JOURNAL OF VIROLOGY, Jan. 1992, p. 132-138 Vol. 66, No. 10022-538X/92/010132-07$02.00/0Copyright © 1992, American Society for Microbiology

Kinetics of Soluble CD4 Binding to Cells Expressing HumanImmunodeficiency Virus Type 1 Envelope Glycoprotein

DIMITER S. DIMITROV,1 KATHLEEN HILLMAN,2 JODY MANISCHEWITZ,2 ROBERT BLUMENTHAL,'AND HANA GOLDING2*

Section on Membrane Structure and Function, National Cancer Institute,' and Division of Virology,Center for Biological Evaluation and Research, Food and Drug Administration,2 Bethesda, Maryland 20892

Received 16 August 1991/Accepted 8 October 1991

The high-affinity interaction between the envelope glycoprotein (gpl2O-gp4l) of the human immunodefi-ciency virus type 1 and its receptor, CD4, is important for viral entry into cells and therapeutical approachesbased on the soluble form of CD4 (sCD4). Using flow cytometry, we studied the kinetics of binding of sCD4 togpl20-gp41 expressed on the cell surface. sCD4 binding was dependent on sCD4 concentration andtemperature and exhibited bimolecular reaction kinetics. Binding was very slow at low sCD4 concentrations(below 0.2 ,ig/ml) and low temperatures (below 13°C) but increased sharply with increasing temperature. Therate constant for association at 37°C (1.5 x 105 M-1 s-1) was 14-fold higher than at 4°C, but the affinity ofsCD4 to membrane-bound gpl2O-gp4l was not significantly affected. The activation energy at highertemperatures (28 to 37°C) was less than at lower temperatures (4 to 13°C). After long periods of incubation, weobserved a decrease of surface-bound sCD4 and gpl20, even at low temperatures, which was attributed tosCD4-induced shedding of gpl20. The rate of gpl20 shedding was much lower than the rate of sCD4 bindingand was dependent on sCD4 concentration and temperature. The finding that sCD4 binding is slow, especiallyat low sCD4 concentrations, can be of critical importance for efficient blocking of viral infection by sCD4 andshould be considered when designing new protocols in the therapy of AIDS patients.

Human immunodeficiency virus type 1 (HIV-1) enterscells by utilizing a high-affinity interaction between the viralglycoprotein (gpl20-gp4l complex) and the cellular receptorCD4 (5, 9, 15, 17-19, 21, 22, 27, 29, 30). The soluble form ofCD4 (sCD4) was found to bind with high affinity to gp120 andto inhibit viral infection and syncytium formation in vitro (2,4, 6, 8, 12, 17, 28, 31). Recently, it was suggested that sCD4may also neutralize HIV-1 by inducing a release (shedding)of the noncovalently bound gpl20 from gp4l (1, 10, 24).Binding of sCD4 to virions and shedding of gp120 correlatedwith the extent of inhibition of HIV-1 infection and de-pended on the concentration of sCD4 and temperature (23).Low sCD4 concentrations (1 pug/ml) completely inhibited theviral infection when the virus was preincubated with sCD4for 2 h at 37°C, whereas sCD4 concentrations greater than 10,ug/ml were required to completely inhibit infection when thevirus was preincubated with sCD4 at 4°C for the same periodof time (23). This observation was attributed to a loweraffinity between HIV-1 and sCD4 and lack of gp120 sheddingat 4°C. However, no attempt was made to compare thekinetics of sCD4 binding at the two different temperatures.

Several studies indicated that binding of gp120 to cell-associated CD4 (3) and sCD4-induced shedding of gp120 (1,10, 24) are slow processes. For example, equilibrium ofrecombinant gp120 (2 ,ug/ml) binding to cellular CD4 at 4°Cwas reached after 4 to 8 h (3). It would be expected that withlower concentrations of gp120, the time required to reachequilibrium would be even longer. sCD4-induced sheddingof gp120 is also a slow process. It takes 2 to 8 h to release asignificant amount of gpl20 from the gpl20-gp4l complex,even at high sCD4 concentrations (1, 10, 24).These findings indicate that inhibition of virus infection

and HIV-1 envelope-mediated syncytium formation by sCD4

* Corresponding author.

might depend on the time of interaction of the envelopeglycoprotein with sCD4 and that differences in virus inacti-vation at 4 versus 37°C could be due to the slow kinetics ofsCD4-gpl20-gp4l interactions, rather than different affin-ities. To test this hypothesis and to further characterize theCD4-gpl2O-gp4l interactions, we designed experiments tomeasure the kinetics of sCD4 binding to the cell surface of aCD4-negative (CD4-) T-cell line expressing the HIV-1 en-velope after infection with the recombinant vaccinia virusvPE16 (7).

