Sequential Roles of Receptor Binding and Low pH in Forming Prehairpin and Hairpin Conformations of a Retroviral Envelope Glycoprotein

A general model has been proposed for the fusion mechanismsof class I viral fusion proteins. According to this model ametastable trimer, anchored in the viral membrane through itstransmembrane domain, transits to a trimeric prehairpin intermediate,anchored at its opposite end in the target membrane throughits fusion peptide. A subsequent refolding event creates a trimerof hairpins (often termed a six-helix bundle) in which the previouslywell-separated transmembrane domain and fusion peptide (andtheir attached membranes) are brought together, thereby drivingmembrane fusion. While there is ample biochemical and structuralinformation on the trimer-of-hairpins conformation of classI viral fusion proteins, less is known about intermediate statesbetween native metastable trimers and the final trimer of hairpins.In this study we analyzed conformational states of the transmembranesubunit (TM), the fusion subunit, of the Env glycoprotein ofthe subtype A avian sarcoma and leukosis virus (ASLV-A). Byanalyzing forms of EnvA TM on mildly denaturing sodium dodecylsulfate gels we identified five conformational states of EnvATM. Following interaction of virions with a soluble form ofthe ASLV-A receptor at 37°C, the metastable form of EnvATM (which migrates at 37 kDa) transits to a 70-kDa and thento a 150-kDa species. Following subsequent exposure to a lowpH (or an elevated temperature or the fusion promoting agentchlorpromazine), an additional set of bands at >150 kDa,and then a final band at 100 kDa, forms. Both an EnvA C-helixpeptide (which inhibits virus fusion and infectivity) and thefusion-inhibitory agent lysophosphatidylcholine inhibit theformation of the >150- and 100-kDa bands. Our data are consistentwith the 70- and 150-kDa bands representing precursor and fullyformed prehairpin conformations of EnvA TM. Our data are alsoconsistent with the >150-kDa bands representing higher-orderoligomers of EnvA TM and with the 100-kDa band representingthe fully formed six-helix bundle. In addition to resolvingfusion-relevant conformational intermediates of EnvA TM, ourdata are compatible with a model in which the EnvA protein isactivated by its receptor (at neutral pH and a temperature greaterthan or equal to room temperature) to form prehairpin conformationsof EnvA TM, and in which subsequent exposure to a low pH isrequired to stabilize the final six-helix bundle, which drivesa later stage of fusion.

INTRODUCTION

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Introduction

The fusion-promoting glycoproteins of orthomyxo-, paramyxo-,filo-, corona-, and retroviruses are grouped as class I fusionproteins because their fusion subunits possess a common postfusionform (12, 19, 20, 57). The structure they have in common consistsof a fusion peptide at or near the N-terminal end, a centraltrimeric coiled coil, a chain reversal segment, a C-terminalregion (in many cases a helix) that packs in the grooves ofthe central coiled coil, a transmembrane domain, and a cytoplasmictail. In the compact postfusion form, the two hydrophobic elements,the fusion peptide and the transmembrane domain, lie in closeproximity. All class I fusion proteins start out as metastabletrimers on the virus surface. In only one case, that of theinfluenza virus hemagglutinin (HA), do we know the atomic structureof the complete ectodomain of the metastable trimer (63). Nonetheless,a common model has emerged for how class I fusion proteins mediatefusion. In essence, the metastable trimer is anchored in theviral membrane through its transmembrane domain. Following atriggering event (interaction with receptors or exposure toa low endosomal pH), the metastable trimer converts to a prehairpinintermediate in which the newly exposed fusion peptide bindsto the target bilayer. The prehairpin is envisioned as a straighttrimeric coiled-coil rod with the fusion peptide at one end(in the target membrane) and the transmembrane domain at theopposite end (in the viral membrane). Through conformationalchanges that essentially fold the prehairpin in half, the twohydrophobic elements (and their attached bilayers) are broughttogether to initiate membrane merger. Support for the commonprehairpin intermediate has come from the use of C-helix peptideinhibitors. C-helix peptides from class I fusion proteins thatfunction at neutral pH (e.g., those of paramyxoviruses, coronaviruses,and retroviruses) inhibit fusion and infectivity (6, 18-20,and references therein). Experiments with C-helix peptide inhibitorsat fusion-arrested states have supplied evidence for prehairpinconformations of human immunodeficiency virus (HIV) Env (44)and a paramyxovirus F protein (52). For both of these proteinsa prehairpin intermediate has been trapped biochemically bycoimmunoprecipitating the fusion proteins with a cognate epitope-taggedC-helix peptide during fusion activation (22, 52).

