JOURNAL OF VIROLOGY, Mar. 1991, p. 1133-1140

Vol. 65, No. 3

0022-538X/91/031133-08$02.00/0 Copyright C) 1991, American Society for Microbiology

Direct Measurement of Soluble CD4 Binding to Human Immunodeficiency Virus Type 1 Virions: gpl20 Dissociation and Its Implications for Virus-Cell Binding and Fusion Reactions and Their Neutralization by Soluble CD4 JOHN P.

MOORE,'*

JANE A.

McKEATING,l WARWICK A.

NORTON,2

AND

QUENTIN J. SATTENTAU3

Chester Beatty Laboratories, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB,l and Department of Applied Mathematics and Theoretical Physics, University of Cambridge, Cambridge CB3 9EW,2 United Kingdom, and Howard Hughes Medical Institute, College of Physicians and Surgeons of Columbia University, New York, New York 100323 Received 10 September 1990/Accepted 28 November 1990

We have analyzed the binding of soluble CD4 (sCD4) to human immunodeficiency virus type 1 (HIV-1) virions (isolates IIIB and RF) at 4 and 37°C by using a combination of gel exclusion chromatography and enzyme-linked immunosorbent assay detection systems. The sCD4 binding curve at 37°C indicates that the affinity of the interaction of sCD4 with gpl20 on the virion surface is indistinguishable from the affinity of sCD4 for the equivalent concentration of soluble gpl20. At 4°C, however, the affinity of sCD4 for virion-bound gpl20 but not for soluble gpl20 is reduced by about 20-fold. Binding of sCD4 (>0.2 ,ug/ml) to virions at 37°C but not 4°C induces the rapid dissociation of a major proportion of gp120 from gp4l on the virion surface. This dissociation requires occupancy by sCD4 of multiple (probably two) binding sites on a gpl2O-gp4l oligomer. At 37°C there are two components to the neutralizing action of sCD4 on HIV-1; reversible, competitive inhibition at low sCD4 concentrations (0.05 ,ug/ml), was half-maximal at approximately 0.15 jig of sCD4 per ml (3 nM) and maximal at 1 to 2 ,ug of sCD4 per ml, and subsequently declined as gpl20 was lost from the virions (cf. Fig. 1 and 2; see Fig. 4). The loss of gpl20 from the virions accounts for the lower ELISA signals at 37°C compared with those at 4°C, since the concentration of gpl20 eluted from the chromatography columns in the virion fraction is in the range where the amount of gpl20 bound by the solid-phase antibody D7324 is proportional to the input concentration. To compare how the binding of sCD4 to virions at 37°C compared with its binding to solubilized, monomeric gpl20 under the same conditions, virions were disrupted with NP40 and then mixed with sCD4 and diluted to the same concentration as that in the column eluate (see Materials and Methods). The concentrations of virion gpl20 in the assays were 5 ng/100 ,ul, the same as that applied to the chromatography columns, or half as much to compensate for the

sCD4 BINDING TO HIV-1 VIRIONS

VOL. 65, 1991

1135

VIRION BOUND gpl2O, X 100

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FIG. 1. Effect of sCD4 on dissociation of gpl20 from virions at 4 and 37°C. sCD4 at the indicated concentrations was incubated with 100-,ul aliquots of HIV-1 IIIB (U, El) or RF (0, 0) virions for 2 h at 4°C (El, 0) or 37°C (D, 0). The reaction mixture was subjected to gel-exclusion chromatography on Sephacryl S-1000; the fraction containing virions was collected, and its gpl20 content was determined. Each data point is derived from a single column, with determination of gpl20 content in triplicate (mean ± standard error of the mean). The data are plotted as percentages of the gpl20 bound on virions in the absence of sCD4 (100%) for each experiment.

approximately 50% of the virion gp120 that was not virion bound and hence not recovered from the columns in the relevant virion fraction. Half-maximal binding of sCD4 was found at sCD4 concentrations of 0.08 to 0.18 ,ug/ml (1.8 to 4.0 nM); the lower values occurred at the higher gpl20 concentration. Soluble, recombinant gp120 (5 ng/100 RI) bound sCD4 half maximally at 0.07 ,ug/ml (1.6 nM) under similar conditions (Fig. 2). Thus, within a factor of 3, binding

of sCD4 to virions at 37°C was indistinguishable from its binding to the equivalent concentration of soluble gp120. We confirmed that sCD4 at 50 ,ug/ml saturated virions at 4°C by disrupting virion-sCD4 complexes with detergent after elution from gel filtration columns at 4°C and then adding additional sCD4 (Fig. 3). There was little increase in sCD4 binding under these conditions, compared with the enhanced binding of sCD4 to gpl20 from virions that were

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J. VIROL.

