The Cytoplasmic Tail Slows the Folding of Human Immunodeficiency Virus Type 1 Env from a Late Prebundle Configuration into the Six-Helix Bundle

Effects of the cytoplasmic tail (CT) of human immunodeficiencyvirus type 1 Env on the process of membrane fusion were investigated.Full-length Env (wild type [WT]) and Env with its CT truncated( ${Delta}$ CT) were expressed on cell surfaces, these cells were fusedto target cells, and the inhibition of fusion by peptides thatprevent Env from folding into a six-helix bundle conformationwas measured. For both X4-tropic and R5-tropic Env proteins, ${Delta}$ CT induced faster fusion kinetics than did the WT, and peptideswere less effective at inhibiting ${Delta}$ CT-induced fusion. We testedthe hypothesis that the inhibitory peptides were less effectiveat inhibiting ${Delta}$ CT-induced fusion because ${Delta}$ CT folds more quicklyinto a six-helix bundle. Early and late intermediates of WT-and ${Delta}$ CT-induced fusion were captured, and the ability of peptidesto block fusion when added at the intermediate stages was quantified.When added at the early intermediate, the peptides were stillless effective at inhibiting ${Delta}$ CT-induced fusion but they wereequally effective at preventing WT- and ${Delta}$ CT-induced fusion whenadded at the late intermediate. We conclude that for both X4-tropicand R5-tropic Env proteins, the CT facilitates conformationalchanges that allow the trimeric coiled coil of prebundles tobecome optimally exposed. But once Env does favorably exposeits coiled coil to inhibitory peptides, the CT hinders subsequentfolding into a six-helix bundle. Because of this facilitationof maximal exposure and hindrance of bundle formation, the coiledcoil is optimally exposed for a longer time for WT than for ${Delta}$ CT. This accounts for the greater peptide inhibition of WT-inducedfusion.

INTRODUCTION

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Introduction

The envelope protein (Env) of human immunodeficiency virus (HIV)is a potential target for vaccines and drug therapies. Blockingformation of the six-helix bundle (6HB) structure of Env hasbeen shown to be an effective means of preventing HIV infection(for a review, see reference 16). As has been observed fromEnv's postfusion structure, its 6HB is formed from three C-terminaland three N-terminal heptad repeat regions of the trimeric transmembranesubunit gp41 (9, 55, 56). The N-terminal segments form a centraltriple-stranded coiled coil, and at a late stage of fusion (42),the C-terminal segments pack, antiparallel to the N segments,into the hydrophobic grooves on the surface of the coiled coil,completing the 6HB. T20, a synthetic 36-residue peptide thatis derived from the C-terminal heptad region, binds to the groovesof the coiled coil and prevents infection by blocking formationof the 6HB (see, for example, references 5, 10, and 15). TheT20 peptide (32) has recently been approved by the Food andDrug Administration and is prescribed under the brand namesFuzeon and Enfuvirtide. Other synthetic peptides that duplicatethe C-terminal heptad repeat regions (e.g., C34) act in a similarmanner. In the native structure of Env, the grooves are notexposed (in fact, the coiled coil may not have yet been formed)but become transiently exposed during the fusion process; inhibitorypeptides can therefore block infection only if they fill thegrooves during this window of exposure.

HIV isolates exhibit considerable variability in their sensitivityto T20 and other 6HB inhibitory peptides (2, 12, 13, 36). Asa rule, isolates that use the chemokine receptor CCR5 as a coreceptorare more resistant to inhibitory peptides than are laboratory-adaptedstrains that use CXCR4 (12, 13). Understanding the molecularbasis for differences in inhibitory peptide sensitivity couldtherefore be important in ensuring effective antiviral therapy.The association between tropism and inhibitory peptide sensitivitymay be related to a much higher binding affinity of R5-tropicEnv to CCR5 than of X4-tropic Env to CXCR4 (see reference 14and references therein). Tropism is primarily determined bythe amino acid sequence of the V3 loop (see, for example, reference53). Studies have shown that replacing the V3 loop of a laboratory-adaptedX4-tropic HIV type 1 (HIV-1) Env protein with that of an R5-tropicprimary isolate yields a chimeric construct that is R5 tropic(12, 13, 26, 27, 29). This chimeric Env protein bound CCR5 withhigh affinity and exhibited resistance to T20, characteristicof the R5-tropic Env protein from which the V3 loop was derived(12, 13, 48). In general, as the affinity between such chimericEnv constructs and chemokine receptors increases, fusion kineticsalso increase and the efficacy of T20 decreases (48). It hasbeen proposed that higher affinity between Env and chemokinereceptors causes quicker folding of Env and therefore fasterfusion kinetics and that quicker folding of Env from prebundleconfigurations into a 6HB shortens the time of exposure of bindingsites, accounting for the greater resistance to T20 (20, 48).

Among enveloped viruses, HIV Env has an unusually long cytoplasmictail (CT), $~$ 150 residues. The CT is thought to modulate severalof the steps in viral entry. When HIV-2 or simian immunodeficiencyvirus (SIV) is grown in tissue culture, a spontaneous truncationof the CT occurs that markedly improves the virus's abilityto grow (8, 25). In addition, the presence of the CT appearsto affect the receptor requirement: HIV-1 or HIV-2 grown incell lines devoid of Env's receptor (CD4), but expressing theappropriate chemokine receptors, tends to naturally developEnv proteins with truncated CTs, as the virus reverts to CD4-independentfusion (17, 35, 37, 49). In fact, a shortened CT is a common—althoughfar from universal—feature of CD4-independent Env (18).

