Conformational Changes in Cell Surface HIV-1 Envelope Glycoproteins Are Triggered by Cooperation between Cell Surface CD4 and Co-receptors*

Philip L. St. J. Jones, Thomas Korte, and Robert BlumenthalDagger

From the Section of Membrane Structure and Function, Laboratory of Experimental and Computational Biology, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have continuously measured CD4-induced conformational changes of cell surface-expressed human immunodeficiency virus type-1 envelope glycoprotein gp120-gp41 in situ using 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid, a fluorescent probe that binds to hydrophobic groups. CD4-expressing human T cell lines induced significant and rapid conformational changes (<1 min delay) in gp120-gp41 from T cell-tropic strains, and little conformational changes in gp120-gp41 from macrophage-tropic strains, with equivalent levels of envelope expression. Conversely, CD4-expressing human macrophages induced significant and rapid conformational changes in gp120-gp41 from macrophage-tropic strains, and little conformational changes in gp120-gp41 from T cell-tropic strains. Thus, the conformational changes undergone by gp120-gp41, which lead to membrane fusion, are highly cooperative and require both receptor and co-receptor. We used a dye transfer assay to show that neither membrane lipid fusion or fusion pore formation can occur with host cells having different tropism from the envelope.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

HIV-11 gains entry into susceptible cells by means of fusion of viral and cellular membranes, which is mediated by the HIV-1 envelope glycoprotein, gp120-gp41 (1). The fusion reaction is triggered by the interaction of gp120-gp41 with host cell surface CD4 (2), and requires co-receptors such as CXCR4 and CCR5 for T-tropic and M-tropic HIV-1 strains, respectively (3-10). We report here new findings, which are relevant to the mechanisms by which the HIV-1 envelope glycoprotein gp120-gp41 mediates fusion of biological membranes. The HIV-1 fusion reaction proceeds along a series of multiple steps before the final event occurs, which results in delivery of the nucleocapsid into the cell (11). In previous studies with influenza hemagglutinin (HA), we have used quantitative fluorescence video microscopy to dissect the process and analyze the molecular interactions taking place in these intermediate steps (12, 13). These include conformational changes in the viral envelope glycoprotein, assembly of envelope glycoproteins into prefusion pore aggregates, insertion of the fusion peptide into target membranes, destabilization of the target membrane, and formation and expansion of the fusion pore.

The first step in the HIV-1 fusion cascade is the triggering of conformational changes in gp120-gp41 (14). We have measured the kinetics of conformational changes of cell surface-expressed gp120-gp41 triggered by interaction with target cell CD4 and co-receptor. The changes in conformation were revealed using the fluorescent probe 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS), which displays little fluorescence in aqueous solution but lights up when bound to hydrophobic groups (15, 16). We used a fluorescence video microscopy set-up equipped with an intensified CCD camera to detect and quantify the changes in fluorescence upon interaction of gp120-gp41-expressing cells and appropriate target cells. To our knowledge, this is the first time conformational changes in gp120-gp41 have been monitored in live cells in situ. We have also examined the effect of appropriate co-receptor on the next step in the fusion cascade, fusion pore formation and dilation.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Culture-- TF228 cells and SupT1 cells were grown in RPMI (10% FBS, 4.5 mg/ml glucose, and 1 mM glutamine) (Life Technologies, Inc.), and HeLa cells were grown in Dulbecco's modified Eagle's medium (10% FBS) (Life Technologies, Inc.). Elutriated monocytes were plated in RPMI (with 4.5 mg/ml glucose, 1 mM glutamine, and 20% FBS). After 5 days to allow the cells to adhere, the medium was changed every 2 days. They were used within 7-14 days of initial plating having differentiated into macrophages.

Expression of Vaccinia Recombinants-- The following strains of vaccinia were used, all of which were derived from the WR strain: vSC60, IIIB/Lai BH8 env, T cell line-tropic; vCB43, Ba-L env, macrophage-tropic; vCB16, IIIB BH8 T-tropic env but with a deletion mutation of the gp120-gp41 cleavage site of the env gene. In addition, the WR strain of vaccinia with no HIV or other foreign gene expression was used as a control in some experiments. To release lyophilized vaccinia from membranes, 0.25 mg/ml trypsin was mixed 1:1 by volume with the virus for 30 min at 37 °C. Released vaccinia was incubated with BJAB cells at a multiplicity of infection of 10 in RPMI, 1% FBS at a density of 2 × 106 cells/ml for 2 h at 37 °C. Then, these BJAB cells were diluted in RPMI, 10% FBS and incubated overnight at 37 °C. In the case of infection of HeLa cells, this protocol was modified so that the total incubation time of HeLa cells with vaccinia virus was 6-7 h and the multiplicity of infection was reduced from 10 to 6. The use of HeLa cells rather than BJAB cells, together with these two modifications in the protocol, produced lower background fluorescence in experiments with bis-ANS. To show that levels of expression were similar in all three strains, samples of infected cells were labeled with an anti-gp41 monoclonal antibody conjugated with fluorescein.