Our laboratory has been studying the fusion mechanism of a modelretrovirus, the subtype A avian sarcoma/leukosis virus (ASLV-A).The Env glycoprotein of ASLV-A (EnvA) is predicted to be a classI fusion protein with structural similarity between its fusionsubunit and those of human T-cell leukemia virus Env and Ebolavirus GP (24, 36). The fusion peptide is sequestered in thenative EnvA trimer but is exposed and binds to target membranesfollowing interaction with the host cell receptor Tva (2) ata temperature greater than or equal to room temperature (T $≥$ RT) (26, 28, 29). Our work indicates that EnvA (on virus particles)can mediate both the target bilayer binding and lipid-mixingstages of fusion at neutral pH and T $≥$ RT (18, 25, 28). Otherwork indicates that reverse transcripts of the virus are notproduced if infected cells are maintained in the continued presenceof agents (e.g., bafilomycin) that raise the pH of endosomes(45; S. Matsuyama, L. Earp, and J. M. White, unpublished data).The latter findings suggest that a low pH is needed for eithera later stage of fusion or viral uncoating.

As part of our ongoing effort to delineate the precise stageof ASLV entry that requires a low pH, we have modified and extendedan analysis (45) of conformational intermediates in the fusionsubunit (the transmembrane subunit [TM]) of EnvA. Our modifiedapproach entailed assessing sequential effects of a minimalsoluble receptor (62) and a low pH on the EnvA TM found on virusparticles. For this analysis we conducted Western blot assaysof viral samples prepared under mildly denaturing conditions.This analysis, in conjunction with specific fusion-inhibitory(e.g., a potent C-helix peptide inhibitor [18]) and fusion-promotingagents, revealed sequential intermediates in the pathway offusion that represent prehairpin and hairpin forms of EnvA.Our findings are consistent with a model in which interactionwith receptor at neutral pH activates EnvA to form prehairpinintermediates and in which a low pH is needed to stabilize afinal six-helix bundle (hairpin) form of EnvA at a later stageof fusion.

MATERIALS AND METHODS

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Materials and Methods

Virus, soluble receptor, and peptide R99. ASLV-A was purified from DF-1 cells chronically infected withRCASBP(A)AP as described previously (18). A soluble form ofthe receptor (sTva) was prepared as described previously (62).Peptide R99 was described previously (18).

Lipids and liposomes. L- ${alpha}$ -Phosphatidylcholine (PC; egg; Avanti Polar Lipids), L- ${alpha}$ -phosphatidylethanolamine(PE; egg; Avanti Polar Lipids), sphingomyelin (Sph; brain; AvantiPolar Lipids), and cholesterol (Chol; Sigma Chemical Co.) werestored as described previously (28). Lipids (1:1:1:1.5 molarPC-PE-Sph-Chol ratio) were mixed, dried under N₂ gas in a glasstube, and lyophilized overnight. After addition of buffer (20mM morpholineethanesulfonic acid, 20 mM HEPES, pH 7.4), thelipid suspension was vortexed, sonicated in a water bath sonicator,and then extruded 25 times through a 0.1-µm-pore-sizeNuclepore filter in an Avanti Mini-Extruder. Liposomes (5.6mM lipid on the basis of the input lipid) were stored at 4°Cand used within 1 week.

Membrane-perturbing reagents. LPC (lysophosphatidylcholine) and CPZ (chlorpromazine) werepurchased from Sigma Chemical Co. Fresh stocks of LPC and CPZ(25 mM in 20 mM morpholineethanesulfonic acid-20 mM HEPES, pH7.4) were prepared just before use.

TM oligomer assay. The TM oligomer assay developed by P. Bates and coworkers wasperformed as described in reference 45, with minor modifications.In brief, 2.5 µl of freshly harvested and freshly concentratedASLV-A and 0.5 µl (300 ng) of sTva were mixed on ice.After 30 min, 2.5 µl of liposomes was added on ice. Forone-step incubations, samples were warmed to 37°C for 30min (unless stated otherwise). For two-step incubations, sampleswere warmed to 37°C for 30 min (at neutral pH) and thenadjusted to pH 5 (unless stated otherwise) with a predeterminedamount of 0.1 M HCl. After 5 min at 37°C, 0.5 µl of1 M Tris (pH 8.0) was added to neutralize low-pH-treated samples.Samples were then treated with buffer to yield final concentrationsof 0.1% sodium dodecyl sulfate (SDS), 2% ß-mercaptoethanol,and 6% glycerol, incubated for 5 min at 37°C, and then loadedonto SDS-7% polyacrylamide gel electrophoresis gels. Followingelectrophoresis, proteins were transferred to nitrocelluloseand probed with a rabbit polyclonal antibody against a peptidecorresponding to the cytoplasmic tail of EnvA (18). Primaryantibody was detected with horseradish peroxidase-conjugatedanti-rabbit antibody (Amersham Biosciences).