In the present study, we investigated fusion activities causedby wild-type (WT) HIV-1 Env and an Env protein in which theCT was deleted ( ${Delta}$ CT). We expressed the R5-tropic and X4-tropicWT and ${Delta}$ CT of HIV-1 in effector cells and fused them to targetcells that express CD4 and appropriate chemokine receptors.By arresting fusion at intermediate stages, we tested whetherdifferences in sensitivity to inhibitory peptides between WT-and ${Delta}$ CT-induced fusions are controlled by folding kinetics; weidentified the transitions between intermediate states thatwere strongly affected by the CT. We found that deleting theCT had two major effects: it increased the rate of fusion, andit rendered Env more resistant to peptides that block 6HB formation.For both WT and ${Delta}$ CT, we created fusion intermediates to stablyexpose the inhibitory peptide binding sites, thereby eliminatingthe window of time for binding as a factor in the inhibitionof fusion. We found that the peptides were equally effectiveat preventing WT-induced fusion and ${Delta}$ CT-induced fusion when addedat an advanced stage. We define the transitory configurationsof gp41 that maximally expose peptide binding sites as "lateprebundle" conformations. We conclude that the time gp41 remainsin these conformations is the major determinant for the differencesin potency of inhibitory peptides to block WT- and ${Delta}$ CT-inducedfusions.

MATERIALS AND METHODS

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Materials and Methods

Cell lines and reagents. The 293T cell line was purchased from the American Type CultureCollection (Manassas, Va.). The TF228.1.16 cell line that stablyexpresses X4-tropic HIV-1 Env (31) was a generous gift fromZ. Jonak (Glaxo SmithKline, Philadelphia, Pa.). HeLaT4⁺ cells(40), provided by R. Axel, and 3T3.T4.CXCR4 and 3T3.T4.CCR5cells, provided by D. Littman (11), were obtained through theAIDS Research and Reference Reagent Program, National Instituteof Allergy and Infectious Diseases, National Institutes of Health.Cells were grown as described previously (42, 44).

Bovine serum albumin and poly-L-lysine were purchased from SigmaChemical Co. (St. Louis, Mo.); lauroyl-lysophosphatidylcholine(LPC) was bought from Avanti Polar Lipids (Alabaster, Ala.).All fluorescent dyes—calcein AM, CMTMR {5 [and 6-]-(((4-chloromethyl)benzoyl)amino)-tetramethylrhodamine},and CMAC (7-amino-4-chloromethylcoumarin)—were purchasedfrom Molecular Probes (Eugene, Oreg.). The HIV gp120 monoclonalantibody (MAb) IgG1b12 (7) was provided by D. Burton and CarlosBarbas, and the 447-52D anti-V3 loop MAb (23) was provided bySusan Zolla-Pazner, both through the AIDS Research and ReferenceReagent Program. The HIV gp41-derived inhibitory peptide C34(sequence: WMEWDREINNYTSLIHSLIEESQNQQEKNEQELL ) was synthesizedby Macromolecular Resources (Fort Collins, Colo.). The untagged5-helix peptide was constructed as previously described (44).It was expressed in Escherichia coli BL21(DE3)/pLysS with amodified pET3a vector (Novagen). The cells were harvested bycentrifugation 4 h postinduction with 0.5 mM isopropyl-ß-D-thiogalactopyranoside(IPTG), and the pellets were resuspended in 50 ml of bufferA (50 mM Tris [pH 8.0], 1 mM EDTA) plus 25% sucrose. The cellswere lysed by sonication and centrifuged (35,000 x g for 30min) to separate the soluble fraction from inclusion bodies.The inclusion bodies were subsequently washed extensively withbuffer A plus 1% Triton X-100. The five-helix peptide was purifieddirectly from the inclusion bodies resuspended in 8 M urea-bufferA. The debris was removed by a 1-h centrifugation at 4°C.The protein was then loaded onto a DEAE-Sepharose column (AmershamPharmacia Biotech) equilibrated with buffer A plus 3 M ureaand eluted with an NaCl gradient (0 to 500 mM) in buffer A plus3 M urea. The five-helix peptide was refolded by step dialysisin buffer A with decreasing amounts of urea at 4°C. Therefolded 5-helix peptide was applied to a MonoQ column (AmershamPharmacia Biotech) equilibrated with buffer A. The protein waseluted from the MonoQ resin with an NaCl gradient in bufferA. The sample was concentrated by ultrafiltration to 5 mg/mland stored at 4°C. The protein was >95% pure as judgedby sodium dodecyl sulfate-polyacrylamide gel electrophoresisand is properly folded in terms of secondary structure and monomericstate (as judged by circular dichroism and sedimentation equilibriumexperiments). Stock solutions of the inhibitory peptides wereprepared in phosphate-buffered saline, aliquoted, and storedat –20°C.

Transient expression of HIV-1 Env. HIV-1 Env was expressed in 293T cells by calcium phosphate transfection.WT and ${Delta}$ CT JRFL Env proteins were expressed with the pCAGGS plasmid(46). For the JRFL ${Delta}$ CT construct, residues C terminal to V708(by standard LAI numbering) were deleted, so that only threeresidues (NRV) of the CT remained. This construct has been describedpreviously (4). The pSRHS vectors encoding the WT and ${Delta}$ CT HXB2Env proteins were obtained from E. Hunter (University of Alabama)(38). In HXB2 ${Delta}$ CT, a 147-residue-long segment was deleted fromthe C terminus of gp41, leaving only four residues, NRVR, inthe cytoplasmic segment. To match the expression densities ofthe WT and ${Delta}$ CT proteins, the amount of ${Delta}$ CT plasmid used for transfectionwas reduced severalfold with respect to that of the WT plasmid(which was usually added at 10 µg/6-cm-diameter dish).The amounts of plasmid used to match WT and ${Delta}$ CT densities onthe positive cells also led to the same percentages of cellsexpressing the WT and ${Delta}$ CT proteins (Table 1). Adding less plasmidto lower the protein density on the surfaces of the transfectedcells also reduced the percentage of cells expressing Env. Surfacedensities therefore could not be lowered too much without greatlyincreasing the percentage of nonexpressing cells. The Env surfaceexpression levels were quantified essentially as described inreference 42, with 447-52D or IgG1b12 MAbs. The 447-52D antibodyrecognizes a short peptide at the apex of the V3 loop of gp120(34, 54) and thus should bind equally well to WT and to conformationallyaltered ${Delta}$ CT (18). Cells were analyzed in an ORTHO Cytoron Absoluteflow cytometer (Ortho Diagnostic Systems, Raritan, N.J.).

