CA2429891A1 - Membrane fusion protein - Google Patents

Abstract

A family of membrane fusion proteins and polynucleotides encoding the proteins have been identified.
The proteins and polynucleotides are derived from the family Reoviridae. Two membrane fusion proteins have been isolated from reoviruses isolated from poikilothermic hosts: the p14 protein from reptilian reovirus (RRV) isolated from python, and the p22 protein from aquareovirus (AQV) isolated from salmon. The genes encoding these proteins have been cloned and sequenced. Analysis of the amino acid sequences of these proteins show that both lack the typical fusion peptide motif found in other membrane fusion proteins.
Expression of these proteins in cells results in cell-cell fusion.

Description

Membrane Fusion Proteins Derived from Reovirus FIELD OF THE INVENTION
The present invention relates to a novel class of proteins that promote membrane fusion. The proteins and the polynucleotides encoding them are derived from the family Reoviridae.
BACKGROUND OF THE INVENTION:
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 w:h:ich 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.
20~ In :Liposome-based delivery systems, liposomes are used to encapsulate b.i.oactive 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 3U 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 ~'indeis 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, multimE=ric, type I membrane proteins, as typified by the influenza virus HA protein, a Iow pH-activated fusion protein, and the Senda:i 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

2;i 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 erxv~eloped virus fusion proteins are believed to function va_a. 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 Sh;e:hel 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 33:4444-4119:8 (1994) ) .
Enveloped virus fusion proteins also trigger cell-cell fusion, resulting ~_n the formation of polykaryons (syncytia). Synthesis of the viral fusion protein inside the infected cell results :i.n 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 Mal. Biol., 13:330-343 (1995); and Grimaldi, Res. Virol., 146:289-293 (1995)).

3 Unlike enveloped viruses, nonenveloped viruses generally do not encode fusion proteins since the absence of a viral membrane preclude; 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 th.e surfacE~ of infected cells and, hence, generally do not induce syncytiurn formation.
Selected members of the family Reoviridae, however, do induce syncytium formation (see Duncan et al., Virology, 212:752-756 (1995), Dunc:an, Virology, 260:316-328 (1999), and references therein). Re~aviridae are a family of nonenveloped viruses containing segmented double-stranded RNA (dsRNA) genomes (see, for examp7_e, Nibert et al., Reoviruses and their replication, I.n: 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 (7.999)). 'The avian reoviruses (ARV) are all fusogenic and induce rapid and extensive cell-cell fusion, resulting in ~~yncytium :Formation in infected cell cultures (see Robertson and Wilcox, Vet. Bull., 56:726-733 (1986)).
Mammalian reo~Jiruses generally are not fusogenic.
However, there: are at least two exceptions. One was isolated from a flying fox and i;s named Nelson Bay virus (NBV) (see Gard and Compans, :T. Virol., 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)).

4 Fusogenic reoviruses have also been isolated from poikilothermic hosts. Two strains of reptilian reoviruses (RRV, a member of the C~rthoreovirus genus) that induce syncytium formation have been isolated from snakes (see Ahne et al., Arch. Virol. 94:1x5-139 (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 pl0 protein from t:wo strai.n;s of ARV and from NBV, and the p15 1~ 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 pl0 and p15 proteins contain fusion peptide motifs (residues 9-24 of ARV and NBV and residues 68-87 of BRV, W099/24582, published May 20, 1999; the p10 protein is referred to as "p11~~ in W099/24582). 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 2:5 hydrophobic than is observed in typical fusion motifs from enveloped viruses. The hydrophobicity of the fusion motif from pl0 is estimated to be about 0.3 to 0.4, in contrast to the typical values of 0.6 1 0.1. Nevertheless, the fusion motif from p10 still contains the heptad repeats seen in more typical fusion motifs; i.e. se',ren-residue sequences in which residues at positions 1 and 4 are apolar; (See Figure 6 which shows the heptad configuration o:f p10). It is generally thought that the conserved apalar residues serve to form the hydrophobic face of

5 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.
SUMMARY OF THE INVENTIONf According to the present invention, a new class of membrane fusion proteins is described which is derived from Reoviridae and. whose amino acid sequence is free of fusion peptide motifs.
In one aspect, members of this class are membrane fusion proteins which are encoded by the genome of the family Reoviridae, which comprise at least one transmembrane domain, and whose amino acid sequence is free of any fusion peptide motifs. A fusion peptide motif (type I) is an amino acid sequence typically 17 to 28 residues long, with a hydrophobicit~r of about 0.6 to 0.'~, and whose content of alanine plus glycine is about 29 to 43%. A fusion peptide motif (type II) is 16 to 20 residues long, has a hydrophobicity 2() value about 0.3 to 0.4, has an alanine plus glycine content of about 29 to 43%; and contains a heptad repeat.
In another aspect, the proteins of the invention are membrane fusion proteins whose amino acid sequences are free of any fusion peptide motifs, as defined above, and which are 2~ related to Reoviridae i.n that they comprise an amino acid sequence which has at least 33% overall identity to the membrane fusion protein encoded by Reoviridae, and comprise a transmembrane domain whose amino acid sequence has at least 60%
amino acid sequence identity to the transmembrane domain of the 30 membrane fusion protein encoded by Reovixidae.

6 In one embodiment, the proteins of the invention are encoded by, or related to, the genome of Orthoreovirus or Aquareovirus, or the genome of a reovirus which naturally infects a poikilothermic host. Specifically, in one embodiment, thE: proteins of the invention are proteins comprising an amino acid sequence of SEQ ID N0:2 or SEQ ID
N0:6. In another embodiment, the proteins of the invention are membrane fusion proteins whose amino acid sequences are free of any fusion peptide motif, as defined above, and which are related to Orthoreovirus or Aquareovirus in that they comprise an amino acid sequence which has at least 33% overall identity to the membrane fusion protein encoded by Orthoreovirus or Aquareovirus, and comprise a transmembrane domain whose amino acid sequence has at lE::ast 60% amino acid sequence identity to the transmembrane domain of the membrane fusion protein encoded by Orthoreovirus or Aquareovirus.
In another embodiment, the proteins of the invention are membrane fusion proteins whose amino acid sequence is free of a fusion peptide motif as defined above, which comprise an amino acid sequence which has at least 33% identity overall to SEQ ID N0:2, and which comprises a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to th.e sequence from residue 3~ to 57 of SEQ ID N0:2.
In another embodiment, the proteins of the invention are membrane fusion proteins whose amino acid sequence is free of a fusion peptide motif as defined above, which comprise an amino acid sequence which has at least 33% identity overall to SEQ ID N0:6, and which comprises a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to the predicted transmembrane sequence 5' LAICCCCCICCTGGLYLV 3' of SEQ ID N0:6,

