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 G protein, the trigger for fusion is acid pH. In contrast, herpesviruses carry multiple envelope proteins on their surface that might contribute to entry in different ways depending on the target cell (Fig. 5.9). It is unclear how conformational changes induced in these different surface proteins upon engagement of their various targets trigger fusion by gB.
Figure 5.19 Conformational changes in class III proteins during fusion. Structure of part of the ectodomain of vesicular stomatitis virus G protein at neutral and acid pH (PDB ID: 5I2S, 5MDM). For simplicity, only one monomer of the trimer is depicted. Domain I is colored green, domain II black, domain III orange, and domain IV (the β-sandwich core) cyan, with the two fusion loops in red. Upon exposure to acid pH, the protein flips to extend the fusion loops toward the target membrane and bring the viral membrane closer. In contrast to other fusion proteins, the conformational changes induced by acid pH in the vesicular stomatitis virus G protein are reversible in solution.
Intracellular Trafficking and Uncoating
Following entry, viral and subviral particles travel within the cell to compartments appropriate for virus genome replication. Transport relies on cellular networks. For enveloped virus particles that fuse at the plasma membrane, the transport cargo is subviral particles, whereas for those that fuse in internal cellular compartments, transport, at least in part, is achieved by processes that move vesicles. At some point during or after transport, uncoating occurs so that the viral genome is released from the viral capsid and can replicate. The genomes of nonenveloped viruses are transferred across the cell membrane by mechanisms different than membrane fusion. For these viruses, the processes of entry and uncoating are tightly linked.
Movement of Viral and Subviral Particles within Cells
Movement of molecules larger than 500 kDa does not occur by passive diffusion, because the cytoplasm is crowded with organelles, high concentrations of proteins, and the cytoskeleton. Rather, viral particles and their components are transported via the cytoskeleton. Such movement can be visualized in live cells by using fluorescently labeled viral proteins (Chapter 2).
The cytoskeleton is a dynamic network of protein filaments that extends throughout the cytoplasm. It is composed of microtubules and actin filaments. Microtubules are organized in a polarized manner, with minus ends situated at the microtubule-organizing center near the nucleus, and plus ends located at the cell periphery (Fig. 5.11). This arrangement permits directed movement of cellular and viral components over long distances. Actin filaments typically assist in virus movement close to the plasma membrane. Techniques to follow the movement of virus particles after entry continue to improve. For example, a combination of technologies such as real-time quantum dots-based single particle tracking with biochemical assays was used to track reovirus particles that enter cells via clathrin-mediated endocytosis. Following internalization, movement of individual particles was slow and dependent on actin, while movement became faster toward the cell interior and dependent on microtubules.
Transport along actin filaments is accomplished by myosin motors, and movement on microtubules is via kinesin and dynein motors. Hydrolysis of adenosine triphosphate (ATP) provides the energy for the motors to move their cargo along cytoskeletal tracks. There are two basic ways for viral or subviral particles to travel within the cell: within a membrane vesicle such as an endosome, which interacts with the cytoskeletal transport machinery; or directly (Fig. 5.11). In the latter case, some form of the virus particle must bind to the transport machinery. After leaving endosomes, the subviral particles derived from adenoviruses and parvoviruses are transported along microtubules to the nucleus. Although adenovirus particles exhibit bidirectional plus- and minus-end-directed microtubule movement, their net movement is toward the nucleus. Adenovirus binding to cells activates two different signal transduction pathways that increase the net velocity of minus-end-directed motility. The signaling pathways are therefore required for efficient delivery of the viral genome to the nucleus. Adenovirus subviral particles are loaded onto microtubules by interaction of the capsid protein, hexon, with dynein. The particles move toward the centrosome and are then released and dock onto nuclear pores, prior to viral genome entry into the nucleus.
A number of different viruses enter the peripheral nervous system and spread to the central nervous system via axons.