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TABLE 1. WT and ${Delta}$ CT expression in transfected cells

Measurements of cell-cell fusion. Fusion was measured by two techniques: two-color fluorescencemicroscopy and flow cytometry. For fluorescence microscopy,as has been described in detail (42-44), effector cells expressingHIV-1 Env were loaded with calcein (green emission) and targetcells expressing CD4 and coreceptor were loaded with CMAC (blueemission). Fusion events were scored as the appearance of cellpairs that were positive for both dyes, normalized by the totalnumber of effector and target (E-T) cells in contact plus fusedcells. The flow cytometry-based assay to measure cell-cell fusioninduced by HIV-1 Env was similar to assays described previously(28, 30, 57). Effector cells loaded with the green cytoplasmicdye calcein AM were laid on top of target cells loaded withthe orange cytoplasmic dye CMTMR. The amounts of loaded calceinand CMTMR were adjusted so that excitation by the 488-nm laserof the flow cytometer yielded comparable emission signals fromthe two dyes. Cells were coincubated at 37°C, washed (toremove the unbound effector cells), and lifted off the dishby brief treatment with 0.5 mg of trypsin per ml and 0.5 mMEDTA in divalent-free phosphate-buffered saline (Gibco BRL).The last step stopped the fusion reaction and, most importantly,dispersed the unfused E-T cell pairs and aggregates that wouldotherwise produce a false double-positive cell population inflow cytometry. Bound but unfused E-T cells were, in practice,the primary cause of background signals. Trypsin cleavage wasstopped by adding an excess of soybean trypsin inhibitor, andcell-cell fusion was quantified as the ratio of the double-positivecell population to the sum of the double-positive cells andthe remaining target (orange) cells. By stopping the fusionreaction at different times and measuring the extents of fusion,we were able to determine the kinetics of fusion by flow cytometrywhich, to our knowledge, is a novel technique for measuringkinetics.

The percentage of fusion determined by flow cytometry was consistentlylower than the percentage determined by fluorescence microscopybecause of the method of calculation. In flow cytometry, thepercentage is normalized by the total number of target cells,whereas in fluorescence microscopy, the percentage is normalizedby the total number of bound target cells, which is always lessthan the total number of target cells. The efficacy of HIV fusioninhibitors under different sets of conditions was parameterizedby determining 50% inhibitory concentrations (IC₅₀s) as describedin reference 43. Briefly, dose-response data were fit with aLangmuir isotherm as follows: F = F₀/(1 + C_i/IC₅₀), where F₀is the extent of fusion in the absence of an inhibitor (takenas 100%) and C_i is the inhibitor concentration. IC₅₀ was chosento minimize least-square deviation.

Fusion intermediates. Coincubating E-T cells for 2.5 h at 23°C, a temperaturethat does not permit fusion, kinetically advances the fusionprocess to a temperature-arrested stage (TAS) at which Env hasundergone conformational changes that allow bundle-blockingpeptides to bind gp41 (44). Adding 0.15 mg of LPC per ml afterreaching the TAS, waiting 3 min at 23°C, warming the cellsto 37°C for 15 min, lowering the temperature to 4°C,and then removing the LPC by washing the cells twice with mediumcontaining bovine serum albumin yields a lipid-arrested stage(LAS) of fusion. When creating the LAS, fusion does not occur.To test the efficacy of C34 or the 5-helix peptide when addedat the LAS, the peptides were added at 4°C, followed bya 30-min incubation at 23°C. Fusion did not occur duringthe incubation. The temperature was then raised to 37°C,and the extent of fusion was measured.

Immunoprecipitation of cell surface-expressed Env. The extent of proteolytic cleavage of gp160 into gp120/gp41was assessed as described previously (1). Briefly, cell surfaceproteins were biotinylated and cells were lysed. The total amountof protein was determined (by a Bio-Rad protein assay kit),samples were adjusted to contain equal concentrations of protein,and Env was immunoprecipitated with HIV immune serum (from G.Spear, Rush University Medical Center). The immune complexeswere separated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. The biotinylated Env was visualized by horseradishperoxidase-conjugated streptavidin (Pierce, Rockford, Ill.)with an ECL kit (Amersham-Pharmacia Biotech, Little Chalfont,Buckinghamshire, United Kingdom).

RESULTS

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Results

Flow cytometry reliably measures the extent and kinetics of cell-cell fusion. In this study, we quantified membrane fusion by flow cytometryand fluorescence microscopy. In initial experiments, we loadedeffector TF228.1.16 cells (stably expressing HIV-1 Env) witha green dye (calcein) and fused them to target HeLaT4⁺ cells(stably expressing CD4 and CXCR4) that were loaded with an orangedye (CMTMR). We chose these E-T cell pairs because we had alreadycharacterized their kinetics and fusion intermediates in considerabledetail by fluorescence microscopy (42, 44). We could thereforecompare fusion obtained by fluorescence microscopy and flowcytometry to ensure that results were independent of methodology.As determined by flow cytometry, incubating these E-T cellstogether at temperatures at or below 23°C did not yieldfusion (e.g., see Fig. 3B); two distinct populations of cells,one green and one orange, were observed. In contrast, coincubatingE-T cells at 37°C yielded a population of cells that werelabeled by both dyes, denoting cell-cell fusion (Fig. 1A, leftside). Typically, about 15% of the HelaT4 cells fused to TF228.1.16cells (as determined by flow cytometry). As expected, a highconcentration of the C34 peptide (added to E-T cells at thetime of coincubation) completely eliminated double-labeled cells,showing that fusion was inhibited (Fig. 1A, right side). Also,effector cells cocultivated with CD4⁺ target cells that expressthe noncognate CCR5 did not produce detectable fusion (datanot shown). Flow cytometry has been used previously in similarmanners to measure extents of fusion (28, 30, 38, 57).