Dye Transfer Assays-- Vaccinia recombinants were expressed in BJAB cells, and the BJAB cells were labeled with the cytosolic dye, calcein. BJAB were incubated with 2 µM calcein-AM (Molecular Probes, Eugene, OR) for 45-60 min, washed, incubated for another 30 min, and washed again. Target cells were incubated with 2 mg/ml 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarboxycyanine perchlorate (DiI; Molecular Probes) in 1:2 diluent C (Sigma) medium for 15 min at room temperature, and then washed. BJAB cells were cocultured with either macrophages or a T cell line, SupT1, at 37 °C for 2 h in a 5% CO2 incubator on glass coverslips. After the incubation period, images of calcein emission (excitation, 494 nm; emission, 517 nm), DiI emission (emission, 565 nm; excitation, 547 nm), and brightfield were recorded using a Diaphot inverted microscope (Nikon, Garden City, NY) through a Fluor 40 objective, a 82000 filter cube (Chroma Technology Corp., Brattleboro, VT), and an intensified CCD camera (Hamamatsu, Hamamatsu-City, Japan). Images were analyzed using MetaMorph (Universal Imaging, West Chester, PA) software to count cells that had transferred dye upon contact.

Measurement of CD4-induced Conformational Changes-- Bis-ANS (Molecular Probes) (excitation, 395 nm; emission, 500 nm) was added to envelope-expressing cells at 2 µg/ml in culture medium without serum. Once steady levels of fluorescence were obtained, usually within 10 min, target cells or soluble CD4 was added. Images were recorded from an intensified CCD camera at 20-30-s intervals using the optical filter cube, consisting of a D380/12 exciter, 400DCLP beam splitter, and D510/40 emitter (Chroma Technology Corp.). Background fluorescence resulting from target cells and medium was low compared with fluorescence of envelope-expressing cells and was subtracted using image analysis software (MetaFluor, Universal Imaging). We used the same gain on the CCD camera, set to measure over its linear range to obtain comparability between experiments.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

It is known that T cell-tropic gp120-gp41 mediates fusion with T cell lines, but not with macrophages, and that macrophage-tropic gp120-gp41 mediates fusion with macrophages, but not with T cell lines (17). We used fluorescence microscopy to investigate the stage of the viral fusion process at which tropism-dependent block of fusion occurs (Fig. 1). Recombinant vaccinia was used to express viral envelope proteins of a T cell-tropic strain, LAI, a macrophage-tropic strain, Ba-L, and an uncleaved gp160 LAI control in BJAB cells, a B lymphocyte cell line. We loaded the envelope-expressing cells with a lipid dye, DiI, and the target cells, SupT1 or macrophages, with the aqueous dye, calcein. Following incubation for 2 h at 37 °C, images of calcein emission, DiI emission, and brightfield microscopy were recorded using an intensified CCD camera. Cell membrane fusion was measured as the percent of target cells that had taken up DiI out of envelope-expressing BJAB cells that were touching target cells, and pore widening as the percent of BJAB cells in contact with target cells that had received the cytosolic dye (18). The results, presented in Fig. 1, show that about 50% of cells expressing T-tropic envelope fuse to cell membranes and form large fusion pores with the T cell lines. Little dye transfer was obtained with the M-tropic strain or with the uncleaved T-tropic strain. By contrast, in macrophages, there was about 20% dye transfer with M-tropic envelope-expressing cells but no effect with either the uncleaved or the T-tropic strains. Control BJAB cells infected with vaccinia wild type WR strain produced less than 1% dye transfer of either calcein or DiI with both SupT1 cells and macrophages. Clearly, cells with mismatched tropism are not capable of either large fusion pore formation or membrane mixing, indicating that the mechanisms of tropism must occur at earlier stages of the fusion process.


