentration (IC50) values than Bori with all three antibodies. These studies suggest that improved contacts between the envelope glycoprotein and CD4 may be a factor in the increased ability of Bori-15 to infect cells expressing low levels of CD4 such as microglia.
MATERIALS AND METHODS
Cells and anti-gp120 antibodies. 293T human embryonic kidney cells, U87 human astroglioma cells, and HeLa cell clones expressing different levels of CD4 were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO-Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics. HOS cells stably expressing CD4 and CCR5 (12, 37), kindly provided by Nathaniel Landau through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (ARRRP), were maintained in DMEM supplemented with serum, mycophenolic acid, xanthine, hypoxanthine, HEPES, and puromycin.
The following MAbs were obtained through the NIH ARRRP: F105 (9, 60) from Marshall Posner and Lisa Cavacini, immunoglobulin IgG1 (IgG1) b12 (7, 54, 55, 65) from Dennis Burton and Carlos Barbas, 17b and 48d (36, 78-80, 90-92) from James Robinson, 2G12 (67, 81) from Hermann Katinger, and 447-52D (10, 22-24) from Susan Zolla Pazner. The sheep polyclonal D7324 Ab (47) was purchased from Aalto BioReagents (Dublin, Ireland).
Production of soluble, monomeric gp120 proteins. Bori and Bori-15 gp120s were generated by introducing a stop codon at the gp120/gp41 cleavage junction in the corresponding envelope expression vectors (72) with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), following the manufacturer's instructions. The oligonucleotides used for amplification (forward, 5'-GGTGCAGAGAGAAAAAAGATAAGCGGCCGCAATAGGAGCTATGTTCC-3'; reverse, 5'-GGAACATAGCTCCTATTGCGGCCGCTTATCTTTTTTCTCTCTGCACC-3') contained the stop codon (in boldface type in the sequences above) as well as a NotI site (underlined). Subsequently, SalI-NotI fragments (gp120 encoding regions) were ligated into the pSI vector (Promega Corp., Madison, WI) downstream of the bacteriophage T7 RNA polymerase promoter. For protein production, 293T cells were infected with the recombinant vaccinia virus vTF1.1, which expresses bacteriophage T7 RNA polymerase (20), for 1 to 2 h at 37°C and then transfected with pSI-Bori or pSI-Bori-15 gp120 vectors by calcium phosphate (Promega) for 4 h at 37°C. After the cells were incubated for 20 to 24 h at 32°C in serum-free DMEM supplemented with rifampicin, supernatants were collected, clarified (3,000 rpm; 10 min), and filtered through 0.2-μm filters. In addition, 0.1% Triton X-100 was added for vaccinia virus inactivation. Soluble gp120 proteins were purified by chromatography with agarose-bound Galanthus nivalis lectin (Vector Laboratories, Burlingame, CA), eluted with 0.5 M methyl--D-mannopyranoside (Sigma, St. Louis, MO), and concentrated with 30- and 50-kDa cutoff centrifugal filter devices (Centricon Plus-80 and Centriplus; Millipore, Bedford, MA). Purity, concentration, and functionality were tested by silver staining, a protein colorimetric assay (Bio-Rad DC Protein Assay, Bio-Rad Laboratories, Hercules, CA), Western blotting, and a solid-phase immunoassay with monomeric BaL gp120 (from Division of AIDS, National Institute of Allergy and Infectious Diseases, through the NIH ARRRP) as standards.
Binding of gp120s to HeLa-CD4 cells. CD4 expression in four HeLa-CD4 cell clones (a gift of David Kabat, Oregon Health Sciences University) (30, 34, 58) was assessed by quantitative flow cytometry. Briefly, cells and five microbead populations which differed in their capacities to bind mouse IgG (Quantum Simply Cellular Kit; Sigma) were stained with mouse anti-CD4 MAb #21 (kindly provided by James Hoxie, University of Pennsylvania) and fluorescein-conjugated goat anti-mouse IgG. Fluorescence was measured using a FACScan instrument (BD, Franklin Lakes, NJ; Flow Cytometry Core, University of Pennsylvania) and analyzed with CellQuest software (BD).
To analyze gp120 binding, HeLa-CD4 cell clones were incubated with soluble gp120s for 1 h at 4°C, washed, and fixed (2% formaldehyde in phosphate-buffered saline [PBS] for 30 min on ice), and bound gp120 was detected with D7324 Ab (47) and fluorescein-conjugated rabbit anti-sheep IgG. Fluorescence intensity was measured and analyzed as described above.
Production of murine leukemia virus (MLV) pseudotypes. 293T cells were cotransfected by calcium phosphate with the pcGP construct that contains MLV gag and pol genes (74) (a gift of Michael Malim, King's College, London, United Kingdom) plus either human CD4 or CCR5 expression vectors or an empty vector as a negative control. After 2 days, supernatants were clarified (3,000 rpm for 10 min) and ultracentrifugated over 20% sucrose in a Sorvall Ultra 80, using an SW41Ti rotor (Beckman, Palo Alto, CA) at 100,000 x g (29,000 rpm) for 2 h at 4°C and again at 150,000 x g (35,000 rpm) for 1.5 h at 4°C. The final pellet was suspended in PBS, aliquoted, and stored at –80°C. To test for CD4 and CCR5 incorporation, we performed Western blotting and an enzyme-linked immunosorbent assay (ELISA). For Western blotting, nitrocellulose membranes were probed with anti-MLV gag goat serum and MAbs against linear epitopes of CD4 (AF-379-NA; R&D Systems, Minneapolis, MN) and CCR5 (CTC8; R&D Systems) (38), followed by peroxidase-conjugated Ab and SuperSignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL). For ELISA, MAbs against conformational epitopes of CD4 (#21) and CCR5 (2D7; BD Pharmingen, San Diego, CA) (12, 16, 89) were used on plate-immobilized MLV particles, followed by peroxidase-conjugated Ab and 1-Step ABTS [2,2<<-azinobis(3-ethylbenzthiazolinesulfonic acid); Pierce] as a substrate. Optical density was measured at 405 nm.
Binding studies by ELISA. CD4- or CCR5-pseudotyped MLV particles were immobilized on ELISA plates, and various concentrations of Bori and Bori-15 gp120s were incubated for 2 h at 37°C. Bound protein was detected with D7324 Ab (47), followed by peroxidase-conjugated rabbit anti-sheep IgG and 1-Step ABTS.
To test the exposure of the gp120 CD4i epitopes recognized by 17b and 48d MAbs, gp120s were captured by immobilized D7324 Ab, and 17b or 48d MAb was used as a primary Ab in the absence or presence of human sCD4 (PerkinElmer, Boston, MA) at 20 μg/ml, followed by peroxidase-conjugated goat anti-human IgG and 1-Step ABTS.
SPR analysis of gp120 interaction kinetics. Interaction analyses in real-time using SPR-based optical biosensor technology were performed with a Biacore 3000 (Biacore, Inc., Piscataway, NJ). Immobilization of ligands (sCD4, 17b, and 48d MAbs and D7324 Ab) to CM5 chips (Biacore) was performed, following the standard amine-coupling procedure. Briefly, carboxyl groups were activated by injection of 35 μl of a solution containing 200 mM EDC (1-ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride) and 50 mM NHS (N-hydroxysuccinimide) at a 5 μl/min flow rate. Subsequently, a ligand (1 to 20 μg/ml in 10 mM sodium acetate buffer, pH 5.0) was flowed over the cell at 25°C and at a 5 μl/min flow rate, until the desired response level (indicative of the ligand density) was achieved. Unbound ligand was washed out, and excess carboxyl groups were capped with 35 μl of 1 M ethanolamine, pH 8.0, at 5 μl/min. A reference surface was prepared by activating and blocking a flow cell in the absence of protein ligand. Surfaces were allowed to equilibrate for at least 1 h. Interaction data (in arbitrary resonance units) were obtained at the highest collection rate (2 Hz).
