Studies on the roles of viral envelope surface proteins in cell attachment and entry

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Lamboo, Lindsey
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The Ebola virus (EBOV) surface glycoprotein (GP) is a trimeric class I viral fusion protein, which plays a key role in viral attachment and entry. The major cell entry mechanism during EBOV infection involves the endosomal pathway, where GP specifically binds to domain C of NPC1 (a cholesterol transporter) in the endosome, and causes fusion between the viral envelope and the endosomal membrane to allow entry of the viral nucleocapsid. Previously published evidence suggests that proteolytic cleavage of EBOV GP by host cell proteases is required before receptor binding can take place, and that luminal acidic pH may also be required to trigger EBOV GP membrane fusion within the endosome. A variety of previous studies have analyzed the structure of class I fusion proteins in their pre- and post-fusion states by crystallography. An experimental cell-free system was developed here that allows further dynamic time- resolved studies of viral attachment and fusion, specifically, the molecular interactions and intermediate fusion structures that may occur between GP and NPC1, using cryo- electron microscopy. The system utilizes the interaction between expressed EBOV virus-like particles (VLPs) and synthetic liposomes displaying an engineered NPC1-C receptor domain, (that contains the specific GP binding site) on their surfaces. Two forms of non-infectious virus-like particles (VLPs) were produced by expression of the EBOV matrix protein VP40 along with either the full-length GP surface protein or a mucin-like domain deleted version. The conditions necessary to adequately cleave GP in purified VLPs, and thus prime them for receptor binding were determined using commercial thermolysin in lieu of cathepsins. A soluble version of luminal domain C of NPC1 was constructed with a 6-his tag for the purpose of purification of the protein 2 by high performance liquid chromatography and for specific binding of purified NPC1-C to the surface of liposomes that were produced with lipids containing a nickel ion complex. This resulted in NPC1-C receptor-decorated liposomes that could be easily purified from unbound receptor by density gradient centrifugation. The interaction and binding of NPC1-C decorated liposomes with purified VP40 GP VLPs was investigated by cryo-electron microscopy. Specific high-affinity binding of these liposomes to GP was demonstrated only after prior thermolysin cleavage of the version of GP that had the mucin-like domain deleted. It was also shown that there was no binding of full-length GP to NPC1-C, irrespective of prior thermolysin treatment. This provides evidence, consistent with previous structural studies of the partial and intact GP trimer, that access of NPC1 to the receptor-binding domain requires proteolytic removal not only of the mucin-like domain, but some small and as yet undefined portion of the GP1 domain as well. The cell-free system described here will allow future experimental studies of attachment and fusion, and investigation of the roles of pH or other factors, in isolation from complicating cellular and viral factors that might also affect viral entry into cells. In addition, the high-throughput screening of candidate antiviral drugs that target the binding and/or fusion mechanisms of viruses will be possible.
ebola, liposomes, NPC1