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FIG. 3. Temperature dependence of fusion promoted by the WT (filled symbols) and ${Delta}$ CT (open symbols) from the JRFL (A) and HXB2 (B) strains. Cells were coincubated for 2.5 (HXB2) or 2 (JRFL) h at the indicated temperature and then analyzed by flow cytometry. The extent of fusion induced by JRFL Env at 37°C was determined after a 1 h of incubation to reduce the formation of large syncytia. The background signal (<2% of the effector cells after a 37°C incubation) was subtracted from the plotted data. The apparent reduced fusion of JRFL Env at 37°C was caused by leakage of calcein from the effector cells and by syncytium formation, as verified by fluorescence microscopy (data not shown).

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FIG. 1. Flow cytometry analysis of fusion between TF228.1.16 (effector) and HeLaT4⁺ (target) cells. (A) Effector cells loaded with green cytoplasmic marker were coincubated with target cells loaded with orange marker for 2.5 h at 37°C either in the presence (right) or in the absence (left) of 300 nM C34 peptide. (B) Kinetics of cell-cell fusion were obtained by varying the time E-T cells were coincubated at 37°C, lowering the temperature to halt further fusion, and then lifting cells off the culture dishes with a trypsin-EDTA solution. (C) Dose-response curves for C34 inhibition measured by flow cytometry (open circles) and microscopy (filled circles) assays. A small background signal not related to fusion (about 1.5% of the total number of effector cells) was subtracted from the data in panels B and C. Unless stated otherwise, error bars represent standard errors of the mean for at least three independent duplicate measurements.

We used the flow cytometry assay in a manner that allowed usto obtain the kinetics of cell-cell fusion (see Materials andMethods). We found that the kinetics were similar to the kineticswe previously obtained for the same cell pairs by using fluorescencemicroscopy as the assay (44): a 20-min lag time and $~$ 2 h to reacha maximal extent of fusion (Fig. 1B). To further validate thequantitative reliability of the flow cytometry assay, we comparedits determination of the dose dependence of inhibition of fusionby C34 peptide against that determined by the fluorescence microscopyassay (43). The two assays were in good quantitative agreement(Fig. 1C). We used both assays to compare fusions induced bythe WT and ${Delta}$ CT proteins.

Fusion kinetics of HIV Env is augmented by deletion of its CT. A flow cytometric analysis showed that for the same concentrationof plasmid in transfection, the ${Delta}$ CT protein was expressed onthe surfaces of cells at significantly higher densities thanwas the WT protein. This was observed for both X4-tropic HXB2Env (Table 1) and R5-tropic JRFL Env (data not shown). A higherdensity is expected because deletion of the tail eliminatesan internalization signal (YXXL) (6).

The extent of fusion induced by the ${Delta}$ CT protein was greater thanthat induced by the WT protein; this would be expected becauseof the higher expression levels of the ${Delta}$ CT protein (Table 1 andreference 39). In order to meaningfully compare fusions inducedby the WT and ${Delta}$ CT proteins, we reduced the amount of ${Delta}$ CT-bearingplasmid used for transfection (see Materials and Methods) untilthe densities of the WT and ${Delta}$ CT plasmids were matched. For HXB2Env, reducing the concentration of the ${Delta}$ CT plasmid fivefold withrespect to that of the WT plasmid (2 µg of ${Delta}$ CT plasmidversus 10 µg of WT plasmid) yielded comparable surfacedensities and similar extents of cell-cell fusion after a 2-hcoincubation at 37°C (Table 1). However, the ${Delta}$ CT plasmidinduced faster fusion kinetics than did the WT plasmid (Fig.2). This was the case for both R5-tropic Env (JRFL, Fig. 2A)as determined by fluorescence-activated cell sorter analysisand X4-tropic Env (HXB2, Fig. 2B) as determined by microscopy.In separate experiments, we found that the temperature thresholdswere comparable for ${Delta}$ CT-induced and WT-induced fusions for bothJRFL (Fig. 3A) and HXB2 (Fig. 3B). The lowest temperature necessaryfor fusion was less for JRFL than for HXB2 (Fig. 3).

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FIG. 2. Kinetics of cell-cell fusion induced by the WT and ${Delta}$ CT. (A) Fusion between 293T cells transiently expressing JRFL WT (filled triangles) or ${Delta}$ CT (open triangles) and target 3T3.CD4.CCR5 cells was monitored by the flow cytometry assay as described in the legend to Fig. 1. Open squares are the background signal, as observed with mock-transfected 293T cells. (B) The kinetics of fusion induced by HXB2 WT (filled symbols) or ${Delta}$ CT (open symbols) transiently expressed in 293T and HeLaT4⁺ cells were measured by fluorescence microscopy. Fusion was induced by coculturing the cells either directly at 37°C (triangles) or after preincubation at 23°C for 2.5 h (circles).