Binding was performed at either 25°C or 37°C in PBS buffer, pH 7.2, with 0.005% P20 surfactant and 1 mg/ml carboxymethyl-dextran (CarboMer, San Diego, CA) to decrease nonspecific interactions. The effects of mass transport and rebinding upon the interactions were evaluated by passing a fixed concentration of gp120s over the chip surface at different flow rates (10 to 100 μl/min); rates of 25 μl/min and 50 μl/min for sCD4 and Abs, respectively, were determined to be optimum (data not shown). Sensorgrams were obtained by injection of gp120s over the ligands for 2-min association and 2-min dissociation phases. Surface regeneration was achieved with short injections (6 to 12 s) of 35 mM NaOH and 1.3 M NaCl solution at 100 μl/min for the sCD4 surface, plus pulses of glycine pH 2.0 and 10 mM HCl for Ab surfaces.
We evaluated the binding of various concentrations of gp120s (up to 1 μM) to ligands at different densities and the effect on Ab binding of preincubating gp120s with saturating concentrations of sCD4. Data analysis was performed using BiaEvaluation 3.0.2 software (Biacore). The responses of a buffer injection and responses from a blank flow cell were subtracted to account for nonspecific binding. Experimental data were fitted to a simple 1:1 binding model. Kinetic parameters (association [kon] and dissociation [koff] rates) were used to estimate the affinity of the interaction through the equilibrium dissociation constant (KD = koff/kon). In addition, to confirm the accuracy of the model, the association and dissociation phases were analyzed as previously described by least-squares fit of the data to the bimolecular model (A + B AB) to obtain kon and koff values (8). Differences in association and dissociation rates between Bori and Bori-15 gp120s were evaluated by Student's t test using SPSS statistical software.
Inhibition of pseudotype infection by TAK-779 and anti-CD4 Abs. Envelope-pseudotyped luciferase viruses were produced as previously described (43) and quantified by p24gag content (NEN, Brussels, Belgium). U87 cells expressing different levels of CCR5 were obtained by cotransfection with 2 μg of a CD4 expression plasmid and 0.3 or 3 μg of CCR5-expression plasmid and plated at 2.5 x 104 cells/well into 96-well plates. The next day, the cells were incubated for 1 h at 37°C with DMEM alone or DMEM containing increasing concentrations of TAK-779 (2, 17) and infected with p24gag-normalized virus inocula. At 2 to 3 days postinfection, chemiluminescence (Luciferase assay kit; Promega) was determined with a LumiCount microplate luminometer (Packard, Meriden, CT) as previously described (43). Experiments were performed in triplicate. Results are expressed as relative light units per second.
HOS cells stably expressing CD4 and CCR5 (plated at 104 cells/well into 96-well plates) were incubated for 1 h at 4°C with medium alone or in medium with anti-CD4 MAbs #21, RPA-T4 (eBioscience, San Diego, CA), SK3 (BD Biosciences), or mouse IgG and infected with pseudotypes for 6 h. Luciferase activity was measured at 2 to 3 days postinfection. Experiments were performed in quadruplicate. The percentages of infection at each Ab concentration (with the value for untreated condition set at 100%) from six to eight experiments were compared by Student's t test. The inhibition curves were also analyzed by nonlinear regression with a sigmoidal dose-response model (variable slopes) with GraphPad Prism software, version 4.02 (GraphPad Software, San Diego, CA). The IC50 values generated under this model were also compared by a t test.
RESULTS
Production of soluble, monomeric gp120s. We previously reported that, in comparison with its parent strain (HIV-1Bori), the envelope glycoprotein of microglia-adapted HIV-1Bori-15 incorporated into pseudotypes, and recombinant viruses featured lower CD4 dependence, greater exposure of CD4i epitopes that partially collocate with the chemokine receptor binding site, and sensitivity to neutralization by sCD4, MAbs directed to CD4i epitopes, and human HIV+ sera (43). To test their affinity for receptors, we produced monomeric gp120s from both viruses as described in Materials and Methods. Silver staining and Western blotting showed that the gp120s were of the expected size for fully glycosylated molecules (data not shown). We assessed protein integrity by evaluating the binding of several Abs to conformational and linear epitopes on D7324 Ab-captured gp120s. Both Bori and Bori-15, as well as control BaL, gp120s reacted similarly with Abs to the CD4 binding site (F105 and IgG1 b12), to CD4i conformational epitopes (17b and 48d), with the glycan-specific 2G12 MAb, and with the V3 linear epitope-specific 447-52D MAb (data not shown). We therefore concluded that the proteins were properly folded and functional.
Equilibrium binding to CD4. We evaluated gp120 binding to CD4 on four HeLa cell clones expressing CD4 at various levels (HeLa-J, 2.5 x 105 Ab binding sites [ABS]/cell; HeLa-P, 1.9 x 105 ABS/cell; HeLa-Q, 2.9 x 104 ABS/cell; HeLa-R, 2.7 x 104 ABS/cell). Bound gp120 was detected with the polyclonal sheep D7324 Ab. At concentrations of as high as 100 nM, we could only detect gp120 binding to the two cell clones expressing high levels of CD4 (HeLa-J and -P) but not to the other two clones (data not shown). Moreover, there was no difference between Bori and Bori-15 in the increase in fluorescence intensity over the negative controls and staining with Abs in the absence of gp120 and with gp120s followed by an IgG of irrelevant specificity (data not shown).
We also generated MLV particles pseudotyped with CD4 or CCR5, as described in Materials and Methods. Analysis by Western blotting and ELISA indicated that CD4 was incorporated into virions at much higher levels than CCR5 (data not shown). This reduced ability of chemokine receptors, compared to CD4, to incorporate into MLV particles was reported previously (73). For a solid-phase binding assay, gp120s were incubated with the immobilized pseudotypes, and detection of bound protein was performed with D7324 Ab. As shown in Fig. 1, both Bori and Bori-15 gp120s bound to the same extent to immobilized MLV-CD4 pseudotypes. There was no detectable binding to MLV-CCR5 pseudotypes (either in the absence or presence of sCD4) or to MLV particles produced in cells lacking CD4 and CCR5 (data not shown). Thus, contrary to our expectations, we were unable to detect any difference in equilibrium binding to CD4 between Bori and Bori-15 gp120s.
Exposure of CD4i epitopes on gp120s. We previously reported that without CD4, the Bori-15 envelope proteins incorporated into pseudotypes had greater exposure of the conformational CD4i epitope recognized by the human 17b Mab than parental Bori; accordingly, Bori-15 pseudotypes were sensitive to 17b MAb neutralization, while Bori pseudotypes were resistant (43). Here, we compared the exposure of 17b and 48d CD4i epitopes on Bori and Bori-15 gp120s by testing the binding of these MAbs to D7324-captured gp120s by ELISA. In the absence of sCD4, Bori-15 bound slightly better than Bori gp120 to 48d MAb, but both proteins bound identically to 17b MAb (data not shown). In addition, sCD4 induction promoted an increase in 48d MAb binding that was more evident for Bori gp120 than for Bori-15 but failed to increase binding of 17b MAb to either gp120 (data not shown). Therefore, equilibrium binding studies again did not reveal differences between parental Bori and microglia-adapted Bori-15 gp120s in the interaction with human Abs directed against CD4i epitopes.
Interaction kinetics of gp120s with sCD4. To measure binding of gp120s to sCD4 in greater detail, we analyzed the interaction in real-time using SPR-based optical biosensor technology. Unlike equilibrium methods, this allows the derivation of component individual rate constants and overall equilibrium rates. We immobilized sCD4 at different densities and compared the binding of gp120s at concentrations up to 1 μM at 25°C and 37°C. Responses obtained with buffer alone (no gp120) and on a blank surface were subtracted to account for nonspecific binding. Representative sensorgrams of the interaction between Bori and Bori-15 gp120s and CD4 are shown in Fig. 2A and 2B, respectively. A control BaL gp120 at the same concentrations was evaluated in all experiments (not shown). Each binding curve was fitted to a simple 1:1 Langmuir binding model using BiaEvaluation, and association (kon) and dissociation (koff) rates were obtained. In addition, linearization of experimental data was performed as previously described (8) to confirm the accuracy of the model. Thus, for the association phase, the sensorgrams were plotted as derivative (dR/dt) versus response (Fig. 2C and 2D) and the slopes of the linear regression analysis for each concentration were used in the –kobs versus concentration plot (Fig. 2E), where the slopes of the linear regression analysis correspond to the apparent kon. For the dissociation phase, we plotted ln(R0/R) versus time, where R0 is the response at the starting point of dissociation and R is the response at any time for the highest concentration of each protein, with the slope corresponding to koff (Fig. 2F). Rates obtained by linearization were similar to those provided by BiaEvaluation.