7 In another embodiment, the amino acid sequences encoded by, or related to, the genome of Reoviridae, comprise neutral or basic proteins, i.e. they have an isoelectric point equal to or greater than 7.
In another embodiment, the proteins of the invention further comprises a positive cluster which consists of at least three positive:Ly charged amino acid residues within a contiguous sequence of at most 25 residues, wherein the contiguous sequence is within at most 100 residues flanking the transmembrane domain at. the C-terminal side.
In another embodiment, the proteins of the invention, particularly those related to p14, further comprise a non-transmembrane domain which comprises a polyproline motif comprising at least 3 contiguous proline residues.
In another er~~odiment, the proteins of the invention further comprise a fatty acylation sequence such as a myristylation consensus sequence.
Another aspect of the invention provides an isolated polynucleotide encoding the proteins of the invention, specifically an isolated polynucleotide comprising SEQ ID NO:1 or SEQ ID N0:5, or degenerate variations thereof which encode the same amino acid sequences, or splice variant nucleotide sequences thereof. The isolated polynucleotide of the invention are expressed by being operatively associated with a promoter, which may be .an inducible promoter.
Another aspect of the invention provides a cell which contains the proteins or polynucleotides of the invention.
Another aspect of the invention provides a liposome which contains the proteins or polynucleotides of the invention. I:n an embodiment where liposomes are used to

8 deliver a bioac:tive drug to a cell, the Iiposomes or proteoliposomes contain both the protein of the invention and the bioactive drug.
Another aspect of the invention provides an antibody reactive against the protein of the invention.
Another aspect of the invention provides a method to promote fusion between two or more membranes, comprising contacting the membranes to be fused with an effective amount of the protein of the invention. In one embodiment, the membranes are cell membranes, liposome membranes or proteoliposome membranes, depending on whether cell-cell, Iiposome-liposome, or cell-liposome fusion is required.
In one embodiment, fusion between cells is effected by expressing the membrane fusion proteins of the invention in the cells) which are to undergo fusion.
In another embodiment, the fusion method of the invention is used to produce heterokaryons, particularly hybridoma cells for the production of monoclonal antibodies, cytokines, or immune modulators. Where the hybridoma cells produce monoclonal antibodies, the membranes are from an immortalized cell and axe antibody-synthesizing cell.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 presents the nucleotide sequence of the polycistronic AQV genome segment 7 (SEQ ID N0:5), and the predicted amino acid sequences of the 5'-proximal ORF encoding the p22 fusion protein (SEQ ID N0:6). The 3'-proximal ORF
encodes the unrelated nonstructural virus protein NS28 (SEQ ID
N0:8).
Figure 2 presents the amino acid sequence of the AQV
;i0 p22 fusion protein (SEQ ID N0:6). The predicted transmembrane

9 domain$ is overlined and labeled. The clusters of positively charged amino acids adrjacent to the transmembrane domains are labeled with a + symbol..
Figure 3 is a schematic depiction of the apolar amino acids within t:he fusion peptide motifs of the p10 proteins from ARV and NBV. The heptad repeats are thought to constitute the hydrophobic faces of amphipathic helices, which are thought to be important f:or interaction of this type of membrane fusion protein with t:he membrane.
DETAILED DESCRIPTION
(A) Proteins A c7.ass of proteins, exemplified by the p14 protein from RRV and t:he p22 protein from AQV, have been identified.
The proteins have membrane fusion properties. Accordingly, this class of proteins, including p14, p22 and related polypeptides, can be used to promote membrane fusion.
In one aspect of the invention, proteins are provided which are encoded by they genome of Reoviridae, and whose amino acid sequence is free of fusion peptide motifs.
The family Rec~viridae includes the genus Orthoreovirus, which includes avian, mammalian and reptilian reoviruses, as well as i~he genus Aquareovirus.
Fus~.on peptide motifs, as defined in the present invention, arE: of two c:Lasses: type T and type II. Type I is found typically in fusion proteins from enveloped proteins.
Motif type I consists o:E an amino acid sequence 16 to 36 residues long,. preferab:Ly 17 to 28 residues long, with a hydrophobicity of about 0.5 to 0.8, preferably about 0.6 to 0.7, and has a content of alanine plus glycine of about 24 to 30~ 44%, preferably 29 to 4:3%.

Motif type II is an atypical motif. It is found in the type of fusion protc=_in exemplified by the p10 protein of ARV and NBV. It consisi~s of an amino acid sequence 15 to 20 residues long, preferab:Ly 16 to 18 residues long, with a 5~ hydrophobicity value about 0.2 to 0.4, preferably about 0.3 to 0.4, has an alanine plus glycine content of about 24 to 44%, preferably about 29 to ~43%; and contains at least one heptad repeat.
In accordance with the present invention, a heptad 1C~ repeat is a seven-residue sequence in which residues at positions 1 and 4 are apolar. The conserved apolar first and fourth residues are thovught to form part of the hydrophobic face of amphipathic helices; thus, in proteins containing them, they are like:Ly to be an essential element for interaction 1E~ between the fusion protein and the membrane.
Hydrophobicity values are determined according to methods known in the art. According to the present invention, the values are calculated using the normalized hydrophobicity scale of Eisenberg. (7.984). Ann. Rev. Biochem. 53:595-623.
2C1 By °'polypeptide" or "protein" is meant any chain of amino acids, .regardless of length or post-translational modification (e. g., glycosylation or phosphorylation). Both terms are used interchangeably in the present application.
In another aspect of the invention, proteins are 2'i provided which are membrane fusion proteins whose amino acid sequences are free of any fusion peptide motifs, as defined above, and which are related to Reoviridae in that they comprise an amina acid. sequence which has at least 33% overall identity with the membrane fusion protein encoded by 3U Reoviridae. 'The proteins also comprise a transmembrane domain whose amino acid sequence has at .Least 60% amino acid sequence identity to the transmembrane domain of the membrane fusion protein encoded by Reovi:ridae. The overall percent identity is also contemplated at at. least 40%, 45%, 50%, 60%,and 75%. The percent identity with t;he transmembrane domain is also contemplated at at least 70%, 75%, 80%, 85%, 90% and 95%.
In one aspect., proteins of the invention are related to Reoviridae in that t~l-.ie proteins comprise an amino acid sequence which has at ?e:ast 33% overall identity with the membrane fusion proteizl encoded by Reoviridae. The 33% overall identity is based on thE: degree of identity between the p10 proteins of ARV (strains. 176 and 138) and of NBV.
As a class, it. is clear that the proteins of the invention tolerate a grE:at deal of sequence variation while retaining their membrane fusion properties. The membrane fusion proteins derived from Reoviridae are thus related by function and by overal:~ structure, rather than by strict overall identity. The ~>roteins are functionally defined in that they have membrane fusion properties which can be readily determined using a simple test. One such test is that exemplified in Examples 4 and 8, where the protein which is to be tested for membrane fusion activity is expressed in cells and the cells examined under the microscope for syncytium formation.
The proteins of the invention are structurally defined in that they are: either encoded by Reoviridae, or are related to Reoviridae fusion proteins mainly at the transmembrane domain. Transmembrane domains are readily recognized in the art. A typical transmembrane domain is described in White, AriIlla. Rev. Physiol., 52:675-697 (1990), as a contiguous sequence of: amino acids averaging 29 residues, with average hydrophobic:ity of 0.7~0.09 and an alanine +
glycine content of 16%~8%.