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Fig. 1.   Dye transfer between BJAB cells (expressing T-tropic, M-tropic, and uncleaved strains of HIV-1 envelope) and SupT1 cells or macrophages. Open bars, DiI transfer from BJAB to targets; shaded bars, calcein transfer from targets to BJAB. a-g, SupT1 cells as targets. a, vSC60, DiI; b, vSC60, calcein; c, vCB43, DiI; d, vCB43, calcein; e, vCB16, DiI; f, vCB16, calcein. Both lipid mixing and fusion pore widening are greater than 50% with the T-tropic envelope and T cell line, but little dye transfer occurs with the M-tropic envelope or the uncleaved gp160. g-l, macrophages as targets. g, vSC60, DiI; h, vSC60, calcein; i, vCB43, DiI; j, vCB43, calcein; k, vCB16, DiI; l, vCB16, calcein. The M-tropic strain shows about 20% dye transfer with macrophages; little transfer occurs with the T-tropic envelope and zero transfer with the uncleaved envelope.

According to current models of HIV-1 fusion, CD4 induces conformational changes in the HIV envelope, resulting in exposure of the hydrophobic fusion domain of gp41 (14, 19). In the case of influenza HA, it has been shown that a low pH-triggered conformational change of HA resulting in an exposure of hydrophobic binding sites could be followed by a significant increase in fluorescence intensity of the fluorophore bis-ANS (16).

Using analytical and quantitative video microscopy, we have been able to monitor CD4-triggered conformational changes in situ in gp120-gp41 expressed on cells. HeLa cells expressing HIV-1LAI (Fig. 2, A-D) and HIV-1Ba-L (Fig. 2, E-H) were incubated in the presence of bis-ANS without target cells (Fig. 2, A, C, E, and G) and 20 min after addition of SupT1 cells (Fig. 2, B, D, F, and H). When no SupT1 cells were added, little bis-ANS fluorescence was seen (Fig. 2A). However, there was a marked increase in intensity of bis-ANS fluorescence in HeLa cells expressing HIV-1LAI gp120-gp41 when SupT1 cells were added, and only those in physical contact with SupT1 cells appeared bright (Fig. 2B). The highest fluorescence intensity was seen at the areas of contact between gp120-gp41-expressing cells and target cells. By contrast, HIV-1Ba-L-expressing cells did not noticeably light up when exposed to SupT1 cells as compared with controls without SupT1 cells (Fig. 2, E and F). In the brightfield images, we can distinguish between the original gp120-gp41-expressing cells and the added SupT1 (Fig. 2, C, D, G, and H). Little fluorescence intensity change was seen in the SupT1 cells, indicating that presumed gp120-gp41-induced conformational changes in cell surface CD4 do not result in increased hydrophobicity of the CD4 molecule.


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Fig. 2.   Bis-ANS fluorescence of HIV envelope-expressing HeLa cells before and after addition of SupT1 cells expressing CD4. Fluorescence images are shown in pseudo-color, which represents increasing intensity in the following order: black, purple, blue, green, yellow, red, and white. A-D, the same field of HeLa cells expressing T-tropic, LAI envelope before (A and C) and 20 min after (B and D) addition of SupT1 cells. There is a large increase in fluorescence of the HeLa cells after exposure to membrane-bound CD4. E-H, by contrast, there is little or no change in fluorescence of HeLa cells expressing M-tropic envelope in response to SupT1 cells. E and G, fluorescence and brightfield images before addition of SupT1 cells; F and H, fluorescence and brightfield images 20 min after addition of SupT1 cells.

The integrated fluorescence from individual gp120-gp41-expressing cells was monitored at 37 °C as a function of time following addition of CD4+ target cells, which are susceptible to fusion with either the T-tropic or M-tropic gp120-gp41-expressing effector cells. The amount of surface expression of gp120-gp41 between the different strains was within 5%, as measured by quantitative video microscopy following staining with fluorescein isothiocyanate-labeled, anti-gp41 monoclonal antibody. Fig. 3A shows that SupT1 cells induce rapid conformational changes in the T-tropic envelope with little or no lag time (less than 1 min). The M-tropic envelope produced a considerably smaller and less rapid increase of bis-ANS fluorescence. Addition of macrophages to HeLa cells expressing the M-tropic strain of HIV-1 produced rapid conformational changes, but with a slightly longer lag time (about 4 min) compared with the T-tropic strain with T cells (Fig. 3B). By contrast, there is no significant difference in the curves of the T-tropic wild type or uncleaved T-tropic strains and the vaccinia WR strain that does not express HIV envelope. Fig. 3C shows that when Mink-CD4 cells, which do not express human accessory molecules, were used as target cells, neither M-tropic or T-tropic strains showed increased bis-ANS fluorescence above that obtained by vaccinia WR strain or the uncleaved strain. This shows that molecules in addition to CD4 are required for the rapid conformational change to be induced by membrane-anchored CD4.