Table 1 summarizes the kinetic rates obtained with various protein concentrations and ligand densities. The major difference observed in binding to CD4 between Bori and Bori-15 gp120s was a threefold-reduced equilibrium dissociation constant for Bori-15 at 37°C. This reflects mainly that Bori-15 had a threefold-lower dissociation rate constant than Bori (Student's t test; P < 0.05), since the association rates were virtually the same. Although limited in magnitude and opposite in direction from changes observed in off rates at 25°C (which were not statistically significant), the difference in off rates was intriguing and could reflect an increased stabilization of the Bori-15-CD4 complex.
Interaction kinetics of gp120s with Abs. We also measured the interaction kinetics of gp120s with the 17b MAb (CD4i epitope) and D7324 Ab (an exposed, well-conserved epitope in the C5 region). Various gp120 concentrations were flowed at 25°C or 37°C in the absence of CD4 over Abs immobilized at different densities. Representative sensorgrams of Bori and Bori-15 gp120s binding to the 17b MAb are shown in Fig. 3A and 3B, respectively. BaL gp120 at the same concentrations was evaluated in all experiments (not shown). Data were fitted to a simple 1:1 binding model with BiaEvaluation, and association and dissociation rates were obtained. Linearization of experimental data was also performed as described above, generating the derivative versus response (Fig. 3C and 3D), the –kobs versus concentration (Fig. 3E), and the ln(R0/R) versus time (Fig. 3F) plots. Rates obtained were similar to those provided by BiaEvaluation.
Kinetic rates are summarized in Table 2. No statistically significant differences between the association rates to 17b of Bori, Bori-15, and BaL gp120s were observed. However, Bori-15 gp120 showed statistically significant 3- and 14-fold-lower dissociation rates from 17b than Bori at 25°C (Student's t test; P < 0.02) and 37°C (P < 0.05), respectively. By contrast, they showed no difference in association rates to or dissociation rates from D7324 Ab (Table 2).
We also estimated the affinity of the interaction between gp120s and 17b MAb by the equilibrium dissociation constant and found that Bori-15 had three- to fourfold-higher affinity than Bori and eight- to ninefold higher affinity than BaL for 17b MAb at both temperatures (Table 2).
Finally, to study the effects of sCD4 induction on 17b MAb binding, gp120s (200 nM) were incubated for at least 1 h at 37°C with increasing concentrations of sCD4. Maximum response at saturating concentrations of sCD4 was similar in all three gp120s (data not shown). Accordingly, the increase in response at saturating concentration of sCD4 (with respect to that in the absence of sCD4) was higher for Bori and BaL (a 7- and 15-fold increase, respectively) than for Bori-15 gp120 (a 3.8-fold increase). Similar results were obtained with the 48d MAb.
Inhibition of infection by TAK-779 and anti-CD4 Abs. A higher affinity between gp120 and CCR5 and higher density of CCR5 on the target cell membrane correlate with decreased sensitivity to inhibition by CCR5 antagonists and fusion inhibitors (63). Thus, to study a surrogate for the affinity between trimeric envelopes and CCR5, we compared the sensitivity of Bori and Bori-15 envelope-pseudotyped viruses to inhibition by CCR5 antagonist TAK-779 on U87 cells expressing various levels of CCR5. Both pseudotypes were less sensitive to TAK-779 in cells expressing high CCR5 levels than in those with low CCR5, but there was no difference in sensitivity between Bori and Bori-15 pseudotypes regardless of CCR5 levels (Fig. 4).
Finally, we evaluated the ability of three different anti-CD4 MAbs (RPA-T4, SK3, and #21) to inhibit infection by Bori and Bori-15 pseudotypes as a surrogate for CD4:trimeric envelope affinity. As shown in Fig. 5, Bori-15 pseudotypes were more resistant to inhibition than Bori pseudotypes, consistent with a higher affinity for CD4 and a greater ability to outcompete the binding of MAbs. Nonlinear regression analysis using a sigmoidal dose-response model (variable slopes) demonstrated that the curves showing inhibition of infection by Bori and Bori-15 pseudotypes were statistically different, since the null hypothesis of a single best-fit curve for the two data sets was rejected for all three antibodies (P values of <0.0001 in all cases). The IC50 values estimated from nonlinear regression analysis were between four- and sixfold higher for Bori-15 than for Bori with all three MAbs (values in parentheses are 95% confidence intervals): RPA-T4, 62.8 ng/ml (24.3 to 162.6) versus 11.4 ng/ml (5.2 to 25.4); SK3, 13.1 ng/ml (8.6 to 19.6) versus 2.2 ng/ml (1.0 to 4.9); and #21, 30.9 ng/ml (21.2 to 45.0) versus 7.5 ng/ml (4.0 to 14.3). In addition, the IC50 values and standard error obtained from nonlinear regression for Bori and Bori-15 with each anti-CD4 antibody were compared by t tests; we found that the differences were statistically significant (RPA-T4, P < 0.01; SK3 and #21, P < 0.001).
These differences between Bori and Bori-15 trimeric envelopes incorporated into pseudotypes correlate with those observed with monomeric gp120s and give credence to the possibility that a difference in CD4 affinity between Bori and Bori-15 underlies, at least in part, the mechanism for increased infectivity of Bori-15 for microglial cells expressing low levels of CD4.
DISCUSSION
Compartmentalization of HIV-1 sequences in the brain in comparison with peripheral tissues has been demonstrated in numerous studies (19, 33, 53, 61, 86), suggesting that there is independent viral evolution within the CNS. Although tissue-specific genetic variation in the CNS might be due to a founder effect, it is more likely that there is specific adaptation to this niche, due to limited trafficking across the blood-brain barrier, together with HIV-1 persistence and replication in long-lived cell types such as microglia and perivascular macrophages (35, 82). Thus, it is reasonable to hypothesize that the low level of CD4 expression of microglia and perivascular macrophages drives the selection of viruses that are particularly fit for this environment (13, 29, 57, 85).
We previously demonstrated that pseudotypes containing the envelope glycoproteins from the in vitro microglia-adapted HIV-1Bori-15 isolate infect cells expressing low levels of CD4 more efficiently than those with parental HIV-1Bori envelope glycoproteins (43). In addition, contrary to Bori, Bori-15 pseudotypes were sensitive to sCD4 neutralization (43). Although the latter could be due to a variation in sCD4-induced gp120 shedding, we hypothesized that Bori and Bori-15 might differ in the affinity for CD4. Here we report that these phenotypic differences are associated with altered binding kinetics between Bori-15 gp120 and CD4. Unlike the results of experiments performed at equilibrium, which showed no difference in binding, real-time studies with SPR-based optical biosensor technology showed that the Bori-15 gp120 has a small but statistically significant lower dissociation rate from CD4 than Bori, leading to a higher affinity of similar magnitude. Equilibrium binding assays require the reactant to be separated from the product, and the equilibrium of the reaction can be disturbed by the washing steps. In biosensor analysis, the complex is detected in the presence of unbound material, avoiding this potential interference and providing alternative information regarding the interaction between monomeric gp120s and CD4 (28, 32, 68).
We found that Bori-15 gp120 bound to CD4 with higher affinity than Bori when the experiments were performed at 37°C but not at 25°C. It is intriguing that while Bori and BaL gp120s have similar KDs at either temperature, Bori-15 gp120 had a faster association combined with a slower dissociation at 37°C than at 25°C, resulting in a substantial increase in affinity (13 fold) for the Bori-15-CD4 interaction between the two temperatures investigated. We believe that the slower dissociation of Bori-15 gp120 from CD4 may indicate a more stable interaction between gp120 and CD4 and prolonged contact time (before dissociating), increasing the probability that the gp120-CD4 complex will establish interactions with additional receptors such as CCR5.
However, it could be argued that this small difference in affinity of the monomeric forms of gp120 may not fully account for the phenotypic differences between Bori and Bori-15 that were noted in the context of viral particles (43, 72). As a surrogate for perhaps a more realistic CD4:trimeric envelope affinity, we evaluated the sensitivity of infection by Bori and Bori-15 envelope-pseudotyped viruses to increasing concentrations of three anti-CD4 MAbs. The inhibition curves showed a lower sensitivity for Bori-15, with statistically significant four- to sixfold-higher IC50 values than Bori. Decreased sensitivity to inhibition of infection by anti-CD4 antibodies has been recently reported for brain-derived HIV envelopes by Peters et al. (56). Additional studies will be required to dissect the role of the individual 8 amino acid changes between Bori and Bori-15 envelopes, especially the four substitutions located in the V1/V2 region, on gp120 conformation and/or flexibility.