In one embodiment, the proteins of the invention are encoded by, or related to, the genome of Orthoreovirus or Aquareovirus or the gexlc~rne of a reovirus which naturally infects a poikilothermic (cold-blooded) host. Orthoreovirus includes the avian and mammalian reoviruses. Aquareovirus are reoviruses which naturally infect fish. Poikilothermic hosts include reptiles such a~; snakes, pythons and fish.
Specifically, one embodiment is directed to proteins comprising an amino acid sequence of SEQ ID N0:2 or SEQ ID
N0:6. In another embodiment, the proteins of the invention are membrane fusion proteicm> whose amino acid sequences are free of any fusion peptide motif:, as defined above, and which are related to Orthoreoviru~~ or Aquareovirus in that they comprise an amino acid sequence which has at least 33% overall identity to the membrane fusion protein encoded by Orthoreovirus or Aquareovirus, and comprise a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to the transmembrane domain of the membrane fusion protein encoded by Orthoreovirus or Aquareovirus. The overall percent identity is also contemplated at at least 40%, 45%, 50%, 60%,and 75%.
The percent identity w:it:h the transmembrane domain is contemplated also at at least 70%, 75%, 80%, 85%, 90% and 95%.
The precise 1~T--terminus of p22 (SEQ ID N0:6) has not been determined. HowevE:r, the N-terminal residue has been shown to be located within the first. Z7 residues of SEQ ID
N0:6. Although precise identification of the initiator codon is not essential to the present invention, the initiator can be readily determined using methods known in the art. For example, a stop codon can be inserted systematically into the first 17 codons, as exp:Lained in Example 5. Alternatively, p22 can be isolated from ce=Lls in which it is expressed, and its N-terminal region micro-sequenced.

In another emx>odiment, the membrane fusion proteins of the invention are neutral or basic; i.e. their isoelectric point (pI) is equal to or greater than 7, preferably greater than 8, and more preferably greater than 9. The pI of a protein can be readily determined using standard methods known in the art.
In another embodiment, the proteins of the invention further comprise a positive cluster which consists of at least three, preferably four, more preferably six, most preferably seven, positively chargE:d amino acid residues within a contiguous sequence of at most 25, preferably 23, more preferably 21, and most preferably 20 residues, wherein the contiguous sequence is within at most 100, preferably at most 90, and more ~>referably at most 80, residues flanking the transmembrane domain at the C-terminal side.
In another embodiment, the proteins of the invention further comprise a non-t:ransmembrane domain which comprises a polyproline motif comprising at least 3, preferably 4, and more preferably 5 contiguous proline residues. In a specific embodiment, the fusion ~?rotein of the invention which comprises the polyproline motif is related to the p14 protein.
In another emlaodiment, the proteins of the invention further comprise a fatty acylation sequence, preferably a myristylation consensus sequence which is: (initiator Met 2C~ removed) Glyl-AA2-AA3-AA.~°AASwAA6-'AA7-Vie- , where ~a,~a,~a.~s~~s are small uncharged residues, AA3 and AA4 are preferably neutral, where AAS is preferably serine or threonine, where AA6 is not proline, and where AA7 and AA8 are preferably basic (Towler et al. Annu. Rev. Biochem. 57:69-99 (1988); Resh.
Biochim. Biophys. Acta. 1451:1-16 (1999)). In a specific embodiment, the fusion ;protein of the invention which comprises the fatty acy:Lation sequence is related to the p14 protein.