RESULTS

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Results

Receptor and a low pH sequentially induce different conformational states of EnvA TM. We set out to compare conformational states of the EnvA TM subunit(the fusion subunit) formed during each step of a two-step protocol(Fig. 1A). In all cases, freshly prepared ASLV particles werepremixed with sTva, a soluble form of the EnvA receptor (62),on ice for 30 min at neutral pH. After the addition of PC-PE-Sph-Cholliposomes, samples were incubated at 37°C for 30 min atneutral pH (step 1). The pH was then adjusted to 5.0, and themixture was incubated for an additional 5 min at 37°C (step2). Samples were then neutralized, mixed with sample bufferto a final concentration of 0.1% SDS, incubated at 37°Cfor 5 min, and analyzed by Western blot assays for forms ofthe TM (fusion) subunit. The EnvA TM subunit from starting virionswas present as a 37-kDa band. This band is identical in sizeto the monomeric band obtained when virions are boiled withsample buffer containing 1% SDS. When virions with preboundreceptor were incubated at 37°C, bands of 70 and 150 kDaappeared sequentially in a time-dependent manner (Fig. 1B, lanes5 to 8). A small amount of the 70-kDa band formed upon warmingin the absence of receptor (lanes 3 and 4). Receptor-inducedformation of the 70- and 150-kDa bands occurred equally wellin the absence (lanes 5 to 8) or presence (lanes 9 to 12) ofliposomes and equally well with 1/400 of the amount of receptor(S. Matsuyama, Z. Chen, and J. M. White, unpublished data).Moreover, incubation of receptor-bound virions for longer times(60 to 90 min) at 37°C at neutral pH did not change thegel pattern seen in lanes 8 and 12 (Matsuyama, Chen, and White,unpublished).

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FIG. 1. Sequential formation of distinct EnvA TM conformational states. (A) Schematic of the procedure used to generate various conformational states of EnvA TM. Unless stated otherwise, virions (ASLV-A) were incubated on ice for 30 min in the presence of receptor (sTva). Liposomes were then added, and the samples were warmed to 37°C at neutral pH for 30 min (step 1). As indicated (step 2), the pH was adjusted to 5.0 for 5 min. Low-pH-treated samples were then reneutralized and processed to visualize EnvA TM as described in Materials and Methods. (B) Virions were subjected to step 1 conditions in the absence of either receptor or liposomes (lanes 1 to 4), with receptor only (lanes 5 to 8), or with receptor and liposomes (lanes 9 to 12) for the indicated times at 37°C and then processed as described above. (C) Receptor-bound virion-liposome complexes from step 1 (panel B, lane 12) were exposed to the indicated pH during step 2 and then processed as described above. The values on the left and right of panels B and C are molecular sizes in kilodaltons.

We note that although equal amounts of virus were loaded perlane, the 70- and 150-kDa bands appeared proportionately moreprominent than the 37-kDa band. Although our ability to detectthe 37-kDa band is variable from experiment to experiment, thisobservation suggests that the cytoplasmic tail epitope of EnvATM is hyperreactive in the 70- and 150-kDa bands in this gelsystem.

We next exposed virus-receptor-liposome complexes from step1 to pH values ranging from 7.4 to 5.0 for 5 min at 37°C(step 2). After treatment at pHs 5.5 and 5.0 a new EnvA TM bandat 100 kDa and a mixture of bands at >150 kDa were clearlyobserved (Fig. 1C, lanes 5 and 6). Smaller amounts of the 100-and >150-kDa bands were seen in samples treated at pH 6.5(lane 3) or 6.0 (lane 4). Bands at 70 and 150 kDa were stillpresent after the two-step protocol. This may be due, in part(see Discussion), to the previously noted apparent hypersensitivityof these forms of EnvA TM to the anti-EnvA cytoplasmic tailantibody on immunoblots. On the basis of the pH dependence observedin Fig. 1C, we used pH 5.0 for the low-pH treatment in subsequentexperiments. Liposomes were present in all subsequent experiments.

To rule out the possibility that any of the higher-molecular-weightbands of EnvA TM (70, 150, 100, or >150 kDa) resulted fromstable association with internal viral components, immunoblotsof samples treated by either step 1 or the sequential step 1-step2 protocol were probed with antibodies against the matrix andcapsid proteins. None of the EnvA species reacted with anti-matrixor anti-capsid antibodies (data not shown).

An elevated temperature can mimic the effects of a low pH at step 2. In the case of influenza virus, an elevated temperature cansubstitute for a low pH to trigger fusion-relevant conformationalchanges (7, 51). Similarly, an elevated temperature can substitutefor the need for the HN protein for SV5-mediated fusion (47).We therefore asked whether an increased temperature could mimicthe effects of a low pH in step 2. Accordingly, complexes weresubjected to the step 1 treatment, followed by incubation for5 min at increasing temperatures (at pH 7.4). As shown in Fig.2, the >150-kDa bands began to form at 42°C and the 100-kDaband began to form at 50°C, suggesting that the >150-kDabands form prior to the 100-kDa band. The >150- and 100-kDabands increased in intensity with increasing temperature.

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FIG. 2. Effects of an increased temperature, in lieu of a low pH, at step 2 on conformational states of EnvA TM. Virions (lane 1) were treated to step 1 conditions (lane 2) and then to the indicated temperature (instead of a low pH) at step 2 (lanes 3 to 10). Lane 11 is a sample of virions treated to the two-step protocol. Samples were processed to visualize EnvA TM as described in the legend to Fig. 1. The values on the right are molecular sizes in kilodaltons.