Numerous studies have investigated the interaction between gp120 and CD4 by SPR (5, 50, 87, 92, 93), but its binding to Abs anti-CD4i epitopes has not received as much attention (27, 92, 93). In our study, Bori-15 gp120 in the absence of CD4 showed a statistically significant, 14-fold-lower dissociation rate than Bori from 17b MAb, although its affinity was only 4-fold higher than that of Bori. By contrast, no difference in kinetic rates or affinity in the interaction with the D7324 Ab was observed, suggesting that there is a specific increase in the exposure of CD4i epitopes in Bori-15 prior to any CD4i conformational change. This correlates with the higher 17b binding and sensitivity to neutralization by 17b MAb that we previously reported for Bori-15, compared to Bori, in the context of trimeric envelopes (43). However, both gp120 proteins bound to 17b and 48d MAbs similarly after CD4 induction. Altogether, these results seem to indicate that the envelope of the microglia-adapted isolate may exist predominantly in a partially triggered conformation.
Dissociation rate, but not association rate or overall affinity, has been shown to correlate with HIV-1 neutralization by Abs directed to the V3 loop of gp120 (84). Rapid dissociation seems to be sufficient to prevent neutralization, while a prolonged contact time due to slow dissociation results in neutralization. In our experiments, we have also found that the reported Bori-15 sensitivity to neutralization by sCD4 and 17b MAb (43) correlates with an apparently slower rate of dissociation of monomeric gp120 from both CD4 and 17b MAb. A relationship between CD4 independence and neutralization sensitivity has been reported in the literature (18, 46, 62). However, HIV-1Bori-15 appears to be at an intermediate state, since neutralization sensitivity coexists with CD4 dependence. The lack of natural occurrence of CD4-independent HIV-1 isolates is likely due to an increased susceptibility to neutralizing antibodies by those viruses primed for coreceptor binding. Since the blood-brain barrier renders the brain less accessible to the immune system, the generation of CD4-independent viruses could theoretically proceed in vivo within the CNS, potentially expanding the target cell population by including CD4-negative cells (such as astrocytes, oligodendrocytes, and neurons).
To date, analysis of cloned viruses and viral envelopes from CNS has failed to reveal evidence for CD4-independent isolates. However, Peters et al. recently published a study concluding that envelopes from primary brain isolates confer lower CD4 dependence and lower sensitivity to inhibition by anti-CD4 antibodies than envelopes derived from peripheral tissues of the same individuals (56). In addition, lower CCR5 and CD4 dependencies have been shown to represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS (25). These findings suggest that the phenotypic differences with the Bori and Bori-15 isolates described by us are actually present in vivo and highlight the relevance of our model. Our current studies suggest that those differences found in vivo may be due to increases in receptor affinity; thus, similar experiments should be done with envelopes derived from primary isolates.
Low levels of CCR5 incorporation into MLV particles hampered our attempt to measure the affinity of gp120s to CCR5. Nevertheless, Bori-15 and Bori pseudotypes were equally sensitive to inhibition by the CCR5 antagonist TAK-779 (2, 17). Since sensitivity to coreceptor antagonists is determined to some extent by the affinity of the gp120-coreceptor interaction (63), our results suggest that there is no difference in the affinity of Bori and Bori-15 envelopes for CCR5. In fact, this would correlate with the lack of difference in CCR5 dependency observed with envelope-pseudotyped viruses in cells expressing various levels of CCR5 (43). Innovative SPR analysis with immobilized CCR5 (11, 31, 69, 75) could provide a definitive answer to this issue.
In summary, our results indicate that the monomeric gp120 derived from a microglia-adapted virus (HIV-1Bori-15) binds to CD4 and to the human 17b MAb with altered kinetics, compared with the gp120 of the primary, peripheral parental virus. These changes may result in prolonged contact time of Bori-15 with the cellular receptors. The neutralization sensitivity and accessibility of CD4i epitopes in Bori-15 gp120 resemble that of a CD4-independent envelope, although its gp120 mediates fusion and infection in a CD4-dependent manner, suggesting a partially triggered conformation. The difference in CD4 binding between Bori and Bori-15 monomeric gp120s as detected via SPR correlates with data showing a difference in sensitivity to inhibition of infection by envelope-pseudotyped viruses with anti-CD4 antibodies. Therefore, the data presented here suggest that the envelope of the microglia-adapted virus has a moderately higher affinity for CD4 than that of the parental, peripheral isolate and that this difference may be responsible in part for the phenotypic differences between the two strains.
ACKNOWLEDGMENTS
We thank David Kabat, Michael Malim, Nathaniel Landau, and the NIH AIDS Research and Reference Reagent Program for providing us with useful reagents. We are grateful to Samantha Soldan (University of Pennsylvania) for her comments on the manuscript.
This work was supported by National Institutes of Health grants NS-27405 and NS-35743 (to F.G.-S.) and NS-47970 (to J.M.-G.).
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Medicine
Microbiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
ABSTRACT
We previously showed that the envelope glycoprotein from an in vitro microglia-adapted human immunodeficiency virus type 1 isolate (HIV-1Bori-15) is able to use lower levels of CD4 for infection and demonstrates greater exposure of the CD4-induced epitope recognized by the 17b monoclonal antibody than the envelope of its parental, peripheral isolate (HIV-1Bori). We investigated whether these phenotypic changes were related to a different interaction of their soluble monomeric gp120 proteins with CD4 or 17b. Equilibrium binding analyses showed no difference between Bori and Bori-15 gp120s. However, kinetic analysis of surface plasmon resonance-based, real-time binding experiments showed that while both proteins have similar association rates, Bori-15 gp120 has a statistically significant, 3-fold-lower dissociation rate from immobilized CD4 than Bori and a statistically significant, 14-fold-lower dissociation rate from 17b than Bori in the absence of soluble CD4. In addition, using the sensitivity to inhibition by anti-CD4 antibodies as a surrogate for CD4:trimeric envelope interaction, we found that Bori-15 envelope-pseudotyped viruses were significantly less sensitive than Bori pseudotypes, with four- to sixfold-higher 50% inhibitory concentration values for the three anti-CD4 antibodies tested. These differences, though small, suggest that adaptation to microglia correlates with the generation of a gp120 that forms a more stable interaction with CD4. Nonetheless, the observation of limited binding changes leaves open the possibility that HIV-1 adaptation to microglia and HIV-associated dementia may be related not only to diminished CD4 dependence but also to changes in other molecular factors involved in the infection process.
INTRODUCTION
Central nervous system (CNS) invasion by human immunodeficiency virus type 1 (HIV-1) often occurs during primary infection, but HIV-associated dementia (HAD) is mostly a late feature in patients who have developed AIDS. Although highly active antiretroviral therapy has dramatically decreased the incidence of HAD, the prevalence of minor cognitive and/or motor disorders is increasing and may continue to pose a significant problem as HIV-positive individuals survive longer (15, 45, 51, 83). HIV encephalitis, the pathological correlate of HAD, is defined by the presence of multinucleated giant cells or syncytia, thought to be the result of fusion among infected and uninfected microglia and brain macrophages (6, 14, 70).
The viral mediators of cell-to-cell fusion are the trimeric spikes formed by noncovalently associated surface protein gp120 and transmembrane protein gp41 present on the surface of HIV-1 virions. The heavily glycosylated gp120 (40, 42) has a core defined by five conserved regions (C1 to C5) and variable loop-like structures (V1/V2, V3, V4, and V5) with high flexibility (36, 48, 64, 90). The gp41 protein contains the fusion peptide (4, 21). Entry into cells requires sequential specific binding of gp120 to CD4 and a chemokine receptor, most commonly CCR5 or CXCR4 (12, 16, 52, 80, 88). Binding to CD4 triggers a conformational change in gp120, primarily involving V1/V2 and V3, which results in the exposure of conserved regions previously folded into the core structure (66, 77-79, 88, 91, 92). These CD4-induced (CD4i) regions include discontinuous epitopes recognized by the human neutralizing monoclonal antibodies (MAbs) 17b and 48d, known to interfere with chemokine receptor binding (36, 77-79, 90-92). Thus, the CD4i conformational change is thought to expose a high-affinity coreceptor binding site that collocates with these epitopes. Additionally, fusion kinetics and entry are determined to some extent by the affinity of the interaction between gp120 and the chemokine receptor (63).