As described herein, the invention also encompasses substantially purified proteins. A "substantially purified protein" as u:~ed herein is defined as a protein that is separated from the environment in which it naturally occurs 5~ and/or that is free of the majority of the proteins that are present in the environr~ient in which it was synthesized. For example, a substantially purified protein is free from cytoplasmic proteins. Those skilled in the art would readily understand that the proteins of the invention may be purified from a natural source, i.e., a Reoviridae-infected cell, or produced by recombinant means.
Useful derivatives of the membrane fusion proteins of the invention,, e.g., fusion-promoting fragments, may be designed using computer-assisted analysis of amino acid sequences to identify probable transmembrane regions, or regions of high positive charge. Parameters such as helix propensity, heptad repeats and average hydrophobicity can reveal potential hydraphobi.c, membrane-interacting fragments which may be used as a basis for selecting useful fusion-2Q promoting fragments and variants.
One aspect of the invention provides sequences that are identical or substantially identical to SEQ ID NOs: 2, 4, 6 and 8. By "amino acid sequence substantially identical" is meant a sequence that is at least 80%, preferably 90%, more 2~a preferably 95% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference by a majority of conservative amino acid substitutions.
Conservative amino acid substitutions are substitutions among amino acids of the same class. These 31) classes include, for example, amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; amino acids having basic side chains, such as lysine, arginine, and histidine; amino acids having acidic side chains, such as aspartic acid and glutamic acid;
and amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleuci.ne, proline, phenylalanine, methionine, tryptophan, and cysteine.
Percent identity is measured using sequence analysis software such as the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705. Amino acid 1C~ sequences are aligned to maximize identity. Gaps may be artificially introduced into the sequence to attain proper alignment. OIlCe the optimal alignment has been set up, the degree of identity is established by recording all of the positions in which the amino acids of both sequences are 1:i identical, relative to the total number of positions.
Proteins having a sequence homologous to SEQ ID Nos:
2, 4, 6 or 8 :include naturally-occurring allelic variants, as well as mutants ar any other non-naturally occurring variants that retain the membrane fusion properties of the polypeptides 2C) of SEQ ID Nos: 2, 4, 6 or 8. As is known in the art, an allelic variant is an alternate form of a polypeptide that is characterized as having a substitution, deletion, or addition of one or more amino acids that retain the biological function of the polypeptide, i.e. the membrane-fusion activity.
2;i Homologs and fragments thereof that do not occur naturally are designed using known methods for identifying regions of the protein that are likely to tolerate amino acid sequence changes and/or deletions. As an example, homologous polypeptides from different species are compared; conserved 3U sequences are identified. The more divergent sequences are the most likely to tolerate sequence changes. Percent identity and similarity among sequences may be analyzed using, as an example, the BLAST homology searching algorithm of Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Alternatively, a particular amino acid residue or sequence within the polypeptide can be mutai~ed in vitro, then the mutant ~~ polypeptides :>creened for their agility to promote membrane fusion.
A skilled person will understand that by following the screening process of this invention, it will be determined without undue experimentation whether a particular protein encoded by Reoviridae, or related to a protein encoded by Reoviridae, has membrane fusion activity. The screening procedure comprises the steps:
(i) Introducing the test protein into a cell susceptible to syncytia formation, either by delivering the protein or by expressing the protein from a polynucleotide encoding it; and (ii) Observing whether membrane fusion has occurred by determining whether syncytia have formed, compared to a negative control where an irrelevant protein is introduced into the cell.
(B) Antibodies One aspect of the invention provides antibodies, both polyclonal and monoclonal. Such antibodies can be employed for diagnostic applications, therapeutic applications, and the like. Preferably, for therapeutic applications, the antibodies employed will. be monoclonal antibodies. Antibodies may be recombinant, e.g., chimeric (e. g., constituted by a variable region of murine origin associated with a human constant region), humanized (a human immunoglobulin constant backbone together witr4 hypervariable region of animal, e.g., murine, origin), and~'or single chain. Both polyclonal and monospecific antibodies may also be in the form of immunoglobulin fragments, e.g., F(ab)~2 or Fab fragments. The antibodies of the invention are of any isotype, e.g., 2gG or IgA, and polyclonal antibodies are of a single isotype or a mixture of isotypes.
Antibodies against the proteins or fragments of the invention are generated by immunization of a mammal with a composition comprising the protein or fragment. Methods to produce polyclonal or monoclonal antibodies are well known in the art. For a review, see "Antibodies, A Laboratory Manual, Cold Spring Harbor Laba:ratory, Eds. E. Harlow and D. Lane (1988), and D..E. Yeltan et al., 1981. Ann. Rev. Biochem.
50:657-680.
Polyclonal antibodies may be obtained, for example, by immunizing three manth old male and female white New Zealand 1~'~ rabbits with the synthetic peptide to which Tyr has been added at the C-terminus in order to couple it, as an antigen, to BSA
by a bisdiazotized benzidine (BDB) linkage by reaction for 2 hours at 4°C. The reaction mixture is dialyzed to remove low molecular weight material, and the retentate is frozen in 2~ liquid nitrogen and stored at -20°C. Animals are immunized with the equivalent of 1 mg of the peptide antigen according to the procedure of Benoit et al. P.N.A.S. USA, 79, 917-921 (1982). At four week intervals, the animals are boosted by injections of 200 ug of the antigen and bled ten to fourteen 2',~ days later. .After the third boost, antiserum is examined for its capacity to bind radioiodinated antigen peptide prepared by the chloramine-T method and then purified by CMC-ion exchange column chromatography. The antibody molecules are then collected from the mammal and isolated to the extent desired by 31) well known techniques ~;uch as, for example, by using DEAE-Sephadex to obtain the IgG fraction.

For monoclonal. antibodies, see Kohler & Milstein (1975) Nature 256:495-497. In general, procedures for preparing monoclonal antibodies involve immunizing an animal with the protein or fracCment (the 'antigen'). The splenocytes of such animals are ext:.racted and fused with a suitable myeloma cell line. After fusion, the resulting hybridoma cells are selectively maintained i.n HAT medium, and then cloned as described in Wands et al.. Gastro-enterology. 80:225-232 (1981).
The hybridoma cells obt=ained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the antigen.
To enhance thE: specificity of the antibody, the antibodies may be purified by immunoaffinity chromatography using solid phase-affixed immunizing polypeptide. The antibody is contacted with the scalid phase-affixed immunizing polypeptide for a period of time sufficient for the polypeptide to immunoreact with the antibody mol.ecuies to form a so:Lid phase-affixed immunocomplex. The bound antibodies are separated from the comp7.ex by standard techniques.
A radioimmunoassay is established with the antisera and serum from subsequent bleeds from the same rabbits. The native protein is recognized by the antibodies on an equimolar basis as compared to the synthetic peptide antigen.
The antibody so produced can be used, inter alia, in diagnostic met=hods and systems to detect the level of fusion protein present in a test sample. In addition, anti-fusion protein antibodies can be used in therapeutic methods, e.g., blocking the occurrence of undesired fusion processes. The anti-fusion protein ant=ibodies can. also be used for the immunoaffinity or affin=ity chromatography purification of such fusion proteins.

Accordingly, o:ne aspect of the invention provides a process for purifying, from a biological sample, a polypeptide or polypeptide derivative of the invention, which involves carrying out antibody-based affinity chromatography with the biological sample, wherein the antibody is a monospecific ant ibody of thf~ invent 1 on .
For use in a purification process of the invention, the antibody is either polyclonal or monospecific, and preferably is of the IgG type. Purified IgG is prepared from an antiserum using standard methods (see, e.g., Coligan et al., Current Protocols in Immunology (1994)John Wiley & Sons, Inc., New York, NY.). Conventional chromatography supports, as well as standard methods for grafting antibodies, are described in, e.g., Antibodies: A Laboratory Manual, D. Lane, E. Harlow, Eds.
(1988) and outlined below.
Briefly, a b:i.ological sample, such as a cell extract containing the membrane fusion protein, preferably in a buffer solution, is applied to a chromatography material, preferably equilibrated with the x>u.ffer used to dilute the biological sample so that the polypeptide or polypeptide derivative of the invention (i.e., the antigen) is allowed to adsorb onto the material. The chromatography material, such as a gel or a resin coupled to an antibody of the invention, is in either a batch form or a column. The unbound components are washed off and the antigen is then eluted with an appropriate elution buffer, such as a glyc:~rie buffer or a buffer containing a chaotropic agent, e.g., guanidine :HC1, or high salt concentration (e. g., 3M MgCl2). Eluted fractions are recovered and the presence of the antigen is detected, e.g., by measuring the absorbance at 280 nm.