It was found that the kinetics of binding of sCD4 tomembrane-associated gp120-gp41 and shedding of gp120strongly depend on sCD4 concentration and temperature. Incontrast, the affinity of sCD4 to gp120-gp4l differed onlyminimally at different temperatures. These findings may beof crucial importance for understanding the mechanisms ofthe initial steps of viral entry into cells, interactions betweeninfected and uninfected cells, and inhibition of viral infectionby sCD4.

MATERIALS AND METHODS

Cells and reagents. The human cell line CEM was obtainedfrom the American Type Culture Collection, Rockville, Md.The CD4- subclone 12E1 was derived from CEM cells byethyl methanesulfonate mutagenesis and negative selectionwith OKT4A plus complement, as previously described (11).The recombinant vaccinia virus, vPE16, containing the geneencoding gpl20-gp41 (7) was provided by P. Earl and B.Moss, National Institute of Allergy and Infectious Diseases,Bethesda, Md. Its infectivity was measured by using a HeLacell plaque assay. The rabbit polyclonal anti-gpl20 serumwas obtained from ABT (Cambridge, Mass.). The fluores-cein isothiocyanate (FITC)-conjugated monoclonal antibodyOKT4 was obtained from Ortho Diagnostics (Raritan, N.J.).The FITC-conjugated goat anti-rabbit and goat anti-mouse

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sCD4 BINDING TO HIV-1 ENVELOPE-EXPRESSING CELLS 133

antibodies (Abs) and sCD4 were obtained from Sigma Chem-ical Co. (St. Louis, Mo.).Measurement of cell surface-bound sCD4 and gpl2O. 12E1

(CD4-) cells were infected with recombinant vaccinia virus(vPE16) at 1 to 10 PFU per cell. After 20 h, 106 cells wereincubated for different periods of time with sCD4 at differentconcentrations and temperatures in 100 ,ul of RPMI 1640containing 1% bovine serum albumin (BSA). The cells werewashed twice with cold phosphate-buffered saline containing0.1% BSA and 0.1% sodium azide, stained with 1 ,ug ofFITC-conjugated OKT4 per ml at 4°C for 1 h, and then fixedwith 1% paraformaldehyde. 12E1 (CD4-) cells, which do notexpress gpl2O-gp41, were used as negative controls fornonspecific binding of sCD4, and 12E1-(gpl20-gp41)+ cellswithout sCD4 were used as negative controls for the FITC-conjugated OKT4 staining. CEM cells, which are CD4-positive (CD4+) cells, were used as positive controls forstaining with FITC-conjugated OKT4. In some experiments,the cells expressing gpl20 were incubated with unconjugatedOKT4 after interaction with sCD4 and then incubated withFITC-conjugated goat anti-mouse Ab (indirect staining).The cell surface-bound sCD4 was measured by using flow

cytometry and analyzed as described before (11). Briefly, thestained cells were analyzed by the Epic Profile (CoulterCounter, Hialeah, Fla.). The relative fluorescence intensity(in fluorescence units [FU]) was calculated from a standardcurve generated by using fluorescent beads with increasingintensities (Flow Cytometry Standards, Research TrianglePark, N.C.). The backgrounds (in FU) due to autofluores-cence and to nonspecific binding of antibodies were sub-tracted from the experimental values (25). The data arepresented as a ratio of FU of the bound sCD4 to FU of thesaturated maximal bound sCD4. In most cases, the maximalbound sCD4 was determined by incubation with 20 ,ug ofsCD4 per ml at 4°C for 2 h, because a further increase in theconcentration of sCD4 or incubation period did not lead toan increase in the amount of bound sCD4 (see Fig. 4, 7, and9).The CEM cell line, expressing about 1.7 x 104 CD4

