Sequential roles of receptor binding and low pH in forming prehairpin and hairpin conformations of a retroviral envelope glycoprotein

Abstract

A general model has been proposed for the fusion mechanisms of class I viral fusion proteins. According to this model a metastable trimer, anchored in the viral membrane through its transmembrane domain, transits to a trimeric prehairpin intermediate, anchored at its opposite end in the target membrane through its fusion peptide. A subsequent refolding event creates a trimer of hairpins (often termed a six-helix bundle) in which the previously well-separated transmembrane domain and fusion peptide (and their attached membranes) are brought together, thereby driving membrane fusion. While there is ample biochemical and structural information on the trimer-of-hairpins conformation of class I viral fusion proteins, less is known about intermediate states between native metastable trimers and the final trimer of hairpins. In this study we analyzed conformational states of the transmembrane subunit (TM), the fusion subunit, of the Env glycoprotein of the subtype A avian sarcoma and leukosis virus (ASLV-A). By analyzing forms of EnvA TM on mildly denaturing sodium dodecyl sulfate gels we identified five conformational states of EnvA TM. Following interaction of virions with a soluble form of the ASLV-A receptor at 37 degrees C, the metastable form of EnvA TM (which migrates at 37 kDa) transits to a 70-kDa and then to a 150-kDa species. Following subsequent exposure to a low pH (or an elevated temperature or the fusion promoting agent chlorpromazine), an additional set of bands at >150 kDa, and then a final band at 100 kDa, forms. Both an EnvA C-helix peptide (which inhibits virus fusion and infectivity) and the fusion-inhibitory agent lysophosphatidylcholine inhibit the formation of the >150- and 100-kDa bands. Our data are consistent with the 70- and 150-kDa bands representing precursor and fully formed prehairpin conformations of EnvA TM. Our data are also consistent with the >150-kDa bands representing higher-order oligomers of EnvA TM and with the 100-kDa band representing the fully formed six-helix bundle. In addition to resolving fusion-relevant conformational intermediates of EnvA TM, our data are compatible with a model in which the EnvA protein is activated by its receptor (at neutral pH and a temperature greater than or equal to room temperature) to form prehairpin conformations of EnvA TM, and in which subsequent exposure to a low pH is required to stabilize the final six-helix bundle, which drives a later stage of fusion.

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.

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.

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.

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.

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.

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.

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.