Since it was possible that the faster kinetics of ${Delta}$ CT-inducedfusion were due to more-efficient cleavage into gp120 and gp41subunits (which would increase the density of Env capable ofinducing fusion), we biotinylated surface-expressed Env, immunoprecipitatedit with pooled human HIV-containing sera, and analyzed the immunecomplexes by Western blotting. We found that both the WT and ${Delta}$ CT gp160 precursors were properly and almost completely cleavedinto two subunits, judging from the relative intensities ofgp160 and gp120 bands (Fig. 4). The extent of cleavage of gp160was, however, somewhat greater for ${Delta}$ CT. This indicates that eitherthe cleavage site within the ectodomain was more exposed orthe time for cleavage during intracellular trafficking was greaterfor ${Delta}$ CT. It had been previously found that MAbs directed againstgp120 epitopes normally exposed by CD4 recognize ${Delta}$ CT much betterthan the WT when CD4 is not present (18). Therefore, the CTappears to modulate the structure of the ectodomain of Env.

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FIG. 4. Efficiency of proteolytic cleavage of cell surface-expressed HXB2 WT and ${Delta}$ CT. The amounts of WT- and ${Delta}$ CT-bearing plasmids used to transfect a 6-cm-diameter dish of 239T cells are indicated above the lanes. For details, see Materials and Methods.

Although we found comparable expression levels by flow cytometry,the apparent levels of ${Delta}$ CT expression revealed by immunoprecipitation(followed by Western blotting) were significantly lower thanthose of the WT. If the ${Delta}$ CT expression densities were lower thanthose of the WT, this would further emphasize that the CT ofEnv slows fusion. Factors other than surface density and proteolyticprocessing of Env appear to render ${Delta}$ CT more fusogenic. In short,the absence of the CT speeds the kinetics of fusion and doesso without appreciably altering the efficiency of proteolyticprocessing.

Deletion of the Env CT reduces the effectiveness of fusion inhibitory peptides. It has been found that an increased rate of HIV Env-inducedfusion tends to correlate with reduced potency of peptides thatinhibit fusion by preventing 6HB formation (48). We thereforedetermined the relative abilities of the C34 and 5-helix peptidesto suppress cell-cell fusion induced by the WT and ${Delta}$ CT proteins.C34 inhibits bundle formation by binding to the N-terminal heptadrepeat region (i.e., the coiled coil) of gp41 (16); the recombinant5-helix peptide has a triple-stranded coiled coil with onlyone vacant groove, and it inhibits bundle formation by bindingto the C-terminal heptad repeats of Env (50, 51). By measuringthe dose dependence of inhibition of fusion, we found that the ${Delta}$ CT protein (Fig. 5, closed symbols) was substantially more resistantto both the C34 (Fig. 5A) and 5-helix (Fig. 5B) peptides thanwas the WT (open symbols). Such was the case for both the X4-tropicHXB2 (triangles) and R5-tropic JRFL (circles) Env proteins.For example, for HXB2, the IC₅₀ was approximately fivefold higherfor C34 (Fig. 5A and 6A) and approximately threefold higherfor the 5-helix peptide for ${Delta}$ CT-induced fusion (Fig. 5B and 7A).Much higher concentrations of the C34 and 5-helix peptides wererequired to inhibit JRFL-induced than HXB2-induced fusion (Fig.5). The fusion kinetics were faster for JRFL than for HXB2 (Fig.2). Our results are in accord with a previous demonstrationthat, compared to the WT, an Env protein with a shortened CT(referred to as "8X" [17, 35]) exhibits faster cell-cell fusionand a reduced ability of T20 to inhibit fusion (20).

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FIG. 5. Inhibition of HIV Env-induced cell-cell fusion by the C34 (A) and 5-helix (B) peptides. 293T cells expressing JRFL (circles) or HXB2 (triangles) were coincubated with appropriate target cells in the presence of varied concentrations of HIV fusion inhibitors for 1 or 2.5 h, respectively. WT-induced fusion (open symbols) was more sensitive to inhibitors than was fusion induced by ${Delta}$ CT (filled symbols). Fusion was quantified by flow cytometry.

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FIG. 6. Efficacy of C34 peptide at sequential intermediate stages of fusion induced by HXB2 WT (open circles) and ${Delta}$ CT (filled circles). Varied concentrations of C34 were added either at the beginning of E-T cell coincubation (A), at the TAS (B), or at the LAS (C). Fusion was then triggered by incubating the cells at 37°C for 2.5 h (A) or for 1 h (B and C). The IC₅₀s obtained by curve fitting (see Materials and Methods) are given in parentheses in the insets. Fusion was quantified by fluorescence microscopy.

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FIG. 7. Relative potency of the 5-helix peptide added at the beginning of E-T cell coincubation (A) or at the LAS (B). The IC₅₀s for the HXB2 WT (open circles) and ${Delta}$ CT (filled circles) are given in parentheses. Details are given in the legend to Fig. 6.

The efficacy with which C34 inhibited fusion decreased whenthe expression level of Env was increased. For HXB2, doublingthe amount of plasmid used for transfection raised the IC₅₀of C34 for ${Delta}$ CT-induced fusion from 12 to 26 nM (Table 2). ForJRFL, tripling the amount of plasmid increased the IC₅₀ of C34against ${Delta}$ CT-induced fusion from $~$ 300 to $~$ 730 nM (Table 2). Thefinding that more C34 is required to inhibit fusion as the densityof Env increases was expected: IC₅₀ is the concentration ofpeptide that blocks 50% of the fusion events, not the concentrationthat prevents 50% of the copies of Env from folding into a 6HB.Because higher expression levels should yield more potentialfusion sites between cell pairs, a higher peptide concentrationshould be needed to block fusion. In summary, we have founda correlation between resistance against inhibitory peptidesand fusion kinetics, as previously reported by others (20, 48),as well as a correlation between peptide inhibition and densityof Env. For the remainder of this study, we primarily used HXB2Env because we had previously characterized intermediate stagesof its fusion in some detail (42, 44).