molecules per cell (16) and stained with FITC-conjugatedOKT4, was used as a reference cell line to estimate theabsolute number of bound sCD4 molecules, since only onesCD4 molecule is bound to one gpl20 molecule. The numberof bound sCD4 molecules under saturation conditions isequal to the number of surface-associated gpl20 molecules.Hence, measurement of surface-bound sCD4 also allowedthe determination of the number of cell surface-associatedgpl20 molecules. The expression of gpl20 molecules wasdependent on the multiplicity of infection with recombinantvaccinia virus (Fig. 1). The average number of gpl20 mole-cules per cell varied from about 2 x 102 to 1.5 x 104. Theexpression of gpl20 also varied widely within the cellpopulation (Fig. 1). Rabbit anti-gpl20 Ab was used tomonitor the expression of gpl20 on cells (in the absence ofsCD4) before and after 4 to 6 h of incubation at 4 and 37°C.No significant changes were found (data not shown).The decrease in the amount of gpl20 at the cell surface

after incubation with high concentrations of sCD4 (gpl20shedding) was determined by staining in parallel with FITC-conjugated OKT4 and with rabbit anti-gpl20 polyclonal Ab.The conditions of staining were essentially the same as thosefor staining with OKT4, except that after the incubation withthe anti-gpl20 Ab, the cells were incubated with goat anti-rabbit FITC-conjugated Ab and then fixed. The use of asecondary Ab does not allow rigorous quantification of thenumber of gpl20 molecules left at the cell surface but does

allow us to make conclusions about relative changes in thesurface gpl20 concentration.

Analysis of sCD4 binding. The bimolecular reaction leadingto binding is represented by sCD4 + R ; sCD4-R, where Ris the "receptor" gpl20-gp41 and sCD4-R is the sCD4 boundto gpl20-gp41. This is typical for hormone-receptor bindingand for the initial stages of antibody-antigen binding (20).sCD4 is in excess, and its concentration is constant. Thetime course of binding is given by the following equations:[sCD4-R] = ([sCD4-R],[sCD4]/([sCD4] + K)[1 - exp(-t/ta)],ta = 1/(Ka[sCD4] + Kd), and K = KdIKa, where [sCD4-R], isthe maximal equilibrium concentration of the surface-boundsCD4, corresponding to the concentration of the initiallyunbound gpl20-gp41 molecules; Ka and Kd are the associa-tion and dissociation rate constants, respectively; t is time,and ta is characteristic time of association. The time periodneeded to reach half of the equilibrium value of the surface-bound sCD4, t112, is equal to 0.69 ta. K = KdJKa is theequilibrium dissociation constant. Affinity is equal to K-1.The procedure to calculate the rate constants from the

experimental data was as follows. (i) The maximal sCD4surface concentration, [sCD4-R],, was experimentally deter-mined as that surface concentration which corresponds tothe saturation in the bound sCD4 (sCD4-R), using sCD4 at 20,ug/ml and incubating 12El-(gpl20-gp41)+ cells for 2 h at 4°C(see Fig. 4, 7, and 9). (ii) The sCD4 surface concentrationsfor different conditions were divided by the maximal equi-librium value. (iii) The affinity was determined by fitting(with SigmaPlot 4.0 computer program) the equilibriumvalues of the surface-bound sCD4 to those given by theequation for bimolecular reaction at t>>ta (the affinitycorresponds to the sCD4 concentration needed to reach halfsaturation of the surface-bound sCD4). (iv) The experimen-tal data for the ratio sCD4-RIsCD4-R, for every sCD4concentration were fitted to the kinetic equation by using theSigmaPlot 4.0 computer program. The time of binding, taIwas calculated from the best fits with correlation coefficientshigher than 0.96. (v) The rate constants Ka and Kd werecalculated from the linear plots of tj-' versus bulk sCD4concentration. (vi) Affinity, K-1, was also calculated as a

ratio of the association and dissociation rate constants.Finally, the activation energy was estimated from the slopeof the dependence -lnKa versus 1IRT (Arrhenius coordi-nates), where R is the gas constant and T is the absolutetemperature. Only the datum points before the beginning ofthe decrease in the concentration of surface-bound sCD4were analyzed for binding.The decrease in the surface-bound sCD4 after long periods

of incubation with sCD4 was approximated by an exponen-tial function, [sCD4-R] = a + bexp(-Kst), where a + b =

[sCD4-R]m and [sCD4-R]m is the maximal equilibrium sur-

face concentration of the bound sCD4, a and b are constants,and Ks is the rate constant of sCD4 release after binding.