Microglial cells and perivascular macrophages support productive viral infection within the brain. Similarly to macrophages from other tissues (39, 41), they express low levels of CD4 (13, 29, 57, 85), as well as CCR5 and CXCR4 (44). Since viruses isolated from the brain are macrophage tropic and use mainly CCR5 (1, 26, 71), it is likely that viral tropism for microglia and macrophages is determined by similar mechanisms (3, 49, 59). Genetic analyses have shown compartmentalization of HIV-1 sequences in the CNS (19, 33, 53, 61, 86), leading to the hypothesis that there is independent viral evolution and potential adaptation to the brain microenvironment.
We previously reported that in vitro adaptation to microglia of the primary peripheral isolate HIV-1Bori generated a virus (HIV-1Bori-15) with an increased ability to replicate in microglia/macrophages and a robust syncytium-forming phenotype, with only four amino acid differences in the V1/V2 region of gp120 being responsible for the phenotypic changes (72, 76). In addition, in the context of trimeric spikes, the envelope glycoprotein of the microglia-adapted virus showed (i) an increased ability to use low levels of CD4 for infection and increased sensitivity to neutralization with soluble CD4 (sCD4) and (ii) greater exposure of the CD4i 17b epitope, with enhanced sensitivity to neutralization by the human 17b MAb (43), suggesting a partially triggered conformation and potential differences in the affinity of the interaction with receptors.
Thus, we tested in this study whether phenotypic differences between the parental and microglia-adapted virus correlated with differences in the affinity for CD4 and the Abs to CD4i epitopes (as a surrogate for CCR5). We produced soluble, monomeric gp120 molecules from HIV-1Bori and HIV-1Bori-15; using surface plasmon resonance (SPR)-based optical biosensor technology, we noted that the gp120 of the microglia-adapted Bori-15 has association rates similar to the parental Bori gp120 but dissociation rates from CD4 and 17b MAb that are statistically significantly lower, resulting in moderately higher affinities. In addition, as a surrogate for CD4-trimeric envelope affinity, we analyzed the infection by envelope-pseudotyped viruses in the presence of three anti-CD4 MAbs, finding that Bori-15 has statistically significant four- to sixfold-higher 50% inhibitory concentration (IC50) values than Bori with all three antibodies. These studies suggest that improved contacts between the envelope glycoprotein and CD4 may be a factor in the increased ability of Bori-15 to infect cells expressing low levels of CD4 such as microglia.
MATERIALS AND METHODS
Cells and anti-gp120 antibodies. 293T human embryonic kidney cells, U87 human astroglioma cells, and HeLa cell clones expressing different levels of CD4 were grown in Dulbecco's modified Eagle's medium (DMEM) (GIBCO-Invitrogen, Carlsbad, CA) supplemented with 10% heat-inactivated fetal bovine serum, glutamine, and antibiotics. HOS cells stably expressing CD4 and CCR5 (12, 37), kindly provided by Nathaniel Landau through the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (ARRRP), were maintained in DMEM supplemented with serum, mycophenolic acid, xanthine, hypoxanthine, HEPES, and puromycin.
The following MAbs were obtained through the NIH ARRRP: F105 (9, 60) from Marshall Posner and Lisa Cavacini, immunoglobulin IgG1 (IgG1) b12 (7, 54, 55, 65) from Dennis Burton and Carlos Barbas, 17b and 48d (36, 78-80, 90-92) from James Robinson, 2G12 (67, 81) from Hermann Katinger, and 447-52D (10, 22-24) from Susan Zolla Pazner. The sheep polyclonal D7324 Ab (47) was purchased from Aalto BioReagents (Dublin, Ireland).
Production of soluble, monomeric gp120 proteins. Bori and Bori-15 gp120s were generated by introducing a stop codon at the gp120/gp41 cleavage junction in the corresponding envelope expression vectors (72) with the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA), following the manufacturer's instructions. The oligonucleotides used for amplification (forward, 5'-GGTGCAGAGAGAAAAAAGATAAGCGGCCGCAATAGGAGCTATGTTCC-3'; reverse, 5'-GGAACATAGCTCCTATTGCGGCCGCTTATCTTTTTTCTCTCTGCACC-3') contained the stop codon (in boldface type in the sequences above) as well as a NotI site (underlined). Subsequently, SalI-NotI fragments (gp120 encoding regions) were ligated into the pSI vector (Promega Corp., Madison, WI) downstream of the bacteriophage T7 RNA polymerase promoter. For protein production, 293T cells were infected with the recombinant vaccinia virus vTF1.1, which expresses bacteriophage T7 RNA polymerase (20), for 1 to 2 h at 37°C and then transfected with pSI-Bori or pSI-Bori-15 gp120 vectors by calcium phosphate (Promega) for 4 h at 37°C. After the cells were incubated for 20 to 24 h at 32°C in serum-free DMEM supplemented with rifampicin, supernatants were collected, clarified (3,000 rpm; 10 min), and filtered through 0.2-μm filters. In addition, 0.1% Triton X-100 was added for vaccinia virus inactivation. Soluble gp120 proteins were purified by chromatography with agarose-bound Galanthus nivalis lectin (Vector Laboratories, Burlingame, CA), eluted with 0.5 M methyl--D-mannopyranoside (Sigma, St. Louis, MO), and concentrated with 30- and 50-kDa cutoff centrifugal filter devices (Centricon Plus-80 and Centriplus; Millipore, Bedford, MA). Purity, concentration, and functionality were tested by silver staining, a protein colorimetric assay (Bio-Rad DC Protein Assay, Bio-Rad Laboratories, Hercules, CA), Western blotting, and a solid-phase immunoassay with monomeric BaL gp120 (from Division of AIDS, National Institute of Allergy and Infectious Diseases, through the NIH ARRRP) as standards.
Binding of gp120s to HeLa-CD4 cells. CD4 expression in four HeLa-CD4 cell clones (a gift of David Kabat, Oregon Health Sciences University) (30, 34, 58) was assessed by quantitative flow cytometry. Briefly, cells and five microbead populations which differed in their capacities to bind mouse IgG (Quantum Simply Cellular Kit; Sigma) were stained with mouse anti-CD4 MAb #21 (kindly provided by James Hoxie, University of Pennsylvania) and fluorescein-conjugated goat anti-mouse IgG. Fluorescence was measured using a FACScan instrument (BD, Franklin Lakes, NJ; Flow Cytometry Core, University of Pennsylvania) and analyzed with CellQuest software (BD).
To analyze gp120 binding, HeLa-CD4 cell clones were incubated with soluble gp120s for 1 h at 4°C, washed, and fixed (2% formaldehyde in phosphate-buffered saline [PBS] for 30 min on ice), and bound gp120 was detected with D7324 Ab (47) and fluorescein-conjugated rabbit anti-sheep IgG. Fluorescence intensity was measured and analyzed as described above.
Production of murine leukemia virus (MLV) pseudotypes. 293T cells were cotransfected by calcium phosphate with the pcGP construct that contains MLV gag and pol genes (74) (a gift of Michael Malim, King's College, London, United Kingdom) plus either human CD4 or CCR5 expression vectors or an empty vector as a negative control. After 2 days, supernatants were clarified (3,000 rpm for 10 min) and ultracentrifugated over 20% sucrose in a Sorvall Ultra 80, using an SW41Ti rotor (Beckman, Palo Alto, CA) at 100,000 x g (29,000 rpm) for 2 h at 4°C and again at 150,000 x g (35,000 rpm) for 1.5 h at 4°C. The final pellet was suspended in PBS, aliquoted, and stored at –80°C. To test for CD4 and CCR5 incorporation, we performed Western blotting and an enzyme-linked immunosorbent assay (ELISA). For Western blotting, nitrocellulose membranes were probed with anti-MLV gag goat serum and MAbs against linear epitopes of CD4 (AF-379-NA; R&D Systems, Minneapolis, MN) and CCR5 (CTC8; R&D Systems) (38), followed by peroxidase-conjugated Ab and SuperSignal West Pico Chemiluminescent substrate (Pierce, Rockford, IL). For ELISA, MAbs against conformational epitopes of CD4 (#21) and CCR5 (2D7; BD Pharmingen, San Diego, CA) (12, 16, 89) were used on plate-immobilized MLV particles, followed by peroxidase-conjugated Ab and 1-Step ABTS [2,2<<-azinobis(3-ethylbenzthiazolinesulfonic acid); Pierce] as a substrate. Optical density was measured at 405 nm.
