Abstract
The membrane-anchored proteins of enveloped viruses form labile spikes on the virion surface, primed to undergo large-scale conformational changes culminating in virus-cell membrane fusion and viral entry. The prefusion form of these envelope glycoproteins thus represents an important molecular target for antiviral intervention. A critical roadblock to this endeavor has been our inability to produce the prefusion envelope glycoprotein trimer for biochemical and structural analysis. Through our studies of the GPC envelope glycoprotein of the hemorrhagic fever arenaviruses, we have shown that GPC is unique among class I viral fusion proteins in that the mature complex retains a stable signal peptide (SSP) in addition to the conventional receptor-binding and transmembrane fusion subunits. In this report we show that the recombinant GPC precursor can be produced as a discrete native-like trimer and that its proteolytic cleavage generates the mature glycoprotein. Proteoliposomes containing the cleaved GPC mediate pH-dependent membrane fusion, a characteristic feature of arenavirus entry. This reaction is inhibited by arenavirus-specific monoclonal antibodies and small-molecule fusion inhibitors. The in vitro reconstitution of GPC-mediated membrane-fusion activity offers unprecedented opportunities for biochemical and structural studies of arenavirus entry and its inhibition. To our knowledge, this report is the first to demonstrate functional reconstitution of membrane fusion by a viral envelope glycoprotein.
Highlights
Entry of enveloped viruses into their host cells requires fusion of the viral and cellular membranes, a process that is mediated by the viral envelope glycoprotein
ST-294 and ST-761 are specific to New World (NW) arenaviruses and ST-161 and The Scripps Research Institute (TSRI) 8C1 are specific to the Old World (OW) LASV
We showed that the rGPCCD trimer binds the JUNV transferrin receptor 1 (TfR1) receptor [12], JUNV-neutralizing Monoclonal Antibodies (MAbs) [28], and small-molecule arenavirus-specific fusion inhibitors [27]
Summary
Entry of enveloped viruses into their host cells requires fusion of the viral and cellular membranes, a process that is mediated by the viral envelope glycoprotein. Class I viral fusion proteins, including those of influenza and human immunodeficiency virus type 1 (HIV-1), are synthesized as inactive precursor glycoproteins that assemble as trimers and are subsequently primed by proteolytic cleavage to generate the mature fusogenic spikes. X-ray crystallographic analyses of the most extensively characterized class I envelope glycoproteins – influenza virus hemagglutinin (HA), HIV-1 envelope glycoprotein (Env) and parainfluenza virus 5 F (PIV5 F) - are based on soluble ectodomain fragments. These studies necessarily exclude information regarding the important role of membrane anchorage in envelope glycoprotein assembly, maintenance of the prefusion state and activation of fusogenic conformational changes. The failure of current HIV-1 vaccines to elicit broadly neutralizing antibodies is largely attributed to our inability to produce the trimeric native Env immunogen in a prefusion conformation [3]
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