RESULTS

Specific binding of sCD4 to individual cells. To quantify theamount of sCD4 bound to cells expressing gpl20-gp41, we

developed an assay based on flow cytometry. We make use

of the fact that a single sCD4 molecule can bind to a singlegpl20 molecule, and this binding does not interfere withsubsequent binding of the OKT4 monoclonal antibody,which recognizes an epitope distant from the CD4-gpl20interaction site. This assay allowed us not only to measure

the number of surface-bound sCD4 molecules but also todetermine the number of gpl20 molecules at the cell surface.

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134 DIMITROV ET AL.

0E 0-nSWu-B 12E1(CD4-) D 12E1 (vPE 16)

Uninfected 3.3 PFU/Cell

1000 1

Is .0%^~ ~ .1 10 100

Log Fluorescence

1 10 100Log Fluorescence

FITC-Goat anti mouse

~FITC-OKT4

sCD4 (2,pg/ml)--FITC-OKT4

FIG. 1. Binding of sCD4 to cells, infected with vaccinia virusgpl2O-gp41 (vPE16) at different PFU, as quantified by flow cytom-etry. CD4- (12E1) cells were either uninfected (B) or infected withrecombinant vaccinia virus (vPE16) at 10 (C), 3.3 (D), or 1.1 (E)PFU per cell. After 20 h, the cells were incubated for 2 h with sCD4(2 ,ug/ml) at 4°C. The cells were then washed twice, incubated with1 ,ug of FITC-conjugated OKT4 (FITC-OKT4) per ml for 1 h, fixedwith 1% cold paraformaldehyde for 10 min, and washed. UninfectedCEM (CD4+) cells were stained with FITC-conjugated OKT4 as a

positive control (A). The vaccinia virus infectivity was determinedby using a HeLa cell plaque assay.

Figure 1 shows examples of flow cytometry data for CEMcells stained with FITC-conjugated OKT4 (Fig. 1A), unin-fected 12E1 (CD4-) cells incubated with sCD4 and thenstained with FITC-conjugated OKT4 (Fig. 1B), and 12E1cells infected with recombinant vaccinia virus (vPE16) atdifferent PFU/cell ratios after incubation with sCD4 andFITC-conjugated OKT4 (Fig. 1C to E). The noninfected12E1 (CD4-) cells did not show appreciable nonspecificfluorescence, when incubated either with FITC-conjugatedOKT4 or with sCD4 (2 ,ug/ml) followed by FITC-conjugatedOKT4 (Fig. 1B). Therefore, there was no discernible bindingof sCD4 to uninfected 12E1 cells. The distribution of thenumber of cells expressing gpl2O-gp4l as a function offluorescence (Fig. 1C) (which is proportional to the numberof gp120 molecules) was wider than that for cells expressingendogenous CD4 (Fig. 1A). This heterogeneity suggests thateven at a high multiplicity of infection, individual cells vary

in their levels ofgpl2O-gp4l expression. Expression of gpl20decreased with decreasing multiplicity of infection with therecombinant vaccinia virus (Fig. 1C, D, and E). It should benoted that the multiplicity of infection of vaccinia virus wasmeasured by using a HeLa cell plaque assay and may not bethe same for 12E1.We next determined whether this sCD4 binding assay is

sensitive to variations in the concentration of sCD4 addedand to the incubation temperature. As can be seen in Fig. 2,