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Fig. 3.   Kinetics of exposure of hydrophobic binding sites measured by bis-ANS emission intensity upon addition of CD4-expressing supT1 cells. In all cases, graphs are mean changes in fluorescence intensity of 25-45 individual cells against time following addition of CD4. Background fluorescence due to host cells and medium has been subtracted, and time zero is the instance when CD4 was added. Circles, T-tropic, vSC60; squares, M-tropic, vCB43; upright triangles, uncleaved, vCB16; upside-down triangles, vaccinia wild type WR strain. A, action of SupT1, a T cell line. The T-tropic envelope shows a rapid conformational change with little or no lag time. The M-tropic envelope shows no significant increase in fluorescence above that due to vaccinia wild type alone, indicating that no significant conformational change takes place. The uncleaved envelope glycoprotein does show a conformational change in envelope but with a long lag time of 10-12 min. B, macrophages as target cells reverse the pattern. Both wild type and uncleaved T-tropic strains have no effect compared with that produced by vaccinia WR alone. The M-tropic strain does produce a steep conformational change, but with a lag time of 3-4 min. C, Mink-CD4 cells as host cells have no significant effect with any of the strains of envelope.

Soluble CD4 (sCD4) alone is sufficient to induce conformational changes in TF228 cells, a BJAB cell line constitutively expressing T-tropic HIV envelope (20). The integrated fluorescence from individual TF228 cells was monitored at different temperatures as a function of time following addition of sCD4 (Fig. 4). There is a considerably longer lag time than obtained for membrane-bound CD4 on SupT1 cells. Soluble CD4 also induced conformational changes in HeLa cells expressing T-tropic and M-tropic envelope with similar lag times as seen with TF228 cells. HeLa cells infected with vaccinia WR strain did not show any significant fluorescence increase within 50 min of incubation at 37 °C. Although it is difficult to compare the effective concentrations of CD4 in soluble and membrane-bound form, even concentrations of sCD4 as high as 10 µg/ml did not reduce the lag times to those seen with CD4+ cells bearing appropriate co-receptors. At reduced temperatures (16 and 25 °C), the lag times of the conformational change increased and the rates decreased, but the conformational change could still be observed (Fig. 4). Since there is no gp120-gp41-induced fusion at the lower temperature (20), we surmise that the CD4-induced conformational change can occur in the absence of fusion. However, at these temperatures, we do not see marked changes in bis-ANS fluorescence when cells expressing both membrane-anchored CD4 and the appropriate co-receptor are added to cells expressing the matched envelope glycoprotein.


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Fig. 4.   Kinetics of exposure of hydrophobic binding sites measured by bis-ANS emission intensity upon addition of soluble CD4 to T-tropic gp120-gp41-expressing cells (TF228). For data processing, see legend to Fig. 3. Circles, 16 °C; squares, 25 °C; filled triangles; 37 °C; hollow triangles, 37 °C, pH 8.4. At 37 °C, there is a rapid conformational change following a lag time of about 900 s. At lower temperatures, the lag time is increased and the rate of fluorescence increase is decreased. No significant differences in lag time or fluorescence increase were detected for an incubation at pH 7.4 and 8.4, 37 °C.

We have performed additional measurements of average fluorescence versus [bis-ANS] before and after adding sCD4. The results shown in Table I indicate that, at [bis-ANS] > 8 µg/ml, no increase of fluorescence is seen upon addition of sCD4, whereas the background still increases. This indicates that the newly exposed binding sites become saturated beyond this concentration. However, our experiments have been carried out at 2 µg/ml [bis-ANS], at which concentration the background fluorescence is low and the major portion of the fluorescence appears as a result of the triggering of conformational changes in gp120-gp41.