Effects of peptide R99 on the formation of different conformational states of EnvA TM. Peptide R99 is a synthetic analogue of the EnvA C helix, whichinhibits viral infectivity, virus-cell fusion, and EnvA-mediatedcell-cell fusion (18). We therefore asked whether peptide R99affects the formation of any of the higher-molecular-weightforms of EnvA TM. To do this we performed step 1 and 2 incubations(Fig. 1A) in the presence or absence of peptide R99. As shownin Fig. 3A (lane 4), peptide R99 did not inhibit the formationof either the 70- or the 150-kDa band at neutral pH (step 1).However, when virions were subjected to step 2 of the procedurein the presence of peptide R99, the >150- and 100-kDa bandsdid not form (Fig. 3A, lane 8). Peptide R99 did not inhibitthe formation of the >150- and 100-kDa bands if virions wereexposed to a low pH in step 2 in the absence of receptor (lane6) or if they were directly warmed to pH 5.0 in the presenceor absence of receptor (lanes 10 and 12). Hence, receptor activationat neutral pH (T $≥$ RT) is a prerequisite for forming the >150-and 100-kDa bands along a pathway that is inhibitable by peptideR99. The ability of an increased temperature (in lieu of a lowpH) at step 2 to induce formation of the >150 and 100-kDabands was also inhibited by peptide R99 (Fig. 3B), albeit apparentlyless effectively than when a low pH was used in step 2 (Fig.3A).

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FIG. 3. Effects of peptide R99 on conformational states of EnvA TM. (A) Virions were incubated under step 1 conditions (lanes 1 to 4) or under step 1 and then step 2 conditions (lanes 5 to 8) or directly warmed at pH 5 (lanes 9 to 12) in the absence (lanes 1, 2, 5, 6, 9, and 10) or presence (lanes 3, 4, 7, 8, 11, and 12) of receptor and in the absence (odd-numbered lanes) or presence (even-numbered lanes) of peptide R99 (50 µg/ml). Liposomes were present in all samples. (B) Receptor-bound virion-liposome complexes that had been subjected to step 1 conditions were treated to increasing temperatures (in lieu of a low pH) at step 2 in the absence (odd-numbered lanes) or presence (even-numbered lanes) of peptide R99 (50 µg/ml). (C) Receptor-bound virion-liposome complexes were subjected to the two-step protocol in the presence of the indicated amount of peptide R99. (D) Peptide R99 (50 µg/ml) was added to samples before step 1 (lane 2), after step 1 but before step 2 (lane 3), or after step 2 (lane 4). Lane 1 contains a sample subjected to the two-step protocol in the absence of peptide R99. Samples were processed to visualize EnvA TM as described in the legend to Fig. 1. The values on the left or right are molecular sizes in kilodaltons.

The experiments described in Fig. 3A and B were performed with50 µg of peptide R99 per ml, a concentration that completelyinhibits the lipid-mixing activity of ASLV-A particles (18).However, as shown in Fig. 3C, concentrations of peptide R99as low as 0.78 µg/ml inhibited the formation of the >150-and 100-kDa bands. We next asked if we could define a pointin the protocol at which the >150- and 100-kDa bands becomerefractory to peptide R99. As shown in Fig. 3D, peptide R99inhibited the formation of the >150- and 100-kDa bands ifit was added either before step 1 (warming of virus-receptorcomplexes at neutral pH) (lane 2) or before step 2 (subsequentincubation at a low pH) (lane 3). If peptide R99 was added afterthe low-pH incubation step (lane 4), formation of the >150-and 100-kDa bands was not inhibited.

Effects of fusion modulators on different conformational states of EnvA TM. Agents that affect membrane curvature can inhibit or promotemembrane fusion (9). We therefore tested the effects of a fusion-inhibitingand a fusion-promoting agent on the formation of the variousEnvA TM conformational states. LPC is a lipid analogue thatinhibits membrane merger in many systems (9, 10, 33). Virionswere treated by the two-step method (Fig. 1A) in the presenceof increasing concentrations of LPC. As shown in Fig. 4A, withconcentrations of LPC of $≥$ 0.25 mM, formation of the 150-, >150-,and 100-kDa bands was increasingly inhibited and the amountof the 37-kDa band increased. When the same experiment was performedin the presence of peptide R99 (lanes 7 to 12), the 150-kDaband, but not the >150- and 100-kDa bands, was protected(compare lanes 11 and 12 with lanes 5 and 6). Hence, LPC inhibitsthe formation of the 150-, >150-, and 100-kDa bands producedduring the two-step protocol. Of these, the 150-kDa speciesis protected from LPC inhibition by peptide R99.

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FIG. 4. Effects of LPC on conformational states of EnvA TM. (A) Receptor-bound virions were mixed with liposomes, the indicated amount of LPC was added in the absence (lanes 1 to 6) or presence (lanes 7 to 12) of peptide R99, and mixtures were then subjected to the two-step protocol. (B) Receptor-bound virions were subjected to step 1 only (lanes 1 to 4) or the two-step protocol (lanes 5 to 8); reaction mixtures contained no inhibitors (lanes 1 and 5), LPC (lanes 2, 4, 6, and 8), or peptide R99 (lanes 3, 4, 7, and 8). (C) LPC was added to the virion-receptor-liposome mixtures at the stages indicated in the absence (lanes 1 to 4) or continual presence of peptide R99 (lanes 5 to 8). Alternatively, R99 was added to the mixture at the indicated stage in the continual presence of LPC (lanes 9 to 12). Samples were then processed as described in the legend to Fig. 1. The values on the left are molecular sizes in kilodaltons.