(C) Polynucleotides, their expression and use:
According to one aspect of the invention, isolated polynucleotides are provided which encode the membrane fusion proteins of the invention. In one embodiment, the polynucleotides are those of SEQ ID NOs: 1 and 5.
The term "isolated polynucleotide" is defined as a polynucleotide removed from the environment in which it naturally occurs. For example, a naturally-occurring DNA
molecule present in the genome of a living virus or as part of a gene bank is not isolated, but the same molecule separated from the remaining part. of the viral genome, as a result of, e.g., a cloning event (amplification), is isolated. Typically, an isolated polynucleot.ide molecule is free from polynucleotide regions (e. g., coding :regions) with which it is immediately contiguous at the 5' or 3' end, in the naturally occurring genome. Such isolated polynucleotides may be part of a vector or a composition and st:i.ll be defined as isolated in that such a vector or composition is not part of the natural environment of such polynucleotide_ The polynucleotide of the invention is either RNA or DNA (cDNA, genomic DNA, or synthetic DNA), or modifications, variants, homologs or fragments thereof. The DNA is either double-stranded or sing7_e-stranded, and, if single-stranded, is either the coding strand or the non-coding (anti-sense) strand.
Any one of the sequence=_s that encode the proteins of the invention as :shown in SEQ ID No: 2, 4, 6 and 8 is (a) a coding sequence, (b) a ribonucleotide sequence derived from transcription of (a), or (c) a coding sequence which uses the redundancy or degeneracy of the genetic code to encode the same polypeptides.

Homologous polynucleotide sequences are defined in a similar manner to homologous amino acid sequences. Preferably, a homologous polynucleotide sequence is one that is at least 45%, more preferably 60%, and most preferably 85% identical to sequence encoding the proteins of the invention, or to the coding sequences of SEQ TD NOs 1 and 5, or to the sequence encoding the proteins of SEQ ID NOs:2, 4, 6 and 8.
Polynucleotides encoding homologous polypeptides or allelic variants are retrieved by polymerase chain reaction (PCR) amplification of genomic viral polynucleotides extracted by conventional methods;' (see Example 2). This involves the use of synthetic oligonucleotide primers matching upstream and downstream of the 5' and 3' ends of the coding region.
Suitable primers are designed according to the nucleotide sequence information provided in SEQ ID Nos:l and 5. The procedure is as follows: a primer is selected which consists of