                              
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Table I
Relative fluorescence of TF228 cells in the presence of bis-ANS

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We have measured the kinetics of a crucial first step in the HIV-1 fusion reaction, namely the triggering of a conformational change in gp120-gp41 by the combination of host cell surface CD4 and co-receptors. We have demonstrated for the first time that fusion mediated by T-tropic or M-tropic gp120-gp41 is blocked at this early stage of the fusion cascade if the appropriate co-receptor is not present on the target cell membrane. Membrane-bound CD4 in the absence of the appropriate co-receptor did not produce the conformational changes in the HIV-1 envelope glycoproteins. A CD4- cell line expressing CCR5 did not induce conformational changes in M-tropic gp120-gp41 (data not shown). Previous studies with conformation-sensitive antibodies indicate that certain epitopes on gp41 are exposed upon treatment with sCD4 (21). Membrane-anchored CD4 expressed on a T cell line that is susceptible to fusion mediated by the T-tropic gp120-gp41 also induced conformational changes in gp120-gp41 detected by those antibodies (14). The most striking observation in this study is that the membrane-anchored CD4 is only able to induce rapid conformational changes in gp120-gp41 if the appropriate co-receptor is expressed on the target cell.

Previously, Korte and Herrmann (16) had shown that the conformational change of influenza HA induced by low pH results in strongly enhanced binding of bis-ANS to influenza virus. The binding is accompanied by a significant increase in fluorescence intensity associated with exposing hydrophobic binding sites. Experiments with influenza virus from which HA ectodomain was removed by digestion with bromelain and with liposomes indicates that the increase of bis-ANS fluorescence of intact virus at low pH is mainly attributable to binding of fluorophore to the influenza HA and that the low fluorescence at pH 7.4 in the presence of intact virus could be assigned to a significant extent to binding to lipid sites (16). Similarly, in our measurements, the low background fluorescence in the absence of appropriate target cells or sCD4 could be assigned to lipid sites as well as to cell surface proteins containing hydrophobic pockets. Our interpretation of the relatively low background in the absence of a conformational change is that bis-ANS does not have much access to these sites as the cells are covered by carbohydrates (glycocalyx), preventing access of bis-ANS to the membrane.

There are several hydrophobic regions on the HIV-1 envelope glycoprotein to which bis-ANS could conceivably bind. These include the amino-terminal fusion domain, a leucine zipper domain, and contact regions between gp41 and gp120 (14, 19, 22, 23). In our assay, it is clear that there is a dramatic increase in exposure of hydrophobic groups after the CD4-induced conformational change occurs (at least a 10-fold increase in fluorescence intensity of bis-ANS). The time course of the conformational change provides no evidence of any biphasic, or multiple phasic effects; instead, it suggests a concerted conformational change leading to exposure of hydrophobic groups. HIV-1LAI is among the laboratory-adapted strains of HIV-1 whose interaction with sCD4 leads to shedding of gp120 (24). Previous work with gp120-gp41 (LAI) had identified conditions of high pH (8.4) or low temperature (16 °C) at which shedding of gp120 does not take place (20). We have shown that under both of these conditions soluble CD4 can induce conformational changes in the envelope, indicating that the bis-ANS changes are not due to shedding (Fig. 4). There is a relatively long lag period in the bis-ANS response with sCD4 as compared with the rapid conformational changes seen with membrane-bound CD4 and appropriate co-receptors. Using an assay based on flow cytometry, Dimitrov et al. (25, 26) measured t1/2 = 3.5 min for binding of sCD4 to gp120-gp41-expressing cells at a concentration of 1 µg/ml and 37 °C. In our bis-ANS experiments, the t1/2 for reaching maximum conformational change is 33 min at 37 °C and 1 µg/ml sCD4 (see Fig. 4). We do not think that the difference is due to bis-ANS binding, since Korte and Herrmann (16) have shown that 80% of the fluorescence change due to binding of bis-ANS to low pH-treated influenza virus occurs within 1 min. Moreover, in the case of addition of SupT1 cells to the gp120-gp41 (LAI)-expressing cells, fluorescence changes were seen within 1 min of cell contact. Therefore, we conclude that binding of sCD4 takes place well before the conformational change occurs. This makes the rapid conformational changes we observed in the presence of sCD4 and co-receptor all the more striking.

The rapidity of the conformational change, which in both macrophages and SupT1 cells peaked within 20 min, compared with the time required for the start of lipid mixing, which takes about 30 min to start,2 shows that there must be a substantial barrier to lipid mixing after the conformational change occurs. Our measurements on influenza HA-expressing cells indicate that fusion pore widening occurs soon after lipid mixing commences (12).