S

Ez

0

1 10 100 1000 1 10 100 1000Log Fluorescence Log Fluorescence

FIG. 2. sCD4 binding to cells expressing gpl20-gp4l at 4 and37°C. CD4- (12E1) cells were infected with recombinant vacciniavirus (vPE16, 10 PFU per cell) and incubated for 30 min withdifferent concentrations of sCD4 at 4°C (A) or 37°C (B). The cellswere then washed twice, incubated with 1 ,ug of OKT4 per ml for 1h, fixed with 1% cold paraformaldehyde for 10 min, washed, andstained with 1 ,ug of FITC-conjugated goat anti-mouse Ab per ml for1 h. The sCD4 concentrations (in micrograms per milliliter) were asfollows: 0 (. ), 0.05 (----), 0.2 ( ), 2.0 ( ), and20(---- -)-

the surface-bound sCD4 increased with increasing sCD4concentration, both at 4°C and at 37°C. It was also apparentthat at a fixed period of incubation time (30 min), binding ofsCD4 at 37°C was higher than at 4°C at all sCD4 concentra-tions. This could be due either to differences in the affinity ofsCD4 to gpl20 at different temperatures, as was previouslysuggested for sCD4 binding to intact virions (23), or todifferences in the kinetics of sCD4 binding. To address thesepossibilities and to further characterize the CD4-gpl2O-gp4linteractions, we studied the kinetics and affinities of sCD4binding to gpl2O-gp4l expressing 12E1 cells using increasingconcentrations of sCD4 over increasing incubation times andat different temperatures.sCD4 binding kinetics: concentration dependence and af-

finity at 4°C. The amount of surface-bound sCD4 increaseddramatically with increasing sCD4 concentrations and incu-bation times (Fig. 3). The time dependence was fitted very

1 .0

0.8

0.6 [0-

0r

,c:C)0.4 .

0.2

0.0

0 50 100 150 200 250 300

TIME (MIN)

FIG. 3. Kinetics of sCD4 binding to cells expressing gpl2O-gp4lat 4°C. CD4- (12E1) cells, expressing HIV-1 gpl2O-gp4l afterinfection with vPE16 (3.3 PFU per cell) were incubated with sCD4at 4°C for the indicated periods of time. The amounts of bound sCD4were quantified by flow cytometry and are presented as a ratio to themaximum bound sCD4 (measured by the amount of bound sCD4 [20,ug/ml]) for 2 h at 4°C. The lines represent least-square fits byexponential curves.

z

E 12E1 (vPE 16)1.1 PFU/Cell

I.

/ \.

J. VIROL.

2 /-tg/ml SC0.2 tLg/ml sC0.05 tLg/ml s(

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sCD4 BINDING TO HIV-1 ENVELOPE-EXPRESSING CELLS 135

1.0

C)

C

zD

0mLuI

C,)

J

L)

0.8

0.6 [

0.4 [

0.2

0.0

0 10 20 30 40 50

sCD4 (,ag/ml)FIG. 4. Equilibrium binding of sCD4 at 4°C to cells expressing

gpl2O-gp4l. CD4- (12E1) cells were infected with 3.3 PFU of therecombinant vaccinia virus (vPE16) per cell and were incubated 20h later for 6 h at 4°C with 0, 0.2, 2, 20, and 50 ,ug of sCD4 per ml. Thesurface-bound sCD4 at equilibrium is fitted (continuous line) withthe equation [sCD4-R]/[sCD4-R], = [sCD4]/([sCD4] + K) (seeMaterials and Methods) with K = 13 nM.

well with exponential curves. At 4°C, equilibrium was

reached only after 2 to 4 h of incubation with 2 ,ug of sCD4per ml and after 4 to 6 h with 0.2 ,ug of sCD4 per ml (notshown). Lower sCD4 concentrations required even longerperiods of time to equilibrate with surface gpl2O-gp4l. Theaffinity of sCD4 to gpl2O-gp41 at 4°C was calculated from theequilibrium values of the surface-bound sCD4, K` = (0.78

