Membrane fusion reactions are common in eukaryotic cells. Membranes are fused intracellularly in processes including endocytosis, organelle formation, inter-organelle traffic, and constitutive and regulated exocytosis. Intercellularly, membrane fusion occurs during sperm-egg fusion and myoblast fusion.
Membrane fusion has been induced artificially by the use of liposomes, in which the cell membrane is fused with the liposomal membrane, and by various chemicals or lipids, which induce cell-cell fusion to produce heterokaryons. Naturally occurring proteins shown to induce fusion of biological membranes are mainly fusion proteins of enveloped viruses.
In liposome-based delivery systems, liposomes are used to encapsulate bioactive molecules inside lipid vesicles for delivery into the cell. However, the polar lipid headgroups oriented on both surfaces of the lipid bilayer, along with an associated water layer, make spontaneous membrane fusion thermodynamically unfavorable.
Various chemicals or lipids have been used to promote membrane fusion. However, these reagents usually exhibit cytotoxic effects (see, for example, Iwamoto et al., in Biol. Pharm. Bull. 19:860-863 (1996) and Mizuguchi et al., in Biochem. Biophys. Res. Commun., 218:402-407 (1996)).
It is generally believed that membrane fusion under physiological conditions is protein-mediated. This has led to the development of liposomes that contain fusion-promoting proteins (proteoliposomes), with decreased cytotoxicity (see, for example, Cheng, Hum. Gene Ther. 7:275-282 (1996); Hara et al., Gene 159:167-174 (1995); and Findeis et al., Trends Biotechnol., 11:202-205 (1993)).
The only proteins conclusively shown to induce intercellular fusion of biological membranes are those of enveloped viruses and two proteins from nonenveloped viruses. All enveloped viruses encode proteins responsible for fusion of the viral envelope with the cell membrane. These viral fusion proteins are essential for infection of susceptible cells. The mechanism of action of fusion proteins from enveloped viruses have served as a paradigm for protein-mediated membrane fusion (see, for example, White, Ann. Rev. Physiol., 52:675-697 (1990); and White, Science, 258:917-924 (1992)).
Most enveloped virus fusion proteins are relatively large, multimeric, type I membrane proteins, as typified by the influenza virus HA protein, a low pH-activated fusion protein, and the Sendai virus F protein, which functions at neutral pH. These are structural proteins of the virus with the majority of the fusion protein oriented on the external surface of the virion to facilitate interactions between the virus particle and the cell membrane.
According to the mechanism of action of fusion proteins from enveloped viruses, fusion of the viral envelope with the cell membrane is mediated by an amphipathic alpha-helical region, referred to as a fusion peptide motif, that is present in the viral fusion protein. This type of fusion peptide motif is typically 17 to 28 residues long, hydrophobic (average hydrophobicity of about 0.6±0.1), and contains a high content of glycine and alanine, typically 36%±7% (White, Annu. Rev. Physiol., 52:675-697 (1990).
All of the enveloped virus fusion proteins are believed to function via extensive conformational changes that, by supplying the energy to overcome the thermodynamic barrier, promote membrane fusion. These conformational changes are frequently mediated by heptad repeat regions that form coiled coil structures (see Skehel and Wiley, Cell, 95:871-874 (1998)). Recognition of the importance of fusion peptide motifs in triggering membrane fusion has resulted in the use of small peptides containing fusion peptide motifs to enhance liposome-cell fusion (see, for example, Muga et al., Biochemistry 33:4444-4448 (1994)).
Enveloped virus, fusion proteins also trigger cell-cell fusion, resulting in the formation of polykaryons (syncytia). Synthesis of the viral fusion protein inside the infected cell results in transport of the fusion protein through the endoplasmic reticulum and Golgi transport system to the cell membrane, an essential step in the assembly and budding of infectious progeny virus particles from the infected cell (Petterson, Curr. Top. Micro. Immunol., 170:67-106 (1991)). The synthesis, transport, and folding of the fusion protein is facilitated by a variety of components, including signal peptides to target the protein to the intracellular transport pathway, glycosylation signals for N-linked carbohydrate addition to the protein, and a transmembrane domain to anchor the protein in the cell membrane. These proteins have been used in reconstituted proteoliposomes (‘virosomes’) for enhanced, protein-mediated liposome-cell fusion in both cell culture and in vivo (see, for example, Ramani et al., FEBS Lett., 404:164-168 (1997); Scheule et al., Am. J. Respir. Cell Mol. Biol., 13:330-343 (1995); and Grimaldi, Res. Virol., 146:289-293 (1995)).