10 to 40, preferably 15 to 25 nucleotides. It is advantageous to select primers containing C and G nucleotides in a proportion sufficient to ensure efficient hybridization; i.e., 2C1 an amount of C and G nucleotides of at least 40%, preferably 50% of the total nucleotide content. A standard PCR reaction contains typically 0.~~ to 5 Units of Taq DNA polymerase per 100 ~L, 20 to 200 ~M deoxynucleotide each, preferably at equivalent concentrations, 0.5 to 2.5 rnM magnesium over the total deoxynucleotide concentration, 105 to 106 target molecules, and about 20 pmol of each primer. About 25 to 50 PCR cycles are performed, with an annealing temperature 15°C to 5°C below the true Tm of th.e primers. A more stringent annealing temperature improves discrimination against incorrectly annealed primers and reduces incorporation of incorrect nucleotides at the 3' end of primers. A denaturation temperature of 95°C to 97°C is typical, although higher temperatures may be appropriate for dematuration of G+C-rich targets. The number of cycles performed depends on the starting concentration of target molecules, though typically more than 40 cycles is not recommended as non-specific background products tend to accumulate.
An alternative method for retrieving polynucleotides encoding homologous po:l.ypeptides or allelic variants is by hybridization screening of a DNA or RNA library. Hybridization procedures are well-known in the art and are described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons Inc., 1994), Silhavy et al. {Silhavy et al. Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, 1984), and Davis et al. (Davis et al. A
Manual for Genetic Engineering: Advanced Bacterial Genetics, Cold Spring Harbor Laboratory Press, 1980)). Important parameters fox' optimizing hybridization conditions are reflected in a. formula used to obtain the critical melting temperature above which two complementary DNA strands separate from each other (Casey tx Davidson, Nucl. Acid Res. (1977) 4:1539). For polynucleotides of about 600 nucleotides or larger, this formula is as follows: Tm = 81.5 + 0.41 x (% G+C) + 16.6 log (canon ior~ concentration) - 0.63 x (% formamide) -600/base number. Under appropriate stringency conditions, hybridization temperature (Th) is approximately 20 to 40°C, 20 to 25°C, or, preferably 30 to 40°C below the calculated Tm.
Those skilled in the art will understand that optimal temperature and salt conditions can be readily determined.
Polynucleotide molecules according to the invention, including RNA, DNA, ox' modifications or combinations thereof, have various applications. A DNA molecule is used, for example, (i) in a process for producing the encoded polypeptide in a recombinant host system, (ii) as part of a gene delivery system, e.g. liposomes, which, upon delivery, becomes expressed and promote membrane fusion, (iii) operably linked to regulatory elements as part of an expression cassette which, when turned on, expresses the polynucleotide and promote membrane fusion, and, (iv) as a probe or primer.
Accordingly, nne aspect of the invention encompasses (i) an expression cassette containing a polynucleotide of the invention placed under the control of the elements required for expression, in particular under the control of an appropriate promoter; (ii) an expression vector containing an expression cassette of the invention; (iii) a procaryoti.c or eucaryotic cell transformed ar tra.nsfected with an expression cassette and/or vector of the invention, as well as (iv) a process for producing a polypeptide or polypeptide derivative encoded by a polynucleotide of the :invention, which involves culturing a procaryotic or eucaryoti.c host cell transformed or transfected with an expression cassette and/or vector of the invention, under conditions that allow expression of the DNA molecule of the invention without t~e~ing toxic to the host cell and, recovering the encoded polypeptide or polypeptide derivative from the host cell culture.
A recombinant expression system is selected from procaryotic arad e~ucaryotic hosts. Since the proteins of the invention promote membrane fusion, host cells are selected which can be maintained and which can express the proteins within tolerable limits of toxicity. Eucaryotic hosts include 2~~ yeast cells (c~.g. , Sacc.haromyces cerevisiae or Pichia pastoris), plant cells, and cells which preferably have a cell wall so that i~he integrity of the host cell is not affected by the fusion activity. A preferred expression system is a procaryotic host such as E. colt. Bacterial and eucaryotic cells are available from a number of different sources including commercial sources to those skilled in the art, e.g., the American Type Culture Collection (ATCC; Rockville, Maryland). Commercial. sources of cells used for recombinant protein expression alsc> provide instructions for usage of the cells.
One ;killed .in the art wauld readily understand that not all vectors and expression control sequences and hosts would be expected to express equally well the polynucleotides of this invention.. Wii:.h the guidelines described below, however, a selection oi: vectors, expression control sequences and hosts may 'be made without undue experimentation and without departing from the scope of this invention.
In selecting a vector, the host must be chosen such that it is not affected by the fusion activity of the expressed membrane fusion protein. In addition, a host must be chosen that is compatible with the vector which is to exist and possibly replicate in it. Considerations are made with respect to the vector copy number, the ability to control the copy number, expression of other proteins such as antibiotic resistance. I:n selecting an expression control sequence, a number of variables are considered. Among the important variables are the relative strength of the sequence (e.g. the 2Q ability to dr~.ve expression under variaus conditions), the ability to control the sequence's function, compatibility between the polynuclec>tide to be expressed and the control sequence (e. g. secondary structures are considered to avoid hairpin structures which prevent efficient transcription). In selecting the host, unicellular hosts are selected which are:
compatible with the sE:lected vector, tolerant of any possible toxic effects of the expressed product, able to express the product efficiently, able to express the product in the desired conformation, easily scaled up, and easy to use for purifying 31) the final product .
The choice of the expression cassette depends on the host system selected a~~ well as the features desired for the expressed polypeptide. Typically, an expression cassette includes a promoter that is functional in the selected host system and can be constitutive or inducible; a ribosome binding site; a start codon (ATG) if necessary; a region encoding a signal peptide; a polyn.ucleotide of the invention; a stop codon; and optionally a. 3t terminal region (translation and/or transcription terminatr~r). The signal peptide encoding region is adjacent to the polynucleotide of the invention and placed in proper reading framF~. The signal peptide-encoding region is homologous or heterologous to the DNA molecule encoding the mature polypeptide and i.s compatible with the secretion apparatus of the host rzs~ed for expression. The open reading frame constituted by the DNA molecule of the invention, solely or together with the signal peptide, is placed under the control of the promoter so that transcription and translation occur in the host system.
Promoters and signal peptide encoding regions are widely known and available to those skilled in the art and include, for example, the promoter of Salmonella typhimurium (and derivatives) that :is inducible by arabinose (promoter araB) and is functional in Gram-negative bacteria such as E.
coli (as described in U.S. Patent No. 5,028,530 and in Cagnon et al., (Cagnon et al., Protein Engineering (1991) 4(7):843));
the promoter of the gene of bacteriophage T7 encoding RNA
polymerase, that is functional in a number of E. coli strains expressing T7 polymerase (described in U.S. Patent No. 4,952,496); UspA lipidation signal peptide; and RlpB
lipidation signal peptide (Takase et al., J. Bact. (1987) 169:5692).
Promoters contemplated for use herein include inducible (e. g., minimal CMV promoter, minimal TK promoter, modified MMLV LTR), constitutive (e. g., chicken alpha-actin promoter, MMLV LTR (non-modified), DHFR), and/or tissue specific promoters .
Inducible promoters contemplated for use in the practice of the present invention comprise transcription regulatory regions that function maximally to promote transcription of mRNA under inducing conditions. Examples of suitable induc:ible promoters include DNA sequences corresponding to: the ~'. co.li lac operator responsive to IPTG
(see Nakamura et al., Cell, 18:1109-1117, 1979); the metallothionein promotEar metal-regulatory-elements responsive to heavy-metal (e. g., zinc) induction (see Evans et al., U.S.
Patent No. 4,870,009), the phage T7lac promoter responsive to IPTG (see Studier et a=L., Meth. Enzymol., 185: 60-89, 1990; and U.S. Patent No. 4,952,96), the heat-shock promoter; the TK
minimal promoter; the CMV minimal promoter; a synthetic promoter; and the like.
The expression cassette is typically part of an expression vector, whi:Yl is selected for its ability to replicate in the chosen expression system. Expression vectors (e. g., plasmid.s or viral. vectors) can be chosen, for example, from those described in Pouwels et al. (Cloning Vectors: A
