Receptor-activated binding of viral fusion proteins to target membranes

Abstract

This chapter describes three assays to monitor receptor-induced association of the envelope glycoprotein (EnvA) of avian sarcoma/leukosis virus (ASLV) with target bilayers: (1) the original assay for monitoring binding of the EnvA ectodomain (EnvA-PI) to target membranes (liposomes), (2) a modified and miniaturized EnvA-PI-liposome binding assay, and (3) an assay to measure binding of intact sarcoma/leukosis virus subtype A (ASLV-A) virus particles to target membranes. These assays are also useful for studying other receptor-activated viral fusion proteins. When one viral glycoprotein and one “simple” host cell receptor are involved, it should be possible to develop assays directly analogous to those described above for studying Tva-induced binding of the EnvA ectodomain (EnvA-PI) to target membranes. A general prerequisite for a fusion protein/target membrane binding assay is a soluble and correctly oligomeric form of the viral fusion protein ectodomain. The simplest host cell receptors that would be amenable to this type of analysis are type I or type II integral membrane proteins. The soluble versions of the ectodomains of these receptors, produced by genetic engineering or proteolytic release, could then be used to trigger the cognate fusion protein. The methodology could, similarly, be applicable to multimembrane-spanning host cell receptors when the functional part of the receptor is tethered at only one end or where an ectodomain loop preserves enough structure to function as a soluble analog, perhaps by generating a cyclic peptide analog of the loop. The same “receptor reagents” could be employed for intact virus particle/target membrane binding assays.

Fig. 1

Proposed model for ASLV-A fusion with target membranes. In the native membrane, EnvA exists in a trimeric, metastable form in which the fusion peptides are buried. The SU domains (not shown for clarity) are thought to act as a clamp, which maintains the TM domains in the metastable state. After engaging its receptor, Tva, at T ≥ 22° (step 1), EnvA undergoes a conformational change in which the fusion peptides are exposed and can penetrate the target membrane. After penetration of the target membrane by the fusion peptides (step 2), trimers may cluster (step 3). Further conformational changes occur in which EnvA begins (step 4) to form, and then forms (step 5) a six-helix bundle, which mediates lipid mixing of the outer leaflets of the target and viral membranes. Action of the fusion peptides and transmembrane domains on the hemifusion diaphragm would then open the fusion pore (step 6). This model is based on similar models for the influenza HA. In the case of HA, the trigger for step 1 is exposure to low pH.

Fig. 2

EnvA-PI-liposome binding procedure I. Biotinylated EnvA-PI is incubated with soluble receptor (sTva47) on ice for 15 min. Liposomes are added and samples are shifted to 37° for 10–30 min. Samples are overlaid with a sucrose step gradient, centrifuged, and fractionated as described in Hernandez et al.¹⁵

Fig. 3

EnvA-PI-liposome binding procedure II. (A) EnvA-PI is incubated with sTva47 on ice for 15 min. Liposomes are added, and samples are incubated at 37° for 10 min. Samples are overlaid with sucrose step gradients and centrifuged, fractionated, and subjected to SDS–PAGE, as described in Methods. (B) In the absence of receptor at pH 7 and 37°, EnvA-PI stays in the bottom of the sucrose gradient (top panel). When incubated with receptor at pH 7 and 37°, EnvA-PI floats to the top fractions of the gradient with liposomes (bottom panel).

Fig. 4

Virus–liposome binding procedure. (A) ASLV-A is incubated with or without sTva47 on ice for 15 min. Liposomes are added, and samples are incubated at 37° for 30 min. Samples are placed on top of a sucrose step gradient and centrifuged, fractionated, and processed as described in Methods. (B) In the absence of receptor at pH 7 and 37°, ASLV-A pellets to the bottom of the gradient (top panel). When incubated with receptor at pH 7 and 37°, ASLV-A is largely retained in the top fraction of the gradient (bottom panel).

Fig. 5

Receptor species for use in liposome binding assays. (1) A type I or type II integral membrane protein with a functional domain that is tethered to the membrane at only one end; (2) a multimembrane-spanning protein that possesses a functional domain tethered to the membrane at one end; (3) a multimembrane-spanning protein with an extracellular loop that retains or can be engineered so as to retain enough structure to serve as a functional domain when cleaved from its transmembrane domains. Arrows indicate possible sites at which to cleave the functional receptor domain from the parent protein. Alternatively, secreted functional domains could be produced by genetic engineering or, where applicable, could be mimicked by synthetic peptides.