0.22) x 108 M- (Fig. 4). This value corresponds to anequilibrium dissociation constant, K, equal to 13 + 3 nM. Itis seen from Fig. 4 that the experimental values for theequilibrium surface-bound sCD4 were very well fitted by atheoretical dependence based on a bimolecular reaction (seethe kinetic equation for sCD4 binding at t>>ta in Materialsand Methods). This is an indication that sCD4 bindingexhibited bimolecular reaction kinetics and that there are nocooperative effects. Another indication for bimolecular ki-netics for sCD4 binding is the proportionality of the inversebinding times (l/ta) to the sCD4 concentration (Fig. 5).Temperature dependence of sCD4 binding kinetics. The

kinetics of sCD4 binding to the same cells at higher temper-atures (13, 18, 28, and 37°C) were faster than that at 4°C (Fig.5 and 6). The time period needed to reach half of theequilibrium value of the bound sCD4 (t1/2 = 0.69ta) de-creased with increasing temperature. At 37 and 4°C, the t1/2swere 10 and 120 min, respectively, with a sCD4 concentra-tion of 0.2 pLg/ml. In contrast, affinity was not significantlyaffected by temperature. The equilibrium dissociation con-stant at 37°C was 11.6 versus 13.0 nM at 4°C (means fromthree experiments).

Unlike the modest change in the affinity of sCD4 binding at37°C versus 4°C, the rate constants Ka and Kd decreasedstrongly with decreasing temperature. The association rateconstant at 37°C [(1.5 + 0.42) x 105 M-1 s-'] was 14-foldhigher than at 4°C (0.11 x 105) (means of three experiments)(see above; also Fig. 6). The dissociation rate constant was

also much smaller at 40C (0.18 x 10-4 s-1) than at 37°C (3.3X lo-4 s-1). Although the equilibrium dissociation constants

0.5

0.4 _

0.3 _

0.2 _

0.1 _

0.0 _

0.0 0.5 1.0 1.5 2.0

sCD4 (a(g/ml)FIG. 5. Temperature dependence of the characteristic times of

binding (ta) of sCD4 to cells expressing gpl2O-gp4l. The bindingtimes were calculated by fitting the data for sCD4 binding kinetics at4, 13, and 37°C by exponential curves. The straight lines areleast-square fits to the inverse binding times (1/ta).

calculated from the ratio of the dissociation and associationrate constants, K = KJ,/Ka (1.6 0.5 nM [at 4°C] and 2.20.8 nM [at 37°C]), were lower than the equilibrium dissoci-ation constants calculated from the data for the surface-bound sCD4 at equilibrium, they also were not significantlyaffected by temperature. The activation energy for highertemperatures (18 to 37°C) was severalfold smaller than forlower temperatures (4 to 18°C) (Fig. 6). 18°C seems to be atransition temperature from low to high activation energy.

Kinetics of sCD4-induced shedding of surface-bound gpl2O.Incubation of cells, expressing moderate amounts of gpl20(after infection with 3.3 PFU of recombinant virus), withhigh concentrations of sCD4 (20 ,ug/ml) at 37°C led to asignificant decrease in the amount of bound sCD4 with time.Such a decrease was not seen when cells were incubated at4°C (Fig. 7). The decrease in the amount of bound sCD4 at37°C was fitted to an exponential function, sCD4-R = a +

bexp(-K,t) with a rate constant, Ks, equal to 9.0 x 10-3

12.5

I--

C)

1-1

4.03.0 3.5

1 000/T (1 /K)

FIG. 6. Temperature dependence of the binding rate constants inArrhenius coordinates. The two straight lines are the least-squarefits to the rate constants at 4, 13, and 18°C and at 18, 28, and 37°C.

O 37 C0

V 13 C0

* 4C

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136 DIMITROV ET AL.

bound sCD4 with time correlated with a decrease in anti-gpl20 Ab staining, implying release (shedding) of the sCD4-

1.0 - gp120 complex. The amount of bound sCD4 left at the cellw 08

surface dependent the time of incubation, sCD4

0.8 concentration, and surface gp120 concentration. At highmultiplicities of infection (10 PFU) and after long periods of

z 0.6 incubation (6 h), a decrease in the surface-bound sCD4 was

observed, even at relatively low sCD4 concentrations (0.20.4 0 ,ug/ml) at 37°C (Fig. 9). Shedding was also seen at 4°C but at

higher concentrations of sCD4 (Fig. 9). However, shedding*4°Co was never complete. It ranged between 20 and 50% reduc-

0.2 4 C tion in the number of surface-bound gpl20 molecules.

