analysis of the leucine zippers from several viral transmembrane proteins, including measles virus F and human immunodeficiency virus type 1 TM, demonstrated that this motif was not important for synthesis, transport (and hence oligomerization), and incorporation into the virus particles, but was absolutely required for fusion.
Figure 5.13 Influenza virus entry. The globular heads of HA trimers mediate binding of the virus to sialic acid-containing cell receptors. The virus-receptor complex is endocytosed, and import of H+ ions into the endosome acidifies the interior. Upon acidification, HA undergoes conformational rearrangements that produce a fusogenic protein. The globular heads (gray) are pulled to the side, exposing the fusion peptides (red), and the loop region of native HA (black) becomes a coiled coil, moving the fusion peptides to the top of the molecule near the cell membrane. This structure is referred to as an extended hairpin. At the viral membrane, an α-helix (purple) packs against the trimer core. The coiled-coil bundles, or hairpins, bring the fusion peptides and the transmembrane domains together, moving the cell and viral membranes close so that fusion can occur. To allow release of vRNP into the cytoplasm, the H+ ions in the acidic endosome are pumped into the particle interior by the M2 ion channel. As a result, vRNP is primed to dissociate from M1 after fusion of the viral and endosomal membranes. The released vRNPs are imported into the nucleus through the nuclear pore complex via a nuclear localization signal-dependent mechanism (see “Import of Influenza Virus Ribonucleoprotein” below). Adapted from Carr CM, Kim PS. 1994. Science 266:234–236, with permission.
The leucine zippers of the three fusion protein monomers form a core three-stranded coiled coil. Following the insertion of the fusion peptide into the target membrane, the C-terminal part of each fusion protein monomer folds around this central coiled coil in what is described as a “hairpin” (Fig. 5.12 and 5.13). In some fusion proteins, like the human immunodeficiency virus type 1 TM, these C-terminal regions also assume an α-helical conformation that “zip up” around the central coiled coil, resulting in a six-helical bundle. The overall structure of the hairpin is strikingly similar in different fusion proteins (Fig. 5.14).
Folding of the fusion protein into this hairpin decreases the distance between the viral and cell membranes, thereby permitting fusion (Fig. 5.13). Synthetic peptides corresponding to these regions inhibit fusion by forming hetero-oligomers with α-helices of the viral protein, thereby obstructing the assembly of the viral α-helices around each other. Such peptides directed against the human immunodeficiency virus type 1 TM regions have even made it to the clinic, though they are not broadly used (Volume II, Chapter 8). Similarly, neutralizing antibodies that target these regions in viral transmembrane proteins could hinder formation of this structure sterically and inhibit fusion.
Alternative Fusion Triggers
Receptor-binding-catalyzed fusion. When fusion occurs at the plasma membrane, such as during entry of members of the retrovirus and paramyxovirus families, it is triggered by binding to one or multiple receptors. In the case of human immunodeficiency virus type 1, like that of influenza virus, binding and fusion are mediated by two subunits of the same surface glycoprotein, SU and TM (Fig. 5.8). Binding of SU to its primary receptor, CD4, induces conformational changes in this subunit that allow it to bind to a coreceptor like CCR5. This binding in turn induces further conformational changes so that the surface subunit no longer conceals the fusion peptide, which can now insert into the target cell membrane (Fig. 5.15). Although fusion activation by other retroviruses relies on binding to a single receptor, their fusion triggers can be quite unusual (Box. 5.2).
Figure 5.14 Conservation of the hairpin structure in class I viral fusion proteins. View from the top and side of the hairpin structures formed by viral fusion proteins following the fusion trigger. The structure shown for HA is the low-pH, or fusogenic, form (see Fig. 5.12). The structure of simian virus 5 F protein is of peptides from the N- and C-terminal heptad repeats. Structures of retroviral TM proteins are derived from interacting N- and C-terminal helices of human immunodeficiency virus type 1 peptides and a peptide from Moloney murine leukemia virus and are presumed to represent the fusogenic forms because of structural similarity to HA. In all three molecules, fusion peptides would be located at the membrane-distal portion (the tops of the molecules in the bottom view). All present fusion peptides to cells on top of a central, three-stranded coiled coil while the C-terminal structures fold to bring the viral membrane close to the target membrane. Data from Baker KA et al. 1999. Mol Cell 3:309–319.
Figure 5.15 Fusion at the plasma membrane. (Top) Model for human immunodeficiency virus type 1 (HIV-1) fusion. Binding of SU to CD4 exposes a high-affinity chemokine receptor-binding site on SU. The SU-chemokine receptor interaction leads to conformational changes in TM that expose the fusion peptide and permit it to insert into the cell membrane, catalyzing fusion in a manner similar to that of influenza virus. For simplicity, all envelope glycoproteins are shown as monomers. (Bottom) Model for paramyxovirus membrane fusion. The attachment protein maintains the fusion protein (F) subunits at a metastable conformation. Binding of the attachment protein to the cell receptor induces conformational changes that in turn induce conformational changes in the F protein, moving the fusion peptide from a buried position to insert in the cell membrane.
In contrast, the functions of receptor binding and fusion are separated into two different surface glycoproteins in the case of paramyxoviruses. The attachment protein can possess both hemagglutinin and neuraminidase (HN), only hemagglutinin (H), or neither (G) activities. Despite the differences in activities and receptor binding, all attachment proteins share a similar structure of an N-terminal transmembrane domain, a stalk and a globular head (similar to influenza virus HA proteins), and form tetramers (in contrast to the HA trimers). Binding of the attachment proteins to receptors on the cell surface brings the viral and cellular membranes into close proximity and triggers the type I integral membrane (F) protein to mediate fusion (Fig. 5.15). Like influenza HA proteins, the active F proteins are homotrimers of two disulfide-linked subunits (F2-F1), produced following cleavage of a protein precursor by proteases in the infected cell. The F1 subunit contains the fusion peptide. In contrast to influenza virus HA1 and the human immunodeficiency virus type 1 SU, the F2 subunit is small and probably incapable of effectively shielding the fusion peptide and maintaining F1 in a metastable conformation. Therefore, is it likely that the virus attachment proteins contribute to these functions. It has been proposed that binding of attachment proteins to cell surface receptors induces conformational changes that are transmitted to the F protein, perhaps via direct protein-protein interaction, ultimately resulting in the exposure of the fusion peptide. This mechanism is supported by findings that not only do F proteins from certain paramyxoviruses, like human parainfluenza virus 3 and Newcastle disease virus, require both HN and F proteins to mediate fusion, but