We next tested the effects of LPC during step 1 of the two-stepprotocol (Fig. 1A). As shown above (for example, Fig. 1B), incubationof receptor-bound virions at neutral pH and 37°C leads tothe formation of 70- and 150-kDa bands (Fig. 4B, lane 1). Formationof the 150-kDa band at neutral pH (i.e., during step 1) wasinhibited by LPC (Fig. 4B, lane 2). Also, peptide R99 protectedthe 150-kDa band from inhibition by LPC during step 1 (Fig.4B, lane 4 versus lane 2). Collectively, the results in Fig.4A and B suggest that LPC has two effects in this system (seebelow and Discussion).

We next sought to determine when LPC had to be added to haveits inhibitory effects. As shown in Fig. 4C, if LPC was addedbefore step 1, the >150- and 100-kDa bands did not form andless of the 150-kDa band formed (Fig. 4C, compare lane 2 withlane 1). If LPC was added after step 1 but before step 2, moreof the 150-kDa band formed but formation of the >150- and100-kDa bands was still inhibited (Fig. 4C, lane 3). AddingLPC after step 2 had no effect on any of the EnvA TM bands (Fig.4C, lane 4). If peptide R99 was present continually (lanes 5to 8), LPC, added before step 1 or before step 2, inhibitedthe formation of the >150- and 100-kDa bands but did notinhibit the formation of the 150-kDa band (Fig. 4C, lanes 6and 7). If peptide R99 was added either before step 1 or beforestep 2, in the continual presence of LPC (lanes 9 to 12), the150-kDa band was protected (Fig. 4C, lanes 10 and 11). If addedafter step 2, peptide R99 was no longer able to protect the150-kDa band (Fig. 4C, lane 12).

We next assessed the effects of CPZ, a reagent that promotesmembrane fusion (38, 42), on the formation of the higher-molecular-weightforms of EnvA TM. In particular we asked if CPZ could promotethe formation of the >150- and 100-kDa bands at neutral pH.Indeed, as shown in Fig. 5A, CPZ induced the formation of the>150- and 100-kDa bands at neutral pH. As with increasingtemperature in lieu of a low pH at step 2 (Fig. 2), the >150-kDabands appeared to form first (Fig. 5A, lanes 2 to 5) and thento chase into the 100-kDa band as the CPZ concentration wasincreased (Fig. 5A, lane 6 compared to lane 5). Interestingly,the presence of CPZ could overcome the inhibitory effect ofpeptide R99 on the formation of the >150- and 100-kDa bands(Fig. 5B, lanes 3 to 5; see Discussion). As expected, CPZ didnot affect the formation of the 70- or 150-kDa bands at neutralpH (Fig. 5A), nor did it affect the formation of any of thehigher-molecular-weight bands formed during the two-step protocol(not shown).

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FIG. 5. Effects of CPZ on conformational states of EnvA TM. (A) Receptor-bound virions were mixed with liposomes, the indicated amount of CPZ was added, and the mixtures were then incubated under step 1 conditions. (B) Samples were subjected to the two-step protocol in the presence (lanes 2 to 5) or absence (lane 1) of peptide R99 and in the presence (lanes 3 to 5) or absence (lanes 1 and 2) of the indicated concentration of CPZ. Samples were then processed as described in the legend to Fig. 1. Lanes 1 and 2 were not adjacent in the original gel. The values on the left or right are molecular sizes in kilodaltons.

Stability of EnvA TM conformational states. We tested the stability of the conformational states of EnvATM when subjected to an elevated temperature, SDS, and urea.As shown in Fig. 6A, the 70- and 150-kDa bands were stable at80°C in 0.1% SDS (lanes 1 to 5) and only modestly diminishedafter boiling (lane 6). The >150- and 100-kDa bands wereless stable at an elevated temperature in 0.1% SDS, with lossbeginning at 65°C. All of the higher-molecular-weight formsof EnvA TM were resistant to SDS at concentrations at leastas high as 2% when samples were incubated at 37°C (Fig.6B, lanes 1 to 5). When samples were boiled in 0.1% SDS, the>150- and 100-kDa bands were not observed (Fig. 6B, lane6). The 70- and 150-kDa bands were observed in samples boiledin 0.1% SDS (Fig. 6B, lane 6) but not in samples boiled in higherconcentrations of SDS (Fig. 6B, lanes 7 to 10). As with increasingtemperature (Fig. 6A), loss of the higher-molecular-weight formsof EnvA TM was accompanied by appearance of the 37-kDa monomerband. All of the higher-molecular-weight forms of EnvA TM werestable in up to at least 4 M urea at 37°C (Fig. 6C).