0.0 a DISCUSSION

0 50 100 150 200 A major finding of this work is that the kinetics of sCD4binding to membrane-bound gp120-gp41 is slow, especially

TIME (MIN) at low sCD4 concentrations (Fig. 3) and temperatures (Fig.FIG. 7. Release of bound sCD4 from the cell surface. CD4- 5). This finding may have important implications for (i) in

cells, expressing HIV-1 gp120-gp41 after infection with 3.3 PFU of vitro assays using sCD4, (ii) mechanisms of HIV-1 bindingvPE16, were incubated with 20,ug of sCD4 per ml at 4 or 37°C for and fusion with cells, and (iii) in vivo pharmacokinetics ofthe indicated periods of time. The amount of bound sCD4 was sCD4quantified by flow cytometry and is presented as a ratio to the *

amaximum bound sCD4, assumed to be the saturated amount of The amount of bound sCD4 increased exponentially withbound sCD4 at 4°C and 20,g/ml of free sCD4. The lines represent time (Fig. 3). This is in agreement with previously publishedleast-square fits to the experimental data by single exponential data on binding of gpl20 to cells expressing CD4 at 4°C (3),dependences. which we fitted by a single exponential function with an

association rate constant equal to 1.8 x 104 M-1 s-1. Giventhe differences in the experimental systems, the agreement

min-' (a = 0.77, b = 0.41). For the same sCD4 concentra- between their data and ours (association rate constant of 1.1tion, the binding time (1/ta) is 3.8 min1. Therefore, under x 104 M-1 s-1 at 4°C (see above) is very good.these conditions (37°C, 20 ,ug of sCD4 per ml) the rate of The temperature affected significantly the rate of sCD4binding was 3.8/0.009, or 422-fold higher than the rate of binding. The binding rate constant was 14-fold higher at 37TCdecrease in the amount of surface-associated sCD4. than at 4°C. It was recently suggested that the affinity of

Theobserveddecrease in the amount ofsurfac soied sCD4 msCD4 to HIV-1 was 20-fold smaller at 4°C than at 37°C andThe observed decrease in the amount of bound sCD4 most that this may have important implications for the mechanismlikely reflected shedding of gp12O-sCD4 complex. To con- ofvrsclfuin23.Ortdyhwshaitspsibeofirm this possibility, cells were stained with rabbit anti-gpl20 of virus-cell fusion (23). Our study shows that it is possible toserum before and after incubation with sCD4 (20 p.g/ml) for reach this conclusion if binding is measured at only one time150 mi. As can be seen in Fig. 8, the decrease of surface- point (for example, Fig. 2). However, the kinetics of sCD4

binding clearly demonstrates that the rate constants and notthe affinity were significantly affected by the temperature(Fig. 5 and 6). The reason for this discrepancy could be due

l to differences in the experimental systems (intact virions

° 10 versus cells) or more likely because the virions were notN 1.0 /c>incubated for sufficiently long periods of time at low sCD4aP concentrations and low temperatures. The lack of a signifi-

0.8 - cant effect of temperature on the affinity of sCD4-gpl2Ointeractions implies that high affinity is not the sole determi-

0.6 nant of fusion. The differences in the activation energies atz low and high temperatures may indicate different mecha-0 nisms of sCD4 binding at both temperatures or reflect

M

0 sCD4 changes in the membrane.o: < lZ0 Another interesting observation is that the amount ofL' 0.2 gp surface-bound sCD4 decreased after long periods of incuba-

tion (Fig. 7 to 10). It was associated with a decrease in the0.0 c amount of surface-bound gpl20 as determined by a poly-

clonal rabbit anti-gpl20 Ab (Fig. 9). We interpret these data0 50 l00 150 200 as a release (shedding) of the sCD4-gpl2O complex from the

TIME (MIN) cell surface. These observations are in agreement withprevious studies using other techniques to quantify the