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TABLE 2. Susceptibility of fusion to C34 peptide depends on Env expression density in control experiments but not from the LAS

The resistance of ${Delta}$ CT and WT proteins to inhibitory peptides is the same at the LAS. We arrested fusion at intermediate stages so that the bindingsites for the C34 and 5-helix peptides on gp41 were exposed.We reasoned that if inhibitory peptides were less effectiveat blocking ${Delta}$ CT-induced fusion because ${Delta}$ CT more rapidly foldedinto a 6HB, addition of the C34 and 5-helix peptides after creatingthe intermediates should inhibit fusion of the WT and ${Delta}$ CT proteinsto the same extent. We verified by fluorescence microscopy thatthe process of fusion between 293T cells transiently expressingHXB2 Env and HeLaT4⁺ cells was kinetically advanced at the TAS(created by preincubation at 23°C for 2.5 h) by showingthat a subsequent exposure to 37°C promoted faster fusionthan that which occurred in control experiments in which thepreincubation was omitted (Fig. 2B). Without prior cell coincubation,the kinetics of fusion was appreciably faster for the ${Delta}$ CT proteinthan for the WT protein (Fig. 2B). C34 blocked WT-induced fusionmore effectively than it blocked ${Delta}$ CT-induced fusion (Fig. 6A).Thus, results obtained by fluorescence microscopy were in accordwith the relative potencies of peptides determined by flow cytometry(Fig. 5). After creation of the TAS, the fusion rates were virtuallyidentical for the WT and ${Delta}$ CT proteins (Fig. 2B, open and closedcircles) but C34 was still more effective at blocking WT fusion(Fig. 6B). C34 added at the TAS was not appreciably more effectiveat blocking WT-induced fusion (Fig. 6B) than when it was addedat the start of coincubation (Fig. 6A), but ${Delta}$ CT-induced fusionwas more effectively inhibited by C34 added at the TAS thanwhen it was added at the initiation of coincubation. (We referto experiments in which E-T cells are continuously coincubatedat 37°C as the control.) Thus, eliminating the time of bindingfor C34 as a factor did not alter the inhibition of fusion forthe WT, but it increased the inhibition for ${Delta}$ CT. In other words,the data indicate that when cells are coincubated immediatelyat 37°C, the WT exists in prebundle configurations longenough for effective binding of C34. But for ${Delta}$ CT, grooves aremore briefly exposed, for a time too short for C34 to bind maximally.It has been shown that fusion is inhibited after T20 is addedand unbound peptide is then removed at the TAS (44). The findingthat C34 was still not as effective at inhibiting ${Delta}$ CT-inducedfusion as at inhibiting WT-induced fusion (Fig. 6B) suggeststhat the grooves of the coiled coil of ${Delta}$ CT were not maximallyexposed at the TAS and that the prebundle configuration of ${Delta}$ CTprotein at the TAS folded into a 6HB more quickly than thatof the WT protein.

The LAS intermediate is a more advanced intermediate than theTAS intermediate (44) (in fact, in this study the LAS intermediatewas generated from the TAS intermediate [see Materials and Methods]).We created the LAS intermediate in order to test the relativesensitivities of WT- and ${Delta}$ CT-induced fusions to the inhibitorypeptides at a stage later than the TAS. If 6HBs do not formprior to pore formation (42, 44; see reference 22 for a counterview),a more advanced intermediate could increase the likelihood thatpeptide binding sites on the gp41prebundle become fully exposed.After creating the LAS intermediate, incubating cells at 37°Cled to fusion. However, this extent was less than that whichoccurs by continuously coincubating E-T cells at 37°C andwas somewhat less for the ${Delta}$ CT protein than for the WT protein:compared to the fusion of control experiments, only $~$ 70% of WT-expressingcells and $~$ 50% of ${Delta}$ CT-expressing cells fused from the state ofthe LAS, as determined by fluorescence microscopy. Adding C34(Fig. 6C) at the LAS inhibited fusion in a dose-dependent mannerwhen the temperature was raised to 37°C. At the LAS, C34(Fig. 6C) was virtually as effective for WT-induced fusion asfor ${Delta}$ CT-induced fusion.

We tested whether the ability of C34 to equally inhibit WT-and ${Delta}$ CT-induced fusions at the LAS is independent of Env density.C34 added after creation of the LAS inhibited fusion (inducedby raising the temperature to 37°C) to the same extent forhigh and low expression levels of ${Delta}$ CT; these IC₅₀s were comparableto that measured for the WT (Table 2). This indicates that ${Delta}$ CTdid not more readily inactivate than the WT during the experimentalmanipulations used to achieve LAS: greater inactivation couldhave caused ${Delta}$ CT to produce a smaller number of fusion-competentsites than did the WT. This in turn could have increased thesusceptibility of ${Delta}$ CT-induced fusion to peptides to the pointthat peptide inhibition became comparable for ${Delta}$ CT and the WTat the LAS. The equal efficacies of inhibitory peptides for ${Delta}$ CT- and WT-induced fusions at the LAS, independent of density,are what one would expect if the time of exposure of bindingsites determined the relative susceptibilities of WT- and ${Delta}$ CT-inducedfusions to inhibitor peptides. We are thus led to the key conclusionthat the potency of inhibition of fusion by C34 is inherentlythe same for the WT and ${Delta}$ CT; the difference between the two iscaused by faster folding of ${Delta}$ CT into 6HBs, limiting the timeduring which C34 can bind to the grooves of the coiled coil.