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FIG. 6. Stability of EnvA TM oligomers. Virus samples treated to the two-step protocol were either warmed at the indicated temperature in sample buffer containing 0.1% SDS for 5 min (A), incubated in sample buffer containing the indicated amount of SDS at either 37°C (lanes 1 to 5) or 100°C (lanes 6 to 10) for 5 min (B), or incubated in the indicated amount of urea at 37°C for 5 min (C). Samples were then electrophoresed and subjected to Western blotting as described in Materials and Methods. The values on the left are molecular sizes in kilodaltons. RT, room temperature.

DISCUSSION

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Discussion

In this study we identified a series of conformational statesof the EnvA TM (fusion) subunit that arise sequentially followinginteraction with receptor at neutral pH (step 1) and then exposureto a low pH (step 2). To do this we analyzed virus samples preparedfor SDS gels with sample buffer containing 0.1% SDS and warmedto 37°C. In native virions the EnvA TM subunit appearedas a 37-kDa band. Following interaction with soluble receptorat neutral pH and 37°C, a 70-kDa band and then a 150-kDaform appeared (Fig. 1B). Following subsequent exposure to apH of <6.0, a group of bands at >150 kDa and then a 100-kDaband formed (Fig. 1C). A C-helix peptide that inhibits virus-cellfusion, EnvA-mediated cell-cell fusion, and virus infection(18) inhibits the formation of the >150- and 100-kDa bands.The >150- and 100-kDa bands can form at neutral pH if, followingreceptor activation (at neutral pH), samples are treated atan elevated temperature (e.g., 50°C) or with the fusion-promotingagent CPZ. LPC, a fusion-inhibitory lipid analogue, inhibitsthe formation of the >150- and 100-kDa bands. The opposingeffects of CPZ and LPC suggest that virus-liposome fusion occursin this system. The promoting effect of CPZ and the inhibitoryeffects of LPC and peptide R99 also indicate that the >150-and 100-kDa bands are relevant to virus fusion. The conditionsthat generate the higher-molecular-weight forms of EnvA andtheir temporal relationships are depicted in Fig. 7A.

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FIG. 7. Working model of the roles of distinct conformational states of EnvA TM in distinct steps of fusion. (A) Conformational states (apparent molecular masses in kilodaltons) of Env A TM detected on mildly denaturing SDS gels and the conditions that promote or inhibit their formation. (B) Model of what the distinct conformations may look like and how they may mediate distinct stages of fusion. The TM subunit of starting virions migrates as a 37-kDa band (drawing a). (In drawing a, the SU subunits, which hold the TM subunit in its metastable state, are shown as gray ovals. For clarity, the SU subunits, which have presumably moved out of the way, are not shown in subsequent drawings.) Incubation of virions with soluble receptor at neutral pH and 37°C (step 1) results in the sequential formation of two new bands of 70 and 150 kDa (drawings b and c). We propose that the 150-kDa band represents the fully formed prehairpin conformation of EnvA TM. The 70-kDa band may be a prehairpin precursor. Upon exposure to a low pH (or an increased temperature or CPZ; step 2), a set of >150-kDa bands and then a 100-kDa band form (drawings e and f). Peptide R99 inhibits the formation of the >150- and 100-kDa bands. LPC inhibits the formation of the 150-, >150-, and 100-kDa bands. We propose that LPC inhibits the formation of the >150- and 100-kDa bands because it inhibits fusion. We propose that LPC inhibits the formation of the 150-kDa band owing to interaction with the exposed fusion peptide (see text). A distinct conformational state corresponding to the peptide R99-inhibitable lipid-mixing stage of fusion (drawing d) has not been detected in our gel system. Hence we designate it L for lipid mixing (see text). (The arrows are not meant to imply irreversibility.) R, receptor.

Model for steps of fusion mediated by distinct conformational states of EnvA TM. We suggest that the 70- and 150-kDa bands that form followingincubation with receptor at 37°C represent, respectively,intermediate and then fully formed EnvA TM prehairpins (drawingsb and c in Fig. 7B). Several observations support this hypothesis.For one, the C-helix analogue peptide R99 does not inhibit theformation of either band. In fact, peptide R99 stabilizes the150-kDa band in the presence of LPC (discussed below). Theseobservations are consistent with our prior finding that peptideR99 does not inhibit the fusion peptide-mediated associationof EnvA with target membranes (18). Further support that the150-kDa band represents the target membrane binding prehairpinis that we have recently observed (Z. Chen and J. M. White,unpublished data) that the receptor concentration needed toform the 150-kDa band (in step 1) is the same as that neededto induce ASLV particles to bind to target membranes (18).