Laboratory Manual 1985, Supp. 1987). Suitable expression vectors can be: purchased from various commercial sources.
Methods for transforming/transfecting host cells with expression vectors are well-known in the art and depend on the host system selected as described in Ausubel et al., (Ausubel et al., Current Protocols in Molecular Biology, John Wiley &
Sons Inc., 1994).
Upon expression, a recombinant polypeptide of the 3CI invention (or a polypeptide derivative) is produced and remains in the intracellular compartment, is secreted/excreted in the extracellular medium or in the periplasmic space, or is embedded in the cellular membrane. The polypeptide is recovered in a substantially purified form from the cell extract or from the supernatant after centrifugation of the recombinant cell culture. Typically, the recombinant polypeptide is purified by antibody-based affinity purification or by other well-known methods that can be readily adapted by a person skilled in the a.rt, such as fusion of the polynucleotide encoding the p~olypepticle or its derivative to a small affinity binding domain. Antibodies useful for purifying by immunoaffinity the polypeptides of the invention are obtained as described below.
The sequence information provided in the present application enables the design of specific nucleotide probes and primers that are used for diagnostic purposes.
Accordingly, one aspeci~. of the invention provides a nucleotide probe or primer having a sequence found in or derived by degeneracy of the genetic code from a coding sequence shown in SEQ ID No:1 or 5.
A primer is a prabe of usually about 10 to about 40 nucleotides that is used to initiate enzymatic polymerization of DNA in an amplification process (e. g., PCR), in an elongation process, or in a reverse transcription method.
Primers used :in diagnostic methods involving PCR are labeled by methods known in the art.
In one embodiment, optionally labeled cDNAs encoding fusion proteins, or fragments thereof, can be employed to probe library(ies) (e. g., cDNA, genomic, and the like) for additional sequences encoding novel fusion proteins. Such screening is 31) typically initially c:-~rried out under low-stringency conditions, which comprise a temperature of less than about 42°C, a formamide concentration of less than about 50%, and a moderate to low salt concentration. Presently preferred screening conditions comprise a temperature of about 37°C, a formamide concentration of about 20%, and a salt concentration of about 5X standard saline citrate (SSC; 20X SSC contains 3M
sodium chloride, 0.3M sodium citrate, pH 7.0). Such conditions will allow the identification of sequences which have a substantial degree of similarity with the probe sequence, without requiring perfect identity for the identification of a stable hybrid. The phrase "substantial similarity" refers to sequences which share at: least 50% identity. Preferably, hybridization conditions will be selected which allow the identification of sequences having at least 70% identity with the probe, while discriminating against sequences which have a lower degree of identity with the probe.
As used herein, a nucleic acid "probe" is single-stranded DNA or RNA, or analogs thereof, that has a sequence of nucleotides that includes at least 14, preferably at least 20, more preferably at least 50, contiguous bases that are the same as (or the complement of) any 14 or more contiguous bases set 2C1 forth in any of SEQ Ib N0:1 or 5. Probes may be labeled by methods well-known in the art, as described hereinafter, and used in various diagnostic kits.
As used herein, the terms "label" and "indicating means" in their various grammatical forms refer to single atoms and molecules that are either directly or indirectly involved in the production of a detectable signal to indicate the presence of a complex. Any label or indicating means can be linked to or incorporated in a nucleic acid probe, an expressed protein, polypeptide fragment, or antibody molecule that is part of an antibody or monoclonal antibody composition of the present invention, or used separately. These atoms or molecules can. be used alone or in conjunction with additional reagents. Such labels <~re themsel.ves well-known in clinical diagnostic chemistry.
The labeling means can be a fluorescent labeling agent that chemically binds to antibodies or antigens without denaturing them to form a fluorochrome (dye) that is a useful immunofluorescent tracer. Suitable fluorescent labeling agents are fluorochromes such as fluorescein isocyanate (FIC), fluorescein i~othiocyant:e (FITC), 5-dimethylamine-1-naphthalenesulfonyl chloride (DANSC), tetramethylrhodamine isothiocyanate (TRITC), lissamine, rhodamine 8200 sulphonyl chloride (RB-200-SC), and the like. A description of immunofluorescence ana:Lysis techniques is found in DeLuca, "Immunofluorescence Analysis", in Antibody As a Tool, Marchalonis et al., Eds., John Wiley & Sons, Ltd., pp. 189-231 (1982) .
In preferred embodiments, the indicating group is an enzyme, such as horseradish peroxidase (HRP), glucose oxidase, and the like. In such cases where the principal indicating group is an enzyme, additional reagents are required to visualize the fact that a receptor-ligand complex (immunoreactant) has formed. Such additional reagents for HRP
include hydrogen peroxide and an oxidation dye precursor such as diaminobenzidine. An additional reagent useful with glucose oxidase is 2,2'-azino-di-(:3-ethyl-benzthiazoline-G-sulfonic acid) (ARTS).
Radioactive elements are also useful labeling agents and are used illustratively herein. An exemplary radiolabeling agent is a radioactive element that produces gamma ray emissions. Elements which emit gamma rays, such as 124I, 125I, 126I, 131I and 5lCr, represent one class of radioactive element indicating groups. Particularly preferred is 1251. Another group of useful labeling means are those elements such as 11C, 18F, 150 and 1.3N which emit positrons. The positrons so emitted produce gamma :rays upon encounters with electrons present in the animal's body. Also useful is a beta emitter, such as 32P, 1.11In or :3H.
The linking of labels to substrate, i.e., labeling of nucleic acid probes, ant:i.bodies, golypeptides, and proteins, is well known in the art. For instance, antibody molecules produced by a hybridoma can be labeled by metabolic incorporation of radioisotope-containing amino acids provided as a component. in the ~~ulture medium. see, for example, Galfre et al., Meth. Enzymol., 73:3-46 (1981). The techniques of protein conjugation or coupling thraugh activated functional groups are particularly applicable. See, fox example, Aurameas et al., Scand. J. Immunol., Vol. 8, Suppl. 7:7-23 (1978), Rodwell et al., Biotech., 3:889-894 (1984), and U.S. Patent No.
4,493,795.
(D) Com ositions_rontaining membrane fusion proteins and polynucleotides As used herein, the composition of the invention contains one or several membrane fusion proteins or derivatives of the invention.
For use in a composition of the invention, according to one embodiment, a me=mbrane fusion protein or derivative thereof is formulated :into or with liposomes, preferably neutral or anionic liposomes, microspheres, ISCOMS, or virus-like-particles (VLPs) to facilitate delivery and/or enhance the immune respon~,e. These compounds are readily available to one skilled in the art; fox example, see Liposomes: A Practical Approach, RCP New Ed, IRL press (1990).
Anionic and neutral liposomes are well-known in the art (see, e.g., Liposome:s: A Practical Approach, RPC New Ed, IRL press (1990), for a detailed description of methods for making liposomes) and are useful for delivering a large range of products, including polynucleotides.
Cationic lipids are also known in the art and are commonly used for drug or gene delivery. Such lipids include LipofectinTM also known as DOTMA (N- [1- (2, 3-dioleyloxy) propyl] -N,N,N-trimethylammonium chlaride), DOTAP (1,2-bis(oleyloxy)-3-(trimethylammanio)propane), DDAB (dimethyldioctadecylammonium bromide), DOGS (dioctadecylamidologlycyl spermine) and cholesterol derivatives such as DC-Chol (3 beta-(N-(N~,N~-dimethyl aminamethane)~-carbamoyl) cholesterol). A description of these cationic lipids can be found in EP 187,702, WO 90/11092, U.S. Patent No. 5,283,185, WO 91/15501, WO 95/26356, and U.S. F.,atent No. 5,527,928. Cationic lipids for delivery of polynucleotides are preferably used in association with a neutral lipid such as DOPE (dioleyl phosphatidylethanolamine), as described in WO 90/11092 as an example.
Formulations cantaining cationic liposomes may optionally contain other transfection-facilitating compounds.
A number of them are described in WO 93/18759,, WO 93/19768, WO
94/25608, and WO 95/02397. They include spermine derivatives useful for facilitating the transport of DNA through the nuclear membrane (see, for example, WO 93/18759) and membrane-permeabilizing compounds such as GALA, Gramicidine S, and cationic bile salts (see, for example, WO 93/19768).
The ,present invention also contemplates therapeutic compositions containing a physiologically tolerable carrier together with a fusion protein, polypeptide fragment thereof, or anti-fusion protein antibody, as described herein, dissolved or dispersed therein as an active ingredient.

