Membrane-induced conformational change during the activation of HIV-1 gp41¹

Abstract

The human immunodeficiency virus type 1 gp41 ectodomain forms a three-hairpin protease-resistant core in the absence of membranes, namely, the putative gp41 fusion-active state. Here, we show that recombinant proteins corresponding to the ectodomain of gp41, but lacking the fusion peptide, bind membranes and consequently undergo a major conformational change. As a result, the protease-resistant core becomes susceptible to proteolytic digestion. Accordingly, synthetic peptides corresponding to the segments that construct this core bind the membrane. It is remarkable that the hetero-oligomer formed by these peptides dissociates upon binding to the membrane. These results are consistent with a model in which, after the three-hairpin conformation is formed, membrane binding induces opening of the gp41 core complex. We speculate that binding of the segments that constructed the core to the viral and cellular membranes could bring the membranes closer together and facilitate their merging.

Introduction

The human immunodeficienicy virus type 1’s (HIV-1) envelope glycoprotein plays a critical role in the virus life cycle, in particular, when HIV-1 enters its host cell. After being synthesized on the rough endoplasmic reticulum (ER), the envelope glycoprotein precursor (gp160) inserts into the lumen of the ER and oligomerizes Earl et al 1990, Earl et al 1991, Otteken et al 1996. The protein is transported subsequently to the Golgi complex, and cleaved by a cellular furin protease into the gp120 and gp41 subunits, which are then transported to the plasma membrane Earl et al 1991, Hallenberger et al 1992, Stein and Engleman 1990.

The first step in HIV-1 infection involves the binding of the viral envelope glycoproteins gp120-gp41 to CD4 Dalgleish et al 1984, Maddon et al 1986, McDougal et al 1986 and subsequently to a coreceptor Alkhatib et al 1996, Doranz et al 1999, Feng et al 1996, Jones et al 1998, Salzwedel et al 2000. As a consequence, gp41 undergoes conformational changes that mediate the fusion between the viral and the cellular membranes or between infected and healthy cells Kowalski et al 1987, Veronese et al 1985. Gallaher and co-workers postulated a model of gp41, identifying a leucine/isoleucine zipper-like sequence (N helix) and an amphipathic helical segment (C helix) in the viral glycoprotein (Gallaher et al., 1989). The gp41 was found to contain a protease-resistant core consisting of these two segments (Lu et al., 1995). Specifically, peptides corresponding to these sequences co-crystallized as a six-helix bundle in which the N and C helices are arranged in a three-hairpin structure Chan et al 1997, Tan et al 1997, Weissenhorn et al 1997. Three N peptides form a coiled-coil, and the C peptides are packed in an anti-parallel manner into hydrophobic grooves on the surface of the coiled-coil. Recently, the solution and crystal structures of the ectodomain of the simian immunodeficiency virus gp41, consisting of those two helices as well as the loop connecting them, confirmed the interplay of the N and C helices Caffrey et al 1998, Yang et al 1999.

A key feature of most viral envelope glycoproteins is the fusion peptide, a stretch of hydrophobic amino acid residues believed to trigger the fusion process. It was proposed that merging of the membranes is initiated by the insertion of the fusion peptide into either the target membrane Harter et al 1989, Pak et al 1994, Stegmann et al 1989, Tsurudome et al 1992, White 1992, the viral membrane Ruigrok et al 1988, Wharton et al 1988, or both Guy et al 1992, Hughson 1995, Stegmann et al 1995. The functional role of the fusion peptide is to bind to and dehydrate the outer bilayer at a localized site, thus reducing the energy barrier for the formation of highly curved, lipidic “stalk” intermediates (reviewed by Durell et al., 1997). In addition to the fusion peptide, other regions in viral glycoproteins were thought to be involved in the membrane fusion event. These include the leucine/isoleucine zipper sequences of Influenza HA (Yu et al., 1994), Sendai F1 (Ghosh & Shai, 1999), and HIV-1 gp41 (Rabenstein & Shin, 1995), and the major immuno-dominant region of gp41 (Santos et al., 1998). However, this is inconsistent with the results of a hydrophobic photolabeling experiment, which suggests that the fusion peptide of influenza virus is the only segment in the ectodomain of the fusion glycoprotein that binds to the membrane (Durrer et al., 1996). Nevertheless, it is important to note that the labeling reagent was attached at carbon 10 of the phospholipid acyl chain as described by Weber & Brunner (1995). The reagent was located deep in the hydrophobic milieu of the membrane, and therefore could not detect the binding of protein segments to the surface of the membrane. Using several biophysical techniques, we found recently that Sendai virus fusion protein comprises two heptad repeats that bind to the surface of the membrane, near the water-membrane interface, rather than penetrating into the hydrophobic milieu of the membrane as the fusion peptide does. These findings may resolve the apparent contradiction regarding the lack of photolabeling of certain regions of the viral glycoprotein and their ability to interact with membranes (Ben-Efraim et al., 1999). In accordance, Epand, Shin and co-workers have shown that a construct corresponding to the ectodomain of influenza hemagglutinin, which lacks the fusion peptide, can induce vesicle aggregation in a pH-dependent manner and is weakly fusogenic (Epand et al., 1999). Furthermore, they showed that the loop region may play a pH-dependent regulatory role (LeDuc et al., 2000). In this study, we used both recombinant proteins and synthetic peptides corresponding to fragments of gp41 (see Figure 1) in a spectroscopic study attempting to determine whether regions in HIV-1 gp41, other than the fusion peptide, may have a direct role in mediating membrane fusion. Our results support the presence of a membrane-induced step in the activation of gp41.