As stated above, LPC (added either before step 1 or before step2) inhibits the formation of the >150- and 100-kDa bands,consistent with an inhibition of fusion. An initially surprisingfinding was that when LPC was added to virion-receptor-liposomemixtures before step 1, formation of the 150-kDa band was alsoinhibited (Fig. 4). Our hypothesis to explain this secondaryeffect of LPC is as follows. Evidence has been presented thatLPC can bind to exposed fusion peptides and inhibit their associationwith target membranes (27, 58). If LPC binds to the exposedEnvA fusion peptide (14, 26, 28), it may sterically inhibitcoiled-coil formation in the N-terminal end of the prehairpin(gray part of protein in drawing b of Fig. 7B). As noted above,peptide R99 can overcome the LPC block to forming the 150-kDaband (Fig. 4). This result suggests that the affinity of peptideR99 for the N-helix coiled coil is greater than the affinityof LPC for the fusion peptide. For these reasons, we proposethat the C-helix analogue peptide R99 can not only bind to theN-terminal end of the fully formed prehairpin (drawing c inFig. 7B) but also stabilize the prehairpin conformation.

When ASLV particles activated by receptor at neutral pH aresubsequently exposed to a low pH, a temperature of >50°C,or CPZ, a 100-kDa band forms. We propose that the 100-kDa bandrepresents the final fully formed EnvA TM hairpin, its six-helixbundle (Fig. 7B, drawing f). This proposal is supported by theobservations that the 100-kDa band is the last species formedunder any of the post receptor activation conditions that wehave explored (a low pH, an elevated temperature, or treatmentwith CPZ) and that formation of the 100-kDa band is inhibitedby the C-helix analogue peptide R99. Interestingly, CPZ is ableto drive the formation of the 100-kDa band in the presence ofR99 (at the concentration of R99 tested; Fig. 5B). Importantly,peptide R99 does not inhibit the formation of the 100-kDa bandif receptor-bound virions (produced and maintained at 4°C)are directly warmed to 37°C at pH 5 or if virions are subjectedto the two-step protocol in the absence of receptor (Fig. 3A).These findings, coupled with the need for sequential activationby receptor at neutral pH, followed by a low-pH shock to overcomethe block to reverse transcript production imposed by bafilomycin(45) (Matsuyama and White, unpublished), suggest that receptoractivation at neutral pH (and T $≥$ RT) is needed to keep EnvAon a productive fusion pathway. We further propose that conversionto the 100-kDa form mediates the final stage of fusion, fusionpore enlargement (drawing f in Fig. 7B; see below).

A set of bands at >150 kDa appear to be intermediates onthe pathway between the 150-kDa band (proposed prehairpin) andthe 100-kDa band (proposed final six-helix bundle). This isevident when either CPZ (Fig. 5A) or an elevated temperature(Fig. 2) is used to drive conformational changes that occurpost receptor activation. We suggest that the >150-kDa bandsmediate the opening of small fusion pores (Fig. 7B, drawinge). The >150-kDa species may be analogous to higher-orderoligomers that form transiently during baculovirus gp64-mediatedfusion (40). Formation of higher-order oligomers has been proposedfor many viral fusion systems (4, 13, 16, 39, 40). Upon fusionpore expansion, the higher-order transient oligomers may disperseand transform into unitary six-helix bundles. The transitionof the >150-kDa bands to the 100-kDa band may drive conversionfrom small, labile fusion pores to large, robust fusion pores,as has recently been described for HIV Env (35, 37).

All of the higher-molecular-weight forms of EnvA TM are stablein at least 2% SDS at 37°C (Fig. 6B, lanes 1 to 5) and inat least 4 M urea at 37°C (Fig. 6C). We were surprised,however, to find that the 70- and 150-kDa bands appear to bemore stable than the >150- and 100-kDa bands if gel samplesare prepared at $≥$ 80°C in 0.1% SDS. Six-helix bundle cores(lacking transmembrane domains and fusion peptides) are highlystable at elevated temperatures in aqueous solutions. However,to our knowledge, there have not been studies comparing thestability of prehairpin and hairpin conformations of intactclass I viral fusion proteins in SDS. The apparent persistenceof the prehairpin forms of EnvA TM (70- and 150-kDa bands) onSDS gels of low-pH-treated samples (which may be exaggeratedby the apparent hyperreactivity of the 70- and 150-kDa bandson our immunoblots; see Results) may indicate that not all EnvATM prehairpins convert to hairpin conformations. Remaining prehairpinsmay help buttress fusion pores, as has recently been modeledfor HIV Env (37).

We have observed that ASLV particles can reach the lipid-mixingstage of fusion with target cells at neutral pH (drawing d inFig. 7B) and that lipid mixing, but not target bilayer association,can be inhibited by peptide R99 (18). However, we have not visualizedan EnvA TM species (on an SDS gel) that is inhibited by peptideR99 and that forms following interaction with receptor at neutralpH and T $≥$ RT. We therefore propose that there is a species,designated L in Fig. 7A, that mediates the lipid-mixing stageof virus-cell fusion. Species L (for lipid mixing) could beone or more of the >150-kDa species that are unstable in0.1% SDS without prior treatment with a low pH, CPZ, or an elevatedtemperature. Alternatively, lipid-mixing species L could bea distinct conformation of EnvA TM, such as that depicted indrawing d of Fig. 7B (which is not stable in 0.1% SDS). As drawn,the C helices in species L make trimeric contacts with eachother (perhaps as in the metastable trimer). Such trimeric contactswould have to be broken so that the three C helices could packindividually into the grooves of the N-terminal end of the coiledcoil (as in drawings e and f). Breaking of such trimeric contacts,and transiting to a symmetric conformation such as that shownin drawing e, could be an energy-requiring step that may befacilitated by a low pH, an elevated temperature, or CPZ. Multiplesequences within the C helix (23, 41), within the disulfide-bondedchain reversal region (36, 54), within the transmembrane (35,59) and juxtamembrane (53) regions, and within the cytoplasmictail (see below), could influence the d-to-e transition.