MOORE ET AL.

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FIG. 3. Saturation of virions by sCD4 at 4°C. T aliquots of IIIB (-, Cl) or RF (0, 0) virions were incutbated for 2 h at 4°C with (0, 0) or without (U, 0) 50 ,ug of sCD4 pe r ml before gel exclusion chromatography. The virion-containing fractions from each pair of runs were pooled and disrupted with 1%1NP40. Aliquots (200 [LI) were incubated for 1 h at room temperature with the sCD4 concentrations indicated and then assayed for gpl20)-bound sCD4.

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eluted from the columns at 4°C in the absence of sCD4 and then disrupted with detergent (Fig. 3). The latter bound sCD4 with high affinity (Fig. 3). A higher con centration of sCD4 is therefore required to saturate gpl20 on virions at 3 7°C. 4°C compared with the concentration required at . at 37C To analyze more completely the differenti,al binding of sCD4 to HIV-1 virions at 4 and 37°C, we recconstructed a theoretical sCD4 binding curve for RF virions at 37°C (Fig. 4) by combining data from Fig. 1 and 2. Thus thwe data shown for sCD4 binding in Fig. 4 are based on the bincding curve for virions at 37°C in Fig. 2 but have been correcte d for the loss of soluble gpl2O-sCD4 complexes with data froim Fig. 1. The resultant curve shows that sCD4 binding to viirions at 37°C was half-maximal at 0.25 ,ug/ml (5.5 nM) and vvas saturating at 1 ,ug/ml (22 nM). The ELISA signal at sEaturation was similar to those for curves derived from disrupted virions containing the same gpl20 concentration (F ig. 2 and 3), suggesting that it reasonably represents sCD4 binding to all the functional gpl20 molecules that were oin the virions before sCD4 addition. The data imply that virion-bound gpl20 has a 10- to 20-fold lower affinity for sCI )4 at 4°C than at 37°C (compare the 4°C curve in Fig. 2 wiith the reconstructed 37°C curve in Fig. 4). In contrast, bin ding of sCD4 to recombinant gpl20 previously captured onto) a solid phase by antibody D7324 (22) was similar at 37 and X4°C, with less than a twofold variation in the sCD4 concentrzation required for half-maximal binding (data not shown). The inherent binding affinity of non-virion-bound gpl20 forr sCD4 is not therefore greatly influenced by small alteratioris in temperature, suggesting that gpl20 on mature viricDns does not behave identically to soluble gpl20 in its bindiing of sCD4 at low temperatures. sCD4 binding to virions, gpl20 loss, and neutr-alization. The relationship between sCD4 binding to HIV-1 viirions at 37°C, its induction of gpl20 dissociation, and its neutiralizing action were analyzed by comparing infectivity inhiibition curves with those for sCD4 binding and gpl20 dissoci ation (Fig. 4). The dose-response curve for sCD4-induced gyp120 dissociation from virions at 37°C was very similar to the recon-

FIG. 4. Comparison of neutralization by sCD4 with sCD4 binding to virions and gp120 dissociation. The data for sCD4-induced gpl20 dissociation from RF virions at 37°C (U) are replotted from Fig. 1 as percentages of the amount of gp120 bound to virions in the

absence of sCD4 (100%). Estimates of the amount of sCD4 bound on the virions at 37°C (A) are derived from the binding curve in Fig. 2, with background-corrected (no sCD4) optical densities at 492 nm multiplied by 100/(percent gp120 shed from virion), which was calculated from Fig. 1. The data are plotted as percentages of the maximum sCD4 bound, which was taken to be the mean of the optical densities at the six highest sCD4 concentrations (0.313 + 0.015, mean + standard error of the mean). Note that the background optical density value in the absence of sCD4 (0.071 ± 0.006) should be added to this value for comparison with other figures. The data for sCD4-mediated inhibition of RF infection (0) were obtained

by removing 10-,ul aliquots from one of the experiments immediately before fractionation of the remainder on Sephacryl S-1000. The log 50% tissue culture infective doses per milliliter corresponding to each of the data points shown were 4.87, 4.87, 4.85, 4.75, 4.62, 4.37,

4.12, 3.62, 3.62, and

Direct measurement of soluble CD4 binding to human immunodeficiency virus type 1 virions: gp120 dissociation and its implications for virus-cell binding and fusion reactions and their neutralization by soluble CD4.

We have analyzed the binding of soluble CD4 (sCD4) to human immunodeficiency virus type 1 (HIV-1) virions (isolates IIIB and RF) at 4 and 37 degrees C...
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