FIG. 8. sCD4-induced shedding of surface-associated gpl20. amount of released gpl20 (1, 10, 24).CD4 (12E1) cells, expressing gpl20-gp41 after infection with 3.3 g2ou shedding exiie an eona pnnePFU of vPE16, were incubated with 20 p.g of sCD4 per ml at 37°C for gpl20 shedding exhibited an exponential dependence on

the indicated periods of time. They were stained with either FITC- time, which may indicate monomolecular reaction kinetics.conjugated OKT4 or with rabbit anti-gpl20 serum followed with This finding is in agreement with recent observations of theFITC-conjugated goat anti-rabbit Ab. The decreases in the amounts kinetics of gpl20 release from infected cells incubated in theof surface-bound sCD4 and surface-associated gpl20 are presented presence of 52 ,ug of sCD4 per ml (10). We fitted the data ofas ratios to their maximal values. Hart et al. (10) by an exponential dependence on time with a

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sCD4 BINDING TO HIV-1 ENVELOPE-EXPRESSING CELLS

0 100 200 300

TIME (MIN)

1.0

0.8

0.6

0.4 [

0.2 [

0.0

0 100 200 300

TIME (MIN)

FIG. 9. Decrease of the amount of surface-bound sCD4 after long periods of incubation. Cells expressing gpl2O-gp4l after infection with10 PFU of vPE16 were incubated for the indicated periods of time with 0.05 (0, 0), 0.2 (V, V), 2 (-, O), or 20 (A, A) of sCD4 per ml at 4(filled symbols) or 37°C (empty symbols).

rate constant, Ks, equal to 4.2 x 10-2 min-'. This value isabout 4.7-fold higher than that calculated from the datashown in Fig. 7. The difference could be due to the higherconcentration of sCD4 used in their study (10) and thedifferent experimental system (infected cepls versus cellsexpressing only gp120-gp4l).The rate of gpl20 shedding was much slower (about

400-fold at 20 ,ug of sCD4 per ml) than the rate of sCD4binding and was dependent on sCD4 concentration andtemperature (Fig. 9). In contrast to previous studies, we

found that sCD4 induces gp120 shedding even at 4°C, butonly after long periods of incubation (6 h) with relatively highsCD4 concentrations and when the initial surface gpl20concentration was high (Fig. 9 and data not shown). Thismay suggest that shedding requires multiple CD4-gpl2O-gp4linteractions and that either sCD4 binding which is needed toinduce shedding was very slow or the shedding itself is slowat low temperatures.The exact molecular mechanism of the interaction be-

tween cell-associated CD4 and membrane-associated gpl20-gp4l leading to binding and fusion and the interaction ofsCD4 with HIV-1 leading to neutralization of viral infectionare still unclear. It was suggested that the interaction of CD4with gpl20-gp41 leads to exposure of the fusion peptide ofgp4l which then inserts into the target membrane, thuspromoting fusion. The same mechanism can lead to inacti-vation if the target membrane is not close to the fusionpeptide. Our experiments demonstrate that high sCD4 con-centrations (20 ptg/ml) lead to much faster (about 400-fold)binding of sCD4 to gpl20-gp41 than shedding of gpl20. Thisimplies that binding and not shedding of gpl20 may be thedominant mechanism of inhibition of HIV-1 infection andinhibition of syncytium formation under conditions whensCD4 concentration changes rapidly. This is, for example,the case in the clinical trials of sCD4 in AIDS patients (14,26), in which the sCD4 concentration in serum sharplydecreases with time after in vivo administration of bolussCD4.The low sCD4 concentrations in serum (lower than 0.2

,ug/ml) achieved in patients with AIDS may also be ineffi-cient because of the slow binding of sCD4. The short half-lifeof sCD4 in serum (15 to 120 min) (4, 14, 26) can make thekinetics of binding even more critical for sCD4 efficiency.These findings may help in explaining the observations thatsCD4 is inefficient in vivo (14, 26, 32), even at initial

concentrations of sCD4 in serum in the range of tens tohundreds of nanograms per milliliter (14, 26), which were

shown to be inhibitory in vitro by preincubation of the viruswith sCD4 (13).

ACKNOWLEDGMENTS

We thank Jeff Hooley and Dave Stephany for help with the flowcytometry and John Weinstein and Thomas Bull for criticallyreviewing the manuscript.

This work was supported by the NIH Intramural AIDS TargetedAntiviral Program.

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