Binding studies by ELISA. CD4- or CCR5-pseudotyped MLV particles were immobilized on ELISA plates, and various concentrations of Bori and Bori-15 gp120s were incubated for 2 h at 37°C. Bound protein was detected with D7324 Ab (47), followed by peroxidase-conjugated rabbit anti-sheep IgG and 1-Step ABTS.
To test the exposure of the gp120 CD4i epitopes recognized by 17b and 48d MAbs, gp120s were captured by immobilized D7324 Ab, and 17b or 48d MAb was used as a primary Ab in the absence or presence of human sCD4 (PerkinElmer, Boston, MA) at 20 μg/ml, followed by peroxidase-conjugated goat anti-human IgG and 1-Step ABTS.
SPR analysis of gp120 interaction kinetics. Interaction analyses in real-time using SPR-based optical biosensor technology were performed with a Biacore 3000 (Biacore, Inc., Piscataway, NJ). Immobilization of ligands (sCD4, 17b, and 48d MAbs and D7324 Ab) to CM5 chips (Biacore) was performed, following the standard amine-coupling procedure. Briefly, carboxyl groups were activated by injection of 35 μl of a solution containing 200 mM EDC (1-ethyl-3[3-dimethylaminopropyl] carbodiimide hydrochloride) and 50 mM NHS (N-hydroxysuccinimide) at a 5 μl/min flow rate. Subsequently, a ligand (1 to 20 μg/ml in 10 mM sodium acetate buffer, pH 5.0) was flowed over the cell at 25°C and at a 5 μl/min flow rate, until the desired response level (indicative of the ligand density) was achieved. Unbound ligand was washed out, and excess carboxyl groups were capped with 35 μl of 1 M ethanolamine, pH 8.0, at 5 μl/min. A reference surface was prepared by activating and blocking a flow cell in the absence of protein ligand. Surfaces were allowed to equilibrate for at least 1 h. Interaction data (in arbitrary resonance units) were obtained at the highest collection rate (2 Hz).
Binding was performed at either 25°C or 37°C in PBS buffer, pH 7.2, with 0.005% P20 surfactant and 1 mg/ml carboxymethyl-dextran (CarboMer, San Diego, CA) to decrease nonspecific interactions. The effects of mass transport and rebinding upon the interactions were evaluated by passing a fixed concentration of gp120s over the chip surface at different flow rates (10 to 100 μl/min); rates of 25 μl/min and 50 μl/min for sCD4 and Abs, respectively, were determined to be optimum (data not shown). Sensorgrams were obtained by injection of gp120s over the ligands for 2-min association and 2-min dissociation phases. Surface regeneration was achieved with short injections (6 to 12 s) of 35 mM NaOH and 1.3 M NaCl solution at 100 μl/min for the sCD4 surface, plus pulses of glycine pH 2.0 and 10 mM HCl for Ab surfaces.
We evaluated the binding of various concentrations of gp120s (up to 1 μM) to ligands at different densities and the effect on Ab binding of preincubating gp120s with saturating concentrations of sCD4. Data analysis was performed using BiaEvaluation 3.0.2 software (Biacore). The responses of a buffer injection and responses from a blank flow cell were subtracted to account for nonspecific binding. Experimental data were fitted to a simple 1:1 binding model. Kinetic parameters (association [kon] and dissociation [koff] rates) were used to estimate the affinity of the interaction through the equilibrium dissociation constant (KD = koff/kon). In addition, to confirm the accuracy of the model, the association and dissociation phases were analyzed as previously described by least-squares fit of the data to the bimolecular model (A + B AB) to obtain kon and koff values (8). Differences in association and dissociation rates between Bori and Bori-15 gp120s were evaluated by Student's t test using SPSS statistical software.
Inhibition of pseudotype infection by TAK-779 and anti-CD4 Abs. Envelope-pseudotyped luciferase viruses were produced as previously described (43) and quantified by p24gag content (NEN, Brussels, Belgium). U87 cells expressing different levels of CCR5 were obtained by cotransfection with 2 μg of a CD4 expression plasmid and 0.3 or 3 μg of CCR5-expression plasmid and plated at 2.5 x 104 cells/well into 96-well plates. The next day, the cells were incubated for 1 h at 37°C with DMEM alone or DMEM containing increasing concentrations of TAK-779 (2, 17) and infected with p24gag-normalized virus inocula. At 2 to 3 days postinfection, chemiluminescence (Luciferase assay kit; Promega) was determined with a LumiCount microplate luminometer (Packard, Meriden, CT) as previously described (43). Experiments were performed in triplicate. Results are expressed as relative light units per second.
HOS cells stably expressing CD4 and CCR5 (plated at 104 cells/well into 96-well plates) were incubated for 1 h at 4°C with medium alone or in medium with anti-CD4 MAbs #21, RPA-T4 (eBioscience, San Diego, CA), SK3 (BD Biosciences), or mouse IgG and infected with pseudotypes for 6 h. Luciferase activity was measured at 2 to 3 days postinfection. Experiments were performed in quadruplicate. The percentages of infection at each Ab concentration (with the value for untreated condition set at 100%) from six to eight experiments were compared by Student's t test. The inhibition curves were also analyzed by nonlinear regression with a sigmoidal dose-response model (variable slopes) with GraphPad Prism software, version 4.02 (GraphPad Software, San Diego, CA). The IC50 values generated under this model were also compared by a t test.
RESULTS
Production of soluble, monomeric gp120s. We previously reported that, in comparison with its parent strain (HIV-1Bori), the envelope glycoprotein of microglia-adapted HIV-1Bori-15 incorporated into pseudotypes, and recombinant viruses featured lower CD4 dependence, greater exposure of CD4i epitopes that partially collocate with the chemokine receptor binding site, and sensitivity to neutralization by sCD4, MAbs directed to CD4i epitopes, and human HIV+ sera (43). To test their affinity for receptors, we produced monomeric gp120s from both viruses as described in Materials and Methods. Silver staining and Western blotting showed that the gp120s were of the expected size for fully glycosylated molecules (data not shown). We assessed protein integrity by evaluating the binding of several Abs to conformational and linear epitopes on D7324 Ab-captured gp120s. Both Bori and Bori-15, as well as control BaL, gp120s reacted similarly with Abs to the CD4 binding site (F105 and IgG1 b12), to CD4i conformational epitopes (17b and 48d), with the glycan-specific 2G12 MAb, and with the V3 linear epitope-specific 447-52D MAb (data not shown). We therefore concluded that the proteins were properly folded and functional.
Equilibrium binding to CD4. We evaluated gp120 binding to CD4 on four HeLa cell clones expressing CD4 at various levels (HeLa-J, 2.5 x 105 Ab binding sites [ABS]/cell; HeLa-P, 1.9 x 105 ABS/cell; HeLa-Q, 2.9 x 104 ABS/cell; HeLa-R, 2.7 x 104 ABS/cell). Bound gp120 was detected with the polyclonal sheep D7324 Ab. At concentrations of as high as 100 nM, we could only detect gp120 binding to the two cell clones expressing high levels of CD4 (HeLa-J and -P) but not to the other two clones (data not shown). Moreover, there was no difference between Bori and Bori-15 in the increase in fluorescence intensity over the negative controls and staining with Abs in the absence of gp120 and with gp120s followed by an IgG of irrelevant specificity (data not shown).