As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents, and reagents, are used interchangeably and represent that the materials are capable of administration to a mammal without the production of undesirable physiological effects such as nausea, dizziness, gastric upset, and the like.
Methods for the preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well known in. the art. Typically such compositions are prepared as injectables either as liquid solutions or suspensions; however, solid forms suitable for solution, or suspension, in liquid prior to use can also be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients that are pharmaceutica=LS.y acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, as well as combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance the effectiveness of the active ingredient.
The therapeutic composition of the present invention can include pharmaceutically acceptable salts of the components therein. Pharmaceutically acceptable nontoxic salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, phosphoric acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, malefic acid, fumaric acid, anthranilic acid, cinnamic acid, naphthalene sulfonic acid, sulfanilic acid, and the like.
Salt=s formed with the free carboxyl groups can also :i be derived from inorganic bases such as, for example, sodium hydroxide, ammonium hydroxide, potassium hydroxide, and the like; and organic bases such as mano-, di-, and tri-alkyl and -aryl amines (e. g., triethylamine, diisopropyl amine, methyl amine, dimeth~,rl amine, and the like), and optionally 1C1 substituted et:hanolamines (e. g., ethanolamine, diethanolamine, and the like).
Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions than contain no materials other than the active 1~i ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline, or both, such as phosphate-buffered saline. Still further, aqueous carricsrs can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, 2C1 polyethylene glycol, and other solutes.
Liquid compositions can also contain liquid phases in addition to and to thE: exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oi:L, and water-oil emulsions.
2:i A therapeutically effective amount is a predetermined amount calculated to achieve the desired effect. The required dosage will v<~ry with the particular treatment and with the duration of desired treatment; however, it is anticipated that dosages between about 10 micrograms and about 1 milligram per 3C) kilogram of body weight per day will be used for therapeutic treatment. In some instances, it may be particularly advantageous to administer such compounds in depot or long-lasting form. A therapeutically effective amount is typically an amount of a fusion protein according to the invention, or polypeptide fragment thereof that, when administered in a ~~ physiological:Ly accept.alble composition, is sufficient to achieve a plasma concenvtration of from about 0.1 ~,g/m1 to about 100 ~g/ml, prE:ferably from about 1.0 ~g/ml to about 50 ~Cg/ml, more preferably at least about 2 ~tg/ml and usually 5 to 10 ~tg/ml. Antibodies are administered in proportionately appropriate amounts in accordance with known practices in this art.
(E) Methods of Use One aspect cf the invention provides methods of using the proteins, polynucleotides and compositions of the invention. Ac;cordingl:y,, methods are provided to promote membrane fusion which ~~omprise contacting the membranes to be fused with an effective amount of the above-described proteins.
Membranes contemplated for fusion in accordance with the present invention :include cell membranes, liposome membranes, proteoliposome membranes, and the like.
In accordance with a still further embodiment of the present invention, there are provided methods for the production of heteroka:ryons, such as B cell or T cell hybridoma cells useful for the production of monoclonal antibodies, cytokines, and. immune modulators, said methods comprising contacting, for example, an immortalized myeloma cell and a primary B cell or T cell in the presence of any one or more of the above-described proteins. Immortalized cells contemplated for use herein. include human or mouse B cell myeloma cells, T
cell myelomas, and the like, and antibody-synthesizing cells contemplated i:or use herein include purified spleen cells from an immunized mammal, and the like.
In accordance with a still further embodiment of the present invention, there are provided methods for the production of liposome-liposome fusions or liposome-cell fusions, said methods comprising contacting lipids suitable for the formation of liposomes and a suitable cell in the presence of one or more proteins as described herein.
In accordance with yet another embodiment of the present invention, there, are provided improved methods for the intracellular delivery of bioactive compounds employing liposomes, they improvement comprising incorporating into said liposomes one or 'more proteins as described herein.
The ability to promote efficient membrane fusion has broad applicability in clinical, industrial, and basic research situations. I'he reovirus fusion proteins could be used as alternatives to chemica7.ly-induced membrane fusion to promote cell-cell fusion, for example, during the production of hybridoma cells for monoclonal antibody production. In this instance, the reovirus fusion proteins would be inducibly expressed from inside a transiently or permanently transfected cell population to trigger fusion of these cells with a target cell population.
The atypical reovirus fusion proteins also have application in enhancing liposome-cell fusion. Liposomes have been developed. as a means to introduce nucleic acids, proteins, and metabolic regulators into cells. Although liposome-cell fusion has been amply d~;monstrated, the unfavourable thermodynamics of membrane fusion contribute to variable efficiencies of fusion and cytotoxicity which lead to the development of proteol.iposomes - 7.iposomes containing specific proteins to promote cell binding and fusion.
Most of the proteoliposome studies reported in the art relate to the use of various enveloped virus fusion proteins. In accordancE= with the present invention, it is possible to take advant<~ge of the novel structural features associated with the invention reovirus fusion proteins for use in proteoliposomes to enhance the intracellular delivery of bioactive compounds (e. g., nucleic acids, proteins or peptides, pharmacological agents, and the like), both in cell culture and in vivo.
The reovirus fusion proteins described herein promote membrane fusion in a diversity of cell types (e. g., fibroblasts and macrophages) from different species (e.g., avian and mammalian, including human) suggesting limited cell receptor-specificity a~~ well as the general applicability of these proteins. It may also be possible to target reovirus fusion protein-containing proteoliposomes to specific cell types by including specific receptor-binding proteins in the liposome membrane. In this instance, the receptor-binding protein would confer targeted cell attachment of the liposome followed by subsequent enhanced lipcasame-cell fusion mediated by the reovirus fusion protein.
The demonstr<it.ed ability of p14 and p22 to induce cell-cell fusion indicates their potential use in the production of heterokaryons, for example, the generation of hybridomas for monoclonal antibody production. The induction of cell-cell fusion is usually triggered using the chemical fusogen polyethylene glycol (PEGS. Although this procedure does trigger cell-cell fusion, toxic effects on cells hamper the efficiency of heterokaryon isolation. It is generally believed that "natural" membrane fusion is mediated by protein-lipid interact: ions, therefore, protein-mediated membrane fusion is likely to be much less cytotoxic than chemically-induced cell fusion.
The demonstrated ability of the small reovirus fusion 5~ proteins to promote ef:f:icient cell-cell fusion indicates their potential use as alternatives to chemical-induced cell fusion.
Expression of p14 or g22 in one population of cells, under the control of a :strong in3r~cible promoter, could trigger fusion with a second cell population, resulting in decreased cytotoxicity and more efficient heterokaryon isolation.
The atypical group of nonenveloped virus fusion proteins described herein represent alternatives to the use of enveloped virus fusion proteins in the protein-mediated enhancement of liposome-cell fusion for the intracellular delivery of bioactive molecules. The potential advantages of the reovirus fusion proteins relate to their unique structural and biological features, From a structural perspectives the small size of the reovirus fusion proteins is the most apparent advantage offered by this system. The large size, post-translational glycosylat.ion, and complex tertiary structure of the enveloped virus fusion proteins makes synthesis and purification of the functional protein using recombinant DNA
approaches and prokaryot,a.c or eukaryotic expression systems problematic.
The majority of studies relating to the use of enveloped virus fusion proteins in proteoliposomes involve the production of 'virus particles which are subsequently purified, solubilized with detergent, and the viral envelopes containing the fusion protein are reconstituted into "virosomes" by removal of the detergent (see Grimaldi in Res. Virol., 146:289-293 (1995) and Ramani et al., FEBS Lett., 404:164-168 (1997)).
Unlike most of the enveloped virus fusion proteins, the reovirus fusion proteins are small membrane proteins. Their small size and simple domain organization suggests that these proteins will be easier and more economical to produce in a functional foam using a diversity of expression and purification protocols. It is also likely that the small size of the reovirus fusion, proteins contributes to less complex protein folding pathways and tertiary structure required for correct protean confoi:mation. As a result, an increased diversity of <;xtraction and solubilization procedures (e. g., choice of detergents and denaturants? should be available to facilitate purification of the functional fusion protein and incorporation into liposomes.
The attractive biological properties of the reovirus fusion proteins relate~i~o their pH-independent fusion mechanism with numerous cell typea. The reavirus fusion proteins function at nE:utral pH, unlike the influenza virus HA protein, simplifying their use in cell culture and in vivo under physiological conditions