The potency of inhibition of WT-induced fusion by C34 was notaffected by creation of the TAS or LAS, but C34 inhibited ${Delta}$ CT-inducedfusion more effectively at the LAS than at the TAS. This indicatesthat the grooves of the coiled coil of ${Delta}$ CT were more exposedat the LAS than at the TAS. Creating the LAS also rendered the5-helix peptide (Fig. 7B) almost equally effective at inhibitingWT- and ${Delta}$ CT-induced fusions. But in contrast to C34, the IC₅₀for inhibition by the 5-helix peptide was significantly higherat the LAS (Fig. 7B) than at initial coincubation (Fig. 7A)for both the WT and ${Delta}$ CT. We infer that the grooves created byN-terminal segments continue to be accessible (and quite possiblybecome more accessible), but the C-terminal segments becomeless accessible as fusion advances toward membrane merger. Asimilar phenomenon has been observed for the fusion proteinof paramyxovirus SV5 (52).

DISCUSSION

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Discussion

Intermediates and peptide inhibitors provide a method for comparing the rate of folding of the WT and mutant Env proteins into a 6HB. Fusion induced by the WT exhibits slower kinetics and greatersusceptibility to inhibition by C34 and the 5-helix peptidethan that induced by ${Delta}$ CT. If the slower fusion kinetics werepredominantly due to the WT more slowly reconfiguring from aprebundle to a bundle, a more potent inhibition of fusion bypeptide would be expected (48). But differences in fusion kineticscan have many different causes, and currently it is not possibleto isolate each of these causes or to determine their relativeimportance, if any, to the fusion process. So the finding that ${Delta}$ CT exhibits faster kinetics of fusion than the WT does not necessarilyaccount for the reduced sensitivity of ${Delta}$ CT to inhibitors of 6HBformation. Our strategy of creating stable intermediates andtesting the potency of peptides against bundle formation atthese stages allows one to bypass these kinetic uncertaintiesand to clearly determine the relative exposure of grooves atprogressive stages of fusion. This methodology should also proveuseful in future studies of the mechanism of inhibition of fusionby other reagents.

Greater resistance to peptide inhibitors is due to faster folding of ${Delta}$ CT than the WT into 6HBs at a late stage of fusion. We found that when inhibitory peptides were added at the TASand then maintained, they were more effective at blocking WT-than ${Delta}$ CT-induced fusion, even though the kinetics of fusion fromthe TAS were the same for the WT and ${Delta}$ CT. The IC₅₀ of C34 forinhibition of WT-induced fusion was relatively independent ofwhether it was added at the beginning of cell coincubation,at the TAS, or at the LAS (Fig. 6). This indicates that thekinetics of refolding of the WT were sufficiently slow, or bindingsites were sufficiently exposed, that C34 could bind accordingto its intrinsic affinity. Maintenance of inhibition also contradictsthe claim that C34 loses potency at advanced fusion intermediatesand that 6HBs form prior to membrane merger (22). Quantitativeretention of fusion inhibition by C34 at the LAS is in accordwith our prior conclusion that copies of Env that participatein fusion do not fold into bundles before membrane merger (44)and that bundle formation is not complete until after pore formation(42).

The advanced intermediate of the LAS had to be achieved in orderfor the inhibitory peptides to equally inhibit WT- and ${Delta}$ CT-inducedfusions. For ${Delta}$ CT, the IC₅₀ progressively decreased as C34 wasadded at the initial E-T cell coincubation at 37°C, at theTAS, and at the LAS. This suggests that binding of C34 to thegrooves of the central coiled coil of ${Delta}$ CT is restricted untilthe LAS is reached and that when fusion is allowed to proceedcontinuously rather than arrested at the LAS, the time of maximalgroove exposure is limited. Taken together, the data indicatethat ${Delta}$ CT (Fig. 8, bottom panels) optimally exposes the groovesof its coiled coil later than does the WT (upper panels), but ${Delta}$ CT more quickly folds into a 6HB from this point. As a result,the grooves are optimally exposed to C34 for longer times forthe WT (upper panels). Thus, our data provide strong supportfor the hypothesis that faster folding of ${Delta}$ CT, compared to thatof the WT, from a late prebundle configuration into a 6HB isthe reason for its greater resistance to the inhibitory peptides.

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FIG. 8. The time from maximal groove exposure to formation of the 6HB is briefer for ${Delta}$ CT. C34 inhibits WT-induced fusion more effectively than ${Delta}$ CT-induced fusion at the TAS, indicating that the grooves of ${Delta}$ CT are less well exposed than those of the WT at this stage. Inhibition of fusion by C34 is the same for the WT and ${Delta}$ CT at the LAS. Once grooves are fully exposed, ${Delta}$ CT more quickly folds into a 6HB, illustrated as shorter transition arrows between the TAS and the LAS and between the LAS and fusion for ${Delta}$ CT. The progression from left to right denotes a reaction coordinate (rather than time) for fusion.

We matched Env densities in order to compare extents and kineticsof fusion induced by the WT and ${Delta}$ CT. But it has been concludedthat mutating the two cysteines of the CT so that Env can nolonger become palmitoylated eliminates association of Env withrafts (3), and thus the WT and ${Delta}$ CT may be distributed differentlyover the cell surfaces. The spatial distribution of Env, andother factors, could significantly affect the time course ofthe protein interactions necessary for fusion. This limits theability to use kinetics of fusion to draw conclusions concerningmechanisms of peptide inhibition of fusion. Our strategy oftesting the potency of peptides at intermediate stages of fusionavoids this limitation.