We also generated MLV particles pseudotyped with CD4 or CCR5, as described in Materials and Methods. Analysis by Western blotting and ELISA indicated that CD4 was incorporated into virions at much higher levels than CCR5 (data not shown). This reduced ability of chemokine receptors, compared to CD4, to incorporate into MLV particles was reported previously (73). For a solid-phase binding assay, gp120s were incubated with the immobilized pseudotypes, and detection of bound protein was performed with D7324 Ab. As shown in Fig. 1, both Bori and Bori-15 gp120s bound to the same extent to immobilized MLV-CD4 pseudotypes. There was no detectable binding to MLV-CCR5 pseudotypes (either in the absence or presence of sCD4) or to MLV particles produced in cells lacking CD4 and CCR5 (data not shown). Thus, contrary to our expectations, we were unable to detect any difference in equilibrium binding to CD4 between Bori and Bori-15 gp120s.
Exposure of CD4i epitopes on gp120s. We previously reported that without CD4, the Bori-15 envelope proteins incorporated into pseudotypes had greater exposure of the conformational CD4i epitope recognized by the human 17b Mab than parental Bori; accordingly, Bori-15 pseudotypes were sensitive to 17b MAb neutralization, while Bori pseudotypes were resistant (43). Here, we compared the exposure of 17b and 48d CD4i epitopes on Bori and Bori-15 gp120s by testing the binding of these MAbs to D7324-captured gp120s by ELISA. In the absence of sCD4, Bori-15 bound slightly better than Bori gp120 to 48d MAb, but both proteins bound identically to 17b MAb (data not shown). In addition, sCD4 induction promoted an increase in 48d MAb binding that was more evident for Bori gp120 than for Bori-15 but failed to increase binding of 17b MAb to either gp120 (data not shown). Therefore, equilibrium binding studies again did not reveal differences between parental Bori and microglia-adapted Bori-15 gp120s in the interaction with human Abs directed against CD4i epitopes.
Interaction kinetics of gp120s with sCD4. To measure binding of gp120s to sCD4 in greater detail, we analyzed the interaction in real-time using SPR-based optical biosensor technology. Unlike equilibrium methods, this allows the derivation of component individual rate constants and overall equilibrium rates. We immobilized sCD4 at different densities and compared the binding of gp120s at concentrations up to 1 μM at 25°C and 37°C. Responses obtained with buffer alone (no gp120) and on a blank surface were subtracted to account for nonspecific binding. Representative sensorgrams of the interaction between Bori and Bori-15 gp120s and CD4 are shown in Fig. 2A and 2B, respectively. A control BaL gp120 at the same concentrations was evaluated in all experiments (not shown). Each binding curve was fitted to a simple 1:1 Langmuir binding model using BiaEvaluation, and association (kon) and dissociation (koff) rates were obtained. In addition, linearization of experimental data was performed as previously described (8) to confirm the accuracy of the model. Thus, for the association phase, the sensorgrams were plotted as derivative (dR/dt) versus response (Fig. 2C and 2D) and the slopes of the linear regression analysis for each concentration were used in the –kobs versus concentration plot (Fig. 2E), where the slopes of the linear regression analysis correspond to the apparent kon. For the dissociation phase, we plotted ln(R0/R) versus time, where R0 is the response at the starting point of dissociation and R is the response at any time for the highest concentration of each protein, with the slope corresponding to koff (Fig. 2F). Rates obtained by linearization were similar to those provided by BiaEvaluation.
Table 1 summarizes the kinetic rates obtained with various protein concentrations and ligand densities. The major difference observed in binding to CD4 between Bori and Bori-15 gp120s was a threefold-reduced equilibrium dissociation constant for Bori-15 at 37°C. This reflects mainly that Bori-15 had a threefold-lower dissociation rate constant than Bori (Student's t test; P < 0.05), since the association rates were virtually the same. Although limited in magnitude and opposite in direction from changes observed in off rates at 25°C (which were not statistically significant), the difference in off rates was intriguing and could reflect an increased stabilization of the Bori-15-CD4 complex.
Interaction kinetics of gp120s with Abs. We also measured the interaction kinetics of gp120s with the 17b MAb (CD4i epitope) and D7324 Ab (an exposed, well-conserved epitope in the C5 region). Various gp120 concentrations were flowed at 25°C or 37°C in the absence of CD4 over Abs immobilized at different densities. Representative sensorgrams of Bori and Bori-15 gp120s binding to the 17b MAb are shown in Fig. 3A and 3B, respectively. BaL gp120 at the same concentrations was evaluated in all experiments (not shown). Data were fitted to a simple 1:1 binding model with BiaEvaluation, and association and dissociation rates were obtained. Linearization of experimental data was also performed as described above, generating the derivative versus response (Fig. 3C and 3D), the –kobs versus concentration (Fig. 3E), and the ln(R0/R) versus time (Fig. 3F) plots. Rates obtained were similar to those provided by BiaEvaluation.
Kinetic rates are summarized in Table 2. No statistically significant differences between the association rates to 17b of Bori, Bori-15, and BaL gp120s were observed. However, Bori-15 gp120 showed statistically significant 3- and 14-fold-lower dissociation rates from 17b than Bori at 25°C (Student's t test; P < 0.02) and 37°C (P < 0.05), respectively. By contrast, they showed no difference in association rates to or dissociation rates from D7324 Ab (Table 2).
We also estimated the affinity of the interaction between gp120s and 17b MAb by the equilibrium dissociation constant and found that Bori-15 had three- to fourfold-higher affinity than Bori and eight- to ninefold higher affinity than BaL for 17b MAb at both temperatures (Table 2).
Finally, to study the effects of sCD4 induction on 17b MAb binding, gp120s (200 nM) were incubated for at least 1 h at 37°C with increasing concentrations of sCD4. Maximum response at saturating concentrations of sCD4 was similar in all three gp120s (data not shown). Accordingly, the increase in response at saturating concentration of sCD4 (with respect to that in the absence of sCD4) was higher for Bori and BaL (a 7- and 15-fold increase, respectively) than for Bori-15 gp120 (a 3.8-fold increase). Similar results were obtained with the 48d MAb.
Inhibition of infection by TAK-779 and anti-CD4 Abs. A higher affinity between gp120 and CCR5 and higher density of CCR5 on the target cell membrane correlate with decreased sensitivity to inhibition by CCR5 antagonists and fusion inhibitors (63). Thus, to study a surrogate for the affinity between trimeric envelopes and CCR5, we compared the sensitivity of Bori and Bori-15 envelope-pseudotyped viruses to inhibition by CCR5 antagonist TAK-779 on U87 cells expressing various levels of CCR5. Both pseudotypes were less sensitive to TAK-779 in cells expressing high CCR5 levels than in those with low CCR5, but there was no difference in sensitivity between Bori and Bori-15 pseudotypes regardless of CCR5 levels (Fig. 4).
Finally, we evaluated the ability of three different anti-CD4 MAbs (RPA-T4, SK3, and #21) to inhibit infection by Bori and Bori-15 pseudotypes as a surrogate for CD4:trimeric envelope affinity. As shown in Fig. 5, Bori-15 pseudotypes were more resistant to inhibition than Bori pseudotypes, consistent with a higher affinity for CD4 and a greater ability to outcompete the binding of MAbs. Nonlinear regression analysis using a sigmoidal dose-response model (variable slopes) demonstrated that the curves showing inhibition of infection by Bori and Bori-15 pseudotypes were statistically different, since the null hypothesis of a single best-fit curve for the two data sets was rejected for all three antibodies (P values of <0.0001 in all cases). The IC50 values estimated from nonlinear regression analysis were between four- and sixfold higher for Bori-15 than for Bori with all three MAbs (values in parentheses are 95% confidence intervals): RPA-T4, 62.8 ng/ml (24.3 to 162.6) versus 11.4 ng/ml (5.2 to 25.4); SK3, 13.1 ng/ml (8.6 to 19.6) versus 2.2 ng/ml (1.0 to 4.9); and #21, 30.9 ng/ml (21.2 to 45.0) versus 7.5 ng/ml (4.0 to 14.3). In addition, the IC50 values and standard error obtained from nonlinear regression for Bori and Bori-15 with each anti-CD4 antibody were compared by t tests; we found that the differences were statistically significant (RPA-T4, P < 0.01; SK3 and #21, P < 0.001).
These differences between Bori and Bori-15 trimeric envelopes incorporated into pseudotypes correlate with those observed with monomeric gp120s and give credence to the possibility that a difference in CD4 affinity between Bori and Bori-15 underlies, at least in part, the mechanism for increased infectivity of Bori-15 for microglial cells expressing low levels of CD4.
