Broad-spectrum antivirals against viral fusion

Abstract

Effective antivirals have been developed against specific viruses, such as HIV, Hepatitis C virus and influenza virus. This 'one bug-one drug' approach to antiviral drug development can be successful, but it may be inadequate for responding to an increasing diversity of viruses that cause significant diseases in humans. The majority of viral pathogens that cause emerging and re-emerging infectious diseases are membrane-enveloped viruses, which require the fusion of viral and cell membranes for virus entry. Therefore, antivirals that target the membrane fusion process represent new paradigms for broad-spectrum antiviral discovery. In this Review, we discuss the mechanisms responsible for the fusion between virus and cell membranes and explore how broad-spectrum antivirals target this process to prevent virus entry.

Figure 1. The fusion process between viral and cellular membranes.

Viral fusion proteins mediate membrane fusion via divergent structures, as shown by the pre- and post-fusion structures of representative class I fusion proteins (haemagglutinin (HA) from Influenza A virus), class II fusion proteins (E protein from Dengue virus) and class III fusion proteins (G protein from Vesicular stomatitis virus). As the fusion intermediates have not been crystallized, purely schematic models consistent with the body of experimental evidence are presented. Free virions harbour one of the three classes of metastable fusion proteins in their pre-fusion conformations. In this state, the fusion peptides (class I) or loops (classes II and III) are buried inside the proteins. Various triggers, such as receptor binding, protease trimming and low pH, induce conformational rearrangements, resulting in the anchoring of the fusion peptides or loops (red triangle at the amino terminus of the fusion protein) in the juxtaposing cellular membrane. Anchoring leads to concurrent formation of complementary amphipathic domains (purple and cyan cylinders) — α-helices in class I proteins and β-sheets in class II proteins — in the pre-hairpin extended intermediates. For simplicity, only one monomer is represented, but the pre-hairpin intermediates are always trimeric. These newly exposed domains are unstable and refold to form more energetically favourable structures. The enthalpy associated with these conformational changes forces mixing of the outer leaflet of the viral membrane with the outer layer of the cellular membrane, resulting in formation of the hemifusion stalk. The inner leaflets of the lipid bilayers then come into contact and begin mixing, opening a pore between viral and cellular membranes as the trimeric structures refold into a highly stable post-fusion conformation. It is likely that the fusion peptides or loops and the transmembrane domains (orange cylinder) interact to some degree to promote the transition from hemifusion stalk to pore formation. Subsequent pore enlargement allows delivery of the viral contents into the target cell cytosol. The bilayer spontaneous curvature (J_S^B) values of the viral and cellular membranes are indicated to highlight the dramatic changes in membrane curvature that occur during the membrane fusion process. The target cell membrane is almost flat (J_S^B ≥ 0), or even negatively curved (J_S^B < 0) when fusion occurs in endosomal membranes, compared with the highly positively curved virion surface (J_S^B >> 0). During membrane fusion, the membrane-bending energetics required to drive the dramatic positive (J_S^B >> 0) to negative (J_S^B << 0) curvature transitions are substantial (Box 2). Adapted with permission from Ref. , Elsevier. PowerPoint slide

Figure 2. Broad-spectrum antivirals targeting fusion proteins.

Small molecules (for example, arbidol) and antiviral peptides (AVPs; most often, α-helical peptides) can interact with the pre-fusion conformations of fusion proteins. These interactions can stabilize or destabilize the fusion proteins, preventing the formation of fusion intermediates. Similarly, inhibitors of enzymes that are specialized in the intramolecular rearrangements of disulfide bonds, such as protein disulfide isomerase (PDI) family of proteins, impair the fine-tuned conformational changes that are required for the subsequent sequence of fusion and thus prevent virus entry. Small fusion inhibitor peptides are AVPs specifically derived from and/or designed to target the hydrophobic domains of fusion proteins. These hydrophobic domains are responsible for the formation of the trimers of hairpins that are necessary to promote progression from the pre-hairpin extended intermediate state to the hemifusion stalk state, and fusion inhibitors therefore impair this progression. PowerPoint slide

Figure 3. Broad-spectrum antivirals targeting viral membranes.

As lipid composition is essential to membrane curvature and fluidity, the removal and addition of lipid species have been evaluated as antiviral strategies. For example, some cationic, amphiphilic antiviral peptides (AVPs) have detergent-like properties at high concentrations and can result in the formation of pores or lead to the micellization of viral membranes. Polyunsaturated endoplasmic reticulum-targeting liposomes (PERLs) have shown potential as broad-spectrum antivirals by depleting cellular and viral membranes of cholesterol; cholesterol depletion reduces the fluidity of the membranes and impairs the negative-curvature transitions that are necessary for the fusion between viral and cellular membranes. Wedge-like or inverted-cone-shaped molecules and some amphiphilic AVPs can increase the spontaneous positive curvature of the viral membrane lipid bilayer, raising the barrier of energy required to power membrane fusion mediated by viral fusion proteins (Box 2). Similarly, membrane-targeting type II photosensitizers generate singlet oxygen within the plane of the viral membrane, and this singlet oxygen oxidizes unsaturated phospholipids and induces changes in the nanoarchitecture of the viral membrane that are not conducive to membrane fusion. The clustering of oxidized phospholipids results in differential lipid packing, reduced fluidity, increased positive curvature, increased area per lipid molecule and reduced membrane thickness. Phospholipid-specific antibodies can target particular phospholipid species that are enriched in some viral membranes (such as phosphatidylserine) and thus block viral attachment and entry. PowerPoint slide