Physiological relevance of the two-step fusion process. We have previously shown that receptor activation can causeASLV to reach the lipid-mixing stage of virus-cell fusion (drawingd in Fig. 7B) at neutral pH (18). The data presented here areconsistent with a need for subsequent exposure to a low pH,presumably after endocytosis, to complete the fusion process(15, 45, 46). Several important questions remain regarding thisnovel two-step fusion process. First, although it is likely,it is still unclear if a low pH is the true cellular triggerfor the second step of fusion. The strongest evidence of a needfor a low pH is that the continued presence of 10 nM bafilomycin,an inhibitor of the endosomal H⁺-ATPase, blocks the productionof reverse transcripts from incoming ASLV-A particles (Matsuyama,Earp, and White, unpublished). However, in addition to its effecton endosomal acidification, bafilomycin can interfere with membranetrafficking events, such as movement from early to late endosomes(3, 11).

A second important question is why a virus would have evolvedto interrupt its fusion process midstream (i.e., at the lipid-mixingstage). Perhaps ASLV evolved the two-step mechanism to deliverviral cores to a specific location in the target cell, for example,below the cortical cytoskeleton and close to the nucleus (32),as a means to increase the efficiency of reverse transcriptionor subsequent steps of replication. Other receptor-activatedviruses (e.g., HIV) appear to have evolved specific proteins(e.g., Nef) to accomplish this topological task (8, 61). A needfor a low pH for virus-cell fusion may also have evolved asa mechanism to prevent extensive cell-cell fusion of infectedEnvA expressing cells at neutral pH. In the case of murine retroviruses,syncytium formation is inhibited by the presence of an extendedcytoplasmic tail sequence that is proteolytically processedby the viral protease, during viral budding, to activate virus-cellfusion activity (1, 5, 34, 43, 49, 60). Interestingly, the fusionactivity of a paramyxovirus F protein that has a long cytoplasmictail was recently shown to be activated either by experimentaltruncation of the cytoplasmic tail or by exposure to a low pH(55). In both cases, specific sequence motifs within the cytoplasmictail appear to influence the fusion phenotype (34, 56, 60).The cytoplasmic tails of other class I viral fusion proteinsalso seem to modulate late stages of fusion (17, 19, 21, 30,31).

A third important question is whether this two-step fusion processis unique to avian retroviruses or whether it is used by otherviruses. It has recently been shown that two other retroviruses,foamy virus (48) and mouse mammary tumor virus (MMTV) (50),show reduced infectivity in the presence of lysosomotropic agents.The receptor for foamy virus is not known, but the receptorfor MMTV is the transferrin receptor (50), whose traffickingpathway through low-pH endosomes has been extensively characterized.It will be interesting to determine if interaction between transferrinreceptor and MMTV Env, at the cell surface at neutral pH, inducesconformational changes in MMTV Env that are prerequisites forsubsequent low-pH-induced conformational changes that mediatelater stages of fusion.

In conclusion, the results of our study have led to a model(Fig. 7) in which receptor and a low pH act sequentially toform distinct conformations of EnvA that mediate specific stagesof fusion. To our knowledge, this represents the first biochemicalidentification of a series of distinct prehairpin and hairpinconformations for a class I viral fusion protein. The majorsignificances of this study are (i) that it lends support tothe notion that avian retroviruses use a novel two-step fusionprocess (45) and (ii) that it provides a framework for futureinvestigations. With this framework in hand, one can evaluatethe precise mechanistic role of a low pH in ASLV-A fusion andentry. Furthermore, our studies suggest that ASLV-A will bean ideal model virus with which to independently study the molecularmechanisms of early and late stages of fusion. Lastly, one shouldbe able to use similar biochemical approaches to test whetherother viruses use this novel two-step fusion process, i.e.,receptor activation at neutral pH followed by a low-pH completionstep.

ACKNOWLEDGMENTS

This work was funded by an NIH grant (AI22470 to J.M.W.).

We thank J. Gruenke, L. Earp, and especially H. Park for helpwith Fig. 7B and H. Park for many helpful comments on the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Department of Cell Biology, UVA Health System, School of Medicine, P.O. Box 800732, Charlottesville, VA 22908-0732. Phone: (434) 924-2593. Fax: (434) 982-3912. E-mail: jw7g{at}virginia.edu.

Present address: Laboratory of Acute Viral Respiratory Infectionsand Cytokines, Department of Virology, National Institute ofInfectious Diseases, Tokyo 208-0011, Japan.

REFERENCES

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