The CT hinders conformational changes required for the prebundle to 6HB transition. Folding kinetics have been suggested as the primary determinantof the greater susceptibility of X4-tropic than R5-tropic Envto peptide inhibitors (48). The evidence was based on the findingthat as V3 loop variants of Env exhibited faster fusion kinetics,resistance of fusion to T20 increased (48). Our results areconsistent with that finding: C34 was a more effective inhibitorof X4-tropic HXB2 than of R5-tropic JRFL (Fig. 5A), and fusionkinetics were faster for JRFL than for HXB2 (Fig. 2). In additionto faster kinetics causing JRFL Env to reside in prebundle configurationsfor shorter times than HXB2, exposure of binding sites may occurlater for JRFL: binding soluble CD4 exposes the coiled coiland C-terminal segments of HXB2 but not of JRFL Env (19). Anotherpotential reason for reduced effectiveness of C34 against JRFLEnv is that the amino acid sequence of C34 is the same as thatof the HXB2 Env sequence, while the corresponding region ofJRFL differs at five positions. However, a C34 derived fromthe sequence of SIV Env effectively inhibits HIV-1 Env (21,41) and about half of the residues are different for HIV-1 andSIV C34 residues. It is therefore expected that C34 binds toHXB2 and JRFL with the same affinity. In addition, an alignmentof helical wheels of HXB2 and JRFL residues shows that noneof their differences occur at a or d positions and it is thesepositions that should fit into the grooves of the coiled coil.

Several lines of evidence suggest that the long CT stabilizesthe native structure of the ectodomain of HIV Env (Fig. 4 andreferences 18, 45, and 58). On the basis of our data, the consequencesof the presence of the CT are not limited to this initial structurebut extend to conformational changes at the various steps offusion. Importantly, the long CT slows, but does not prevent,the major conformational change necessary for the transitionto the 6HB structure from a prebundle conformation in whichgrooves are maximally exposed. We envision that deletion ofthe CT increases the configurational freedom of Env during thefusion process. At some point in the fusion process, severaltrimers of Env probably come together to form a fusion complex. ${Delta}$ CT may have a significantly higher mobility than the WT becausethe CT is large and/or because removing the palmitoyls of CTreduces interactions with the inner monolayer of the plasmamembrane. A higher mobility of ${Delta}$ CT could shorten the time requiredfor cluster formation. The reduced time of groove exposure of ${Delta}$ CT may thus be caused by faster folding of individual trimersinto a 6HB bundle and/or by faster association among individualtrimers. The finding that inhibition of fusion by C34 at theLAS is independent of Env density (Table 2) and the same forthe WT and ${Delta}$ CT suggests that Env trimers have already complexedamong themselves into a fusion complex at the LAS and that thenumber of trimers within a complex is independent of surfacedensity.

The kinetics of the WT and ${Delta}$ CT from the TAS were the same forHXB2 Env (Fig. 2) but faster for ${Delta}$ CT than for the WT in controlexperiments. This suggests that the kinetic advantages conferredby deleting the CT are used in early, rather than in late, stepsof fusion. Because deleting the CT exposes chemokine bindingsites of Env (18), it would be expected that ${Delta}$ CT more quicklyengages CXCR4 than does the WT. Faster engagement of CXCR4 maybe the reason ${Delta}$ CT speeds up the early, pre-TAS steps. However,C34 was still more potent in inhibiting WT-induced fusion fromthe TAS, showing that rank ordering the rate of fusion for variantsof Env does not reliably allow one to predict, or account for,the sequence of potency of inhibition of fusion by bundle-blockingpeptides. In other words, differences in kinetics of fusionbetween variants can be caused by steps that are unrelated tobundle formation.

In comparing C34 and the 5-helix peptide, we found that as thefusion process progressed, inhibition by C34 remained the sameor increased whereas inhibition by the 5-helix peptide decreased.Less accessibility of the C-terminal segments of Env to the5-helix peptide as fusion proceeded could have resulted fromthe relatively large size of the peptide. Reduced inhibitionof fusion at the LAS has also been observed for peptides directedagainst the groove-fitting C-terminal portion of SV5 F (52).

Interactions of the CT with viral and cellular components may influence membrane fusion. In the viral setting, the CT of HIV-1 Env interacts with Gag(45, 58). If the HIV-1 protease does not cleave Gag and Gag-Polpolyprotein precursors, the virus does not mature and remainsnoninfectious (24, 33, 47). But deleting the CT renders thevirus infectious even if Gag and Gag-Pol remain uncleaved (45,58). It has been concluded that the interaction between theCT and Gag of the immature virus prevents the virus from fusingto membranes; deleting the CT eliminates the interactions andallows fusion to proceed (45, 58). Our data suggest that deletionof the CT inherently improves the ability of the virus to fuseto and therefore to infect cells, independent of advantagesconferred by dissociation of Env from Gag.

What molecular occurrence might account for the observationthat eliminating the CT causes the grooves of the prebundleto be exposed for less time? In folding into a 6HB from a prebundleconfiguration, the three membrane-spanning domains of a gp41trimer must separate from each other and move toward the fusionpeptides inserted in the target membrane. We propose that theinteractions between the long CT and viral or cellular components,or the large size of the CT, or a combination of these factors,reduce the freedom of movements of membrane-spanning domains.Deleting the CT eliminates these restrictions. Quicker foldingof individual trimers from prebundles to bundles and/or anyinteractions between trimers would thus be facilitated by deletionof the CT.

ACKNOWLEDGMENTS

We thank Greg Spear for use of his flow cytometer and SofyaBrener for steady and excellent technical assistance. We thankEric Hunter for critical reading of the manuscript and insightfulcomments. He also kindly provided the WT and ${Delta}$ CT plasmids forHXB2.

This study was supported by National Institutes of Health grantsGM27367, GM54787, and AI42382.

FOOTNOTES

* Corresponding author. Mailing address: Department of Molecular Biophysics and Physiology, Rush University Medical Center, 1653 W. Congress Pkwy., Chicago, IL 60612. Phone: (312) 942-6753. Fax: (312) 942-8711. E-mail: fcohen{at}rush.edu.

REFERENCES

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