Since sCD4 by itself can produce the conformational changes in gp120-gp41, why does membrane-anchored CD4 require the appropriate co-receptor to produce this effect? Obviously, sCD4 has little restriction upon the orientation it is able to adopt and can reach any exposed cell surface gp120-gp41. The diagrams in Fig. 5 illustrate how binding of sCD4 to cell surface gp120-gp41 might lead to conformational changes in the envelope glycoprotein. Membrane-anchored CD4, on the other hand, does not have similar accessibility to cell surface gp120-gp41. Fig. 5B illustrates that membrane-anchored CD4 on cells lacking the appropriate co-receptor might attach at a few contact sites leading to stable association between cells. This association might lead to conformational changes in very few gp120-gp41 molecules not detected by current techniques. However, when the appropriate co-receptor is present on the surface of CD4+ cells, cooperative interaction between CD4, gp120-gp41, and co-receptor in forming tertiary complexes in the contact area will result in massive recruitment of the complexes and conformational transitions in a large number of gp120-gp41 molecules. Lapham et al. (27) have provided convincing evidence for associations between cell surface CXCR4 and CD4, which are enhanced by adding recombinant T-tropic gp120. Similar results were obtained on the association between CCR5, CD4, and the M-tropic gp120 (28, 29). Self-association of proteins in membranes is influenced by a number of factors that do not apply to proteins in solution, including high local concentration of the protein, restrictions on tilting and vertical movement, and presence of other proteins at high concentrations (excluded volume effect). Calculations indicate that these factors alone can increase the likelihood of forming oligomers by many orders of magnitude relative to the probability of oligomerization of monomers tumbling freely in an experimentally realistic volume (30). In this manner, massive recruitment of gp120-gp41-CD4-co-receptor complexes at 37 °C, a phenomenon analogous to capping of cell surface receptors (31), could give rise to the striking changes in bis-ANS fluorescence we observe when gp120-gp41 expressing cells touch CD4+ cells bearing the appropriate co-receptor.


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Fig. 5.   A model for conformational changes induced in gp120-gp41 by sCD4, or by membrane-anchored CD4+ co-receptor. The objects are color-coded as follows: gray, unmodified gp120-gp41; blue, gp120-gp41 that has undergone a conformational change; orange, sCD4 or membrane-anchored CD4; yellow, co-receptor; green, cell membrane. Although it is known that gp120-gp41 is assembled as a dimer or higher, it is shown for convenience as a monomer. See "Results" for further explanation.

Our technique for continuous measurement of CD4-induced conformational changes in the HIV-1 envelope has an advantage over immunofluorescence techniques in that no fixation is required and the kinetics of the transitions can be measured. It is an assay system that can test the ability of therapeutic agents to interfere with interactions between gp120, CD4, and accessory molecules. It is quick, direct, quantitative, and does not require preincubation steps at temperatures below 37 °C. Potentially, the assay could be used to monitor conformational changes in gp120-gp41 on intact virions. The molecular structures (or scaffolds) that regulate membrane fusion are made up of an assembly of viral envelope glycoproteins, receptors, and co-receptors. They are responsible for bringing the viral membrane close to the target cell membrane and creating the architecture that enables lipid bilayers to merge. We have developed experimental systems based on fluorescence video microscopy to study such scaffolds and test hypotheses concerning mechanisms of HIV-1 envelope glycoprotein-mediated membrane fusion.

    ACKNOWLEDGEMENTS

We thank Drs. Edward Berger and Chris Broder for the HIV-1 envelope glycoprotein vaccinia recombinants, Dr. Phil Murphy for the CCR5+ CD4- cell line, Dr. Paul Clapham for the Mink-CD4 cell line, and Dr. Zdenka Jonak for the TF228 line. We are also grateful to Drs. Dimiter Dimitrov, Anu Puri, Isabel Munoz-Barroso, and Peter Hug for careful reading of the manuscript and helpful suggestions.

    FOOTNOTES

* This work was supported by the AIDS Intramural Targeted Antiviral Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed: NCI-FCRDC, Bldg. 469, Rm. 213, P. O. Box B, Miller Dr., Frederick, MD 21702-1201. Tel.: 301-846-1446; Fax: 301-846-6192; E-mail: blumen{at}helix.nih.gov.

1 The abbreviations used are: HIV, human immunodeficiency virus; M-tropic, macrophage-tropic; T-tropic, T cell-tropic; HA, hemagglutinin; FBS, fetal bovine serum; bis-ANS, 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid; DiI, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarboxycyanine perchlorate.

2 P. L. St. J. Jones, unpublished observations.

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Top
Abstract
Introduction
Procedures
Results
Discussion
References

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