Glycosylphosphatidylinositol-anchored influenza virus HA induces hemifusion. (Left) Model of the steps of fusion mediated by wild-type HA. (Right) Effect on fusion of an altered form of HA lacking the transmembrane and cytoplasmic domains. Data from Melikyan GB et al. 1995. J Cell Biol 131:679–691.
In contrast to the fusion peptides of class I fusion proteins, fusion loops do not require proteolytic cleavage to be liberated and to be able to insert into membranes. Instead, proteolytic cleavage is required for the conformational change of the second envelope protein, E2 for alphaviruses and prM for flaviviruses (Chapter 13), that shield the fusion loop until the virus particles are delivered in endosomes. Although this cleavage occurs at the Golgi, the process differs for the two virus families. Flavivirus particles bud into the endoplasmic reticulum and are released after passage through the Golgi network, which has a reduced pH. During this process, the E proteins assume the conformation seen on mature particles (Fig. 5.18A). The prM protein is cleaved to pr and M, but the pr fragment continues to shield the fusion loop until the particle is released from the cell, where the pH is neutral. Endocytosis by the target cells returns the virus particles to acid pH, which triggers fusion. On the other hand, alphavirus particles assemble at the plasma membrane and processing of the E2 proteins occurs in the Golgi but prior to their incorporation into particles.
Figure 5.17 SNARE-mediated fusion. The change of syntaxin (t-SNARE, purple) from a closed (not shown) to an open conformation allows SNAP-25 (synaptosome-associated protein of 25 kDa, light and dark orange) and VAMP (vesicle-associated membrane protein, v-SNARE, cyan) helices to “zip up” from their N terminus to their C terminus. The initial “zipping” of the amino-terminal half of SNARE proteins brings the two membranes into nanometer proximity. Completion of assembly of the C termini releases the largest amount of free energy estimated for a protein complex formation and coincides with completion of the fusion process. A number of additional proteins regulate the process (not shown). (PDB ID: 1SCF)
Figure 5.18 Conformational changes in class II proteins during fusion. (A) Ninety dimers of dengue virus envelope glycoprotein E tile the surface of the virus particle. (Inset) Structure of the ectodomains of the dengue protein E dimer is shown at neutral pH (PDB ID: 3J27). Domain I folds into a β-sandwich and is colored orange, domain II cyan/blue, domain III black, and the stem gray. The fusion loop is located at the tip of domain II (red). (B) At low pH, the dimers are disrupted; the proteins extend so that the fusion loop inserts into the target membrane and reorganize into trimers. The glycoprotein then undergoes further conformational rearrangements, folding domain II against domain I, which brings the viral and cell membranes together, allowing fusion. (Inset) Structure of part of the ectodomain of dengue virus E protein at acid pH (based on X-ray crystallographic data; PDB ID: 1OK8), with domains colored as in panel A.
DISCUSSION
Sex and the fusion protein
Gamete adhesion and fusion. Chlamydomonas gamete fusion is used as an example. Cilia on the gamete surface adhere to each other, bringing the two gametes together and activating the formation of mating structures on each gamete. The mating structures bind to each other via cell-specific adhesion proteins, and the HAP2 protein mediates membrane fusion between the two cells. (Inset) Models of the hairpin conformation of HAP2 proteins based on viral class II fusion proteins and structure of Chlamydomonas HAP2 trimer (PDB ID: 6DBS). Monomers are colored gray, yellow, or multiple colors to match Fig. 5.18.
Sex distinguishes eukaryotes from other organisms. Meiotic division produces two haploid germ cells, gametes, that must subsequently meet and merge to produce a cell with a new diploid genome. The processes of “meeting and merging” are poorly understood but are analogous to those of viral surface proteins binding to and fusing with their specific target cells.
Insight into the gamete fusion process came from the identification and subsequent structural studies of proteins from the plants Arabidopsis thaliana and Lilium longiflorum. The protein was named HAP2 (for hapless) because mutant Arabidopsis plants lacking it did not produce fertile male gametes. Orthologues of HAP2 were subsequently discovered in the parasite Plasmodium falciparum, the green alga Chlamydomonas reinhardtii, the invertebrate animals Hydra and Apis (honeybee), and additional eukaryotic species.
The function of these proteins was not obvious, for their primary amino acid sequence did not resemble that of any other protein. However, X-ray crystallography revealed that HAP2 can adopt a trimeric structure that resembles that of class II viral fusion proteins in the fusion active state (see Fig. 5.18). Like the hairpin conformation of the viral fusion proteins, the HAP2 domain II (cyan) that contains the fusion loops (red) is extended toward the target membrane, while domain III (black) is folded against domain I (orange) to bring the membranes of the fusion partners into close proximity.
Several questions remain: What mediates specific attachment of gametes to each other? What is the fusion trigger? What protein fulfills this function in other eukaryotes, including mammals? And how did it arise? Given that HAP2 is ancient, presumably emerging at the same time as sexual reproduction, one possibility is that it was coopted from viruses reproducing in the precursors of sex cells. HAP2 would not be the only example of a viral fusion protein diverted by the host organism. Syncytins, proteins that are critical for the development of the placenta, are related to retroviral class I fusion proteins and were acquired independently in several mammalian lineages. For all the harm that viruses impart on their various host species, it appears that life as we know it might be quite different without them. Sex, for one thing, might be absent.
Fédry J, Liu Y, Péhau-Arnaudet G, Pei J, Li W, Tortorici MA, Traincard F, Meola A, Bricogne G, Grishin NV, Snell WJ, Rey FA, Krey T. 2017. The ancient gamete fusogen HAP2 is a eukaryotic class II fusion protein. Cell 168:904–915.e10.
Feng J, Dong X, Pinello J, Zhang J, Lu C, Iacob RE, Engen JR, Snell WJ, Springer TA. 2018. Fusion surface structure, function, and dynamics of gamete fusogen HAP2. eLife 7:397772.
Class III Fusion Proteins
This class is exemplified by the G protein of the rhabdovirus vesicular stomatitis virus and the gB proteins of herpesviruses. Under most conditions, the vesicular stomatitis virus G protein crystallizes as a trimer. The structural organization, shared by class III fusion proteins, is rather complex, with three domains (I to III) nested around a β-sandwich core and a long C-terminal extension (Fig. 5.19). The transition to the fusion active state consists of rotation of domains I and II and refolding of domain III to form a hairpin, similar to that described for class I fusion proteins, and projects two fusion loops toward the target membrane. In the case of vesicular stomatitis virus