Unlike enveloped viruses, nonenveloped viruses generally do not encode fusion proteins since the absence of a viral membrane precludes membrane fusion-mediated entry. Because progeny virus particles of nonenveloped viruses do not need to acquire a lipid envelope, these viruses usually do not bud from infected cells but, rather, are released by cell lysis. As a result, nonenveloped viruses do not express fusion proteins on the surface of infected cells and, hence, generally do not induce syncytium formation.
Selected members of the family Reoviridae, however, do induce syncytium formation (see Duncan et al., Virology, 212:752-756 (1995), Duncan, Virology, 260:316-328 (1999), and references therein). Reoviridae are a family of nonenveloped viruses containing segmented double-stranded RNA (dsRNA) genomes (see, for example, Nibert et al., Reoviruses and their replication, In: Fundamental Virology, 3rd Edition, B. N. Fields, D. M. Knipe and P. M. Howley (Eds), Lippincott-Raven Press, NY (1996)).
Of the family Reoviridae, the genus Orthoreovirus contains two distinct subgroups typified by the prototypical avian and the mammalian reoviruses (see Duncan, Virology, 260:316-328 (1999)). The avian reoviruses (ARV) are all fusogenic and induce rapid and extensive cell-cell fusion, resulting in syncytium formation in infected cell cultures (see Robertson and Wilcox, Vet. Bull., 56:726-733 (1986)).
Mammalian reoviruses generally are not fusogenic. However, there are at least two exceptions. One was isolated from a flying fox and is named Nelson Bay virus (NBV) (see Gard and Compans, J. Viral., 6:100-106 (1970)). The other was isolated from a baboon and is referred to as Baboon Reovirus (BRV) (see Duncan et al., Virology, 212:752-756 (1995)).
Fusogenic reoviruses have also been isolated from poikilothermic hosts. Two strains of reptilian reoviruses (RRV, a member of the Orthoreovirus genus) that induce syncytium formation have been isolated from snakes (see Ahne et al., Arch. Virol. 94:135-139 (1987), and Vieler et al., Arch. Virol. 138:341-344 (1994)). In addition, members of the genus Aquareovirus (AQV) that infect exclusively piscine (fish) host species, also induce cell-cell fusion (see Samal et al., J. Virol. 64:5235-5240 (1990)). These viruses together represent the few known examples of nonenveloped viruses capable of inducing membrane fusion.
To date, two membrane fusion proteins have been identified and sequenced from nonenveloped viruses: the p10 protein from two strains of ARV and from NBV, and the p15 protein from BRV. The p10 proteins share 33% amino acid identity between ARV and NBV and are clearly homologous proteins. The p15 protein from BRV is not homologous to p10 and appears to belong to a different class. The amino acid sequences of both the p10 and p15 proteins contain fusion peptide motifs (residues 9-24 of ARV and NBV and residues 68-87 of BRV, WO99/24582, published May 20, 1999; the p10 protein is referred to as “p11” in WO99/24582). The fusion peptide motif from the p10 protein (Shmulevitz and Duncan, EMBO J., 19:902-912 (2000)), however, is atypical in that it is much less hydrophobic than is observed in typical fusion motifs from enveloped viruses. The hydrophobicity of the fusion motif from p10 is estimated to be about 0.3 to 0.4, in contrast to the typical values of 0.6±0.1. Nevertheless, the fusion motif from p10 still contains the heptad repeats seen in more typical fusion motifs; i.e. seven-residue sequences in which residues at positions 1 and 4 are apolar; (See FIG. 6 which shows the heptad configuration of p10). It is generally thought that the conserved apolar residues serve to form the hydrophobic face of amphipathic helices which are important for membrane-interactive properties. The presence of heptad repeats in p10 and p15 suggests that these proteins promote membrane fusion by a mechanism similar to that of membrane fusion proteins from enveloped viruses.