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°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.The fusion-promoting glycoproteins of orthomyxo-, paramyxo-, filo-, corona-, and retroviruses are grouped as class I fusion proteins because their fusion subunits possess a common postfusion form (12,19,20,57). The structure they have in common consists of a fusion peptide at or near the Nterminal end, a central trimeric coiled coil, a chain reversal segment, a C-terminal region (in many cases a helix) that packs in the grooves of the central coiled coil, a transmembrane domain, and a cytoplasmic tail. In the compact postfusion form, the two hydrophobic elements, the fusion peptide and the transmemb...

Refolding of a paramyxovirus F protein from prefusion to postfusion conformations observed by liposome binding and electron microscopy

“…and Ping Yuan, unpublished observations). For other viral fusion proteins, such as influenza virus HA (35,36), avian sarcoma and leukosis virus Env (37,38), or severe acute respiratory syndrome coronavirus S (39), heat or urea have been shown to act as a surrogate trigger for protein refolding. However, for Semliki forest virus E1 protein and tick-borne encephalitis virus E protein, heat or urea cannot supplant low pH as a trigger (40,41).…”

Cysteines Flanking the Internal Fusion Peptide Are Required for the Avian Sarcoma/Leukosis Virus Glycoprotein To Mediate the Lipid Mixing Stage of Fusion with High Efficiency

“…No fusion was observed when sTva was omitted from the virus-liposome mixture, and a peptide (R99) corresponding to the C-helix region of the EnvA TM subunit, which inhibits fusion and infectivity (6,15), inhibited lipid mixing. Thus, the known conditions for ASLV-A Env (EnvA)-mediated fusion (dependence on preincubation with the receptor and subsequent exposure to a low pH [11,12,14], and inhibition by the C-helix peptide) are observed using this assay.…”

Na + /H + exchanger type 1 is a receptor for pathogenic subgroup J avian leukosis virus

“…A two-step entry mechanism in which Env-receptor interaction renders the viral glycoprotein sensitive to low-pH-induced activation has been proposed for ALV-A and ALV-B (6). Current models propose that, in the first step, the receptor triggers structural rearrangement and insertion of the fusion peptide into the target membrane (21,37,38). Peptide inhibitors bind to this form, suggesting that it is a prehairpin intermediate (21,37).…”

“…Current models propose that, in the first step, the receptor triggers structural rearrangement and insertion of the fusion peptide into the target membrane (21,37,38). Peptide inhibitors bind to this form, suggesting that it is a prehairpin intermediate (21,37). Low pH appears to act later and promote steps leading to membrane fusion, perhaps by driving six-helix bundle formation (21,37,38).…”

“…Peptide inhibitors bind to this form, suggesting that it is a prehairpin intermediate (21,37). Low pH appears to act later and promote steps leading to membrane fusion, perhaps by driving six-helix bundle formation (21,37,38). It will be important to determine whether EnvJ, whose sequence is quite divergent and has only 32% and 55% identity in the SU and TM subunits, respectively, compared with the other ALV Env proteins, utilizes a similar two-step entry mechanism.…”