DISCUSSION
Compartmentalization of HIV-1 sequences in the brain in comparison with peripheral tissues has been demonstrated in numerous studies (19, 33, 53, 61, 86), suggesting that there is independent viral evolution within the CNS. Although tissue-specific genetic variation in the CNS might be due to a founder effect, it is more likely that there is specific adaptation to this niche, due to limited trafficking across the blood-brain barrier, together with HIV-1 persistence and replication in long-lived cell types such as microglia and perivascular macrophages (35, 82). Thus, it is reasonable to hypothesize that the low level of CD4 expression of microglia and perivascular macrophages drives the selection of viruses that are particularly fit for this environment (13, 29, 57, 85).
We previously demonstrated that pseudotypes containing the envelope glycoproteins from the in vitro microglia-adapted HIV-1Bori-15 isolate infect cells expressing low levels of CD4 more efficiently than those with parental HIV-1Bori envelope glycoproteins (43). In addition, contrary to Bori, Bori-15 pseudotypes were sensitive to sCD4 neutralization (43). Although the latter could be due to a variation in sCD4-induced gp120 shedding, we hypothesized that Bori and Bori-15 might differ in the affinity for CD4. Here we report that these phenotypic differences are associated with altered binding kinetics between Bori-15 gp120 and CD4. Unlike the results of experiments performed at equilibrium, which showed no difference in binding, real-time studies with SPR-based optical biosensor technology showed that the Bori-15 gp120 has a small but statistically significant lower dissociation rate from CD4 than Bori, leading to a higher affinity of similar magnitude. Equilibrium binding assays require the reactant to be separated from the product, and the equilibrium of the reaction can be disturbed by the washing steps. In biosensor analysis, the complex is detected in the presence of unbound material, avoiding this potential interference and providing alternative information regarding the interaction between monomeric gp120s and CD4 (28, 32, 68).
We found that Bori-15 gp120 bound to CD4 with higher affinity than Bori when the experiments were performed at 37°C but not at 25°C. It is intriguing that while Bori and BaL gp120s have similar KDs at either temperature, Bori-15 gp120 had a faster association combined with a slower dissociation at 37°C than at 25°C, resulting in a substantial increase in affinity (13 fold) for the Bori-15-CD4 interaction between the two temperatures investigated. We believe that the slower dissociation of Bori-15 gp120 from CD4 may indicate a more stable interaction between gp120 and CD4 and prolonged contact time (before dissociating), increasing the probability that the gp120-CD4 complex will establish interactions with additional receptors such as CCR5.
However, it could be argued that this small difference in affinity of the monomeric forms of gp120 may not fully account for the phenotypic differences between Bori and Bori-15 that were noted in the context of viral particles (43, 72). As a surrogate for perhaps a more realistic CD4:trimeric envelope affinity, we evaluated the sensitivity of infection by Bori and Bori-15 envelope-pseudotyped viruses to increasing concentrations of three anti-CD4 MAbs. The inhibition curves showed a lower sensitivity for Bori-15, with statistically significant four- to sixfold-higher IC50 values than Bori. Decreased sensitivity to inhibition of infection by anti-CD4 antibodies has been recently reported for brain-derived HIV envelopes by Peters et al. (56). Additional studies will be required to dissect the role of the individual 8 amino acid changes between Bori and Bori-15 envelopes, especially the four substitutions located in the V1/V2 region, on gp120 conformation and/or flexibility.
Numerous studies have investigated the interaction between gp120 and CD4 by SPR (5, 50, 87, 92, 93), but its binding to Abs anti-CD4i epitopes has not received as much attention (27, 92, 93). In our study, Bori-15 gp120 in the absence of CD4 showed a statistically significant, 14-fold-lower dissociation rate than Bori from 17b MAb, although its affinity was only 4-fold higher than that of Bori. By contrast, no difference in kinetic rates or affinity in the interaction with the D7324 Ab was observed, suggesting that there is a specific increase in the exposure of CD4i epitopes in Bori-15 prior to any CD4i conformational change. This correlates with the higher 17b binding and sensitivity to neutralization by 17b MAb that we previously reported for Bori-15, compared to Bori, in the context of trimeric envelopes (43). However, both gp120 proteins bound to 17b and 48d MAbs similarly after CD4 induction. Altogether, these results seem to indicate that the envelope of the microglia-adapted isolate may exist predominantly in a partially triggered conformation.
Dissociation rate, but not association rate or overall affinity, has been shown to correlate with HIV-1 neutralization by Abs directed to the V3 loop of gp120 (84). Rapid dissociation seems to be sufficient to prevent neutralization, while a prolonged contact time due to slow dissociation results in neutralization. In our experiments, we have also found that the reported Bori-15 sensitivity to neutralization by sCD4 and 17b MAb (43) correlates with an apparently slower rate of dissociation of monomeric gp120 from both CD4 and 17b MAb. A relationship between CD4 independence and neutralization sensitivity has been reported in the literature (18, 46, 62). However, HIV-1Bori-15 appears to be at an intermediate state, since neutralization sensitivity coexists with CD4 dependence. The lack of natural occurrence of CD4-independent HIV-1 isolates is likely due to an increased susceptibility to neutralizing antibodies by those viruses primed for coreceptor binding. Since the blood-brain barrier renders the brain less accessible to the immune system, the generation of CD4-independent viruses could theoretically proceed in vivo within the CNS, potentially expanding the target cell population by including CD4-negative cells (such as astrocytes, oligodendrocytes, and neurons).
To date, analysis of cloned viruses and viral envelopes from CNS has failed to reveal evidence for CD4-independent isolates. However, Peters et al. recently published a study concluding that envelopes from primary brain isolates confer lower CD4 dependence and lower sensitivity to inhibition by anti-CD4 antibodies than envelopes derived from peripheral tissues of the same individuals (56). In addition, lower CCR5 and CD4 dependencies have been shown to represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS (25). These findings suggest that the phenotypic differences with the Bori and Bori-15 isolates described by us are actually present in vivo and highlight the relevance of our model. Our current studies suggest that those differences found in vivo may be due to increases in receptor affinity; thus, similar experiments should be done with envelopes derived from primary isolates.
Low levels of CCR5 incorporation into MLV particles hampered our attempt to measure the affinity of gp120s to CCR5. Nevertheless, Bori-15 and Bori pseudotypes were equally sensitive to inhibition by the CCR5 antagonist TAK-779 (2, 17). Since sensitivity to coreceptor antagonists is determined to some extent by the affinity of the gp120-coreceptor interaction (63), our results suggest that there is no difference in the affinity of Bori and Bori-15 envelopes for CCR5. In fact, this would correlate with the lack of difference in CCR5 dependency observed with envelope-pseudotyped viruses in cells expressing various levels of CCR5 (43). Innovative SPR analysis with immobilized CCR5 (11, 31, 69, 75) could provide a definitive answer to this issue.
In summary, our results indicate that the monomeric gp120 derived from a microglia-adapted virus (HIV-1Bori-15) binds to CD4 and to the human 17b MAb with altered kinetics, compared with the gp120 of the primary, peripheral parental virus. These changes may result in prolonged contact time of Bori-15 with the cellular receptors. The neutralization sensitivity and accessibility of CD4i epitopes in Bori-15 gp120 resemble that of a CD4-independent envelope, although its gp120 mediates fusion and infection in a CD4-dependent manner, suggesting a partially triggered conformation. The difference in CD4 binding between Bori and Bori-15 monomeric gp120s as detected via SPR correlates with data showing a difference in sensitivity to inhibition of infection by envelope-pseudotyped viruses with anti-CD4 antibodies. Therefore, the data presented here suggest that the envelope of the microglia-adapted virus has a moderately higher affinity for CD4 than that of the parental, peripheral isolate and that this difference may be responsible in part for the phenotypic differences between the two strains.
ACKNOWLEDGMENTS
We thank David Kabat, Michael Malim, Nathaniel Landau, and the NIH AIDS Research and Reference Reagent Program for providing us with useful reagents. We are grateful to Samantha Soldan (University of Pennsylvania) for her comments on the manuscript.
This work was supported by National Institutes of Health grants NS-27405 and NS-35743 (to F.G.-S.) and NS-47970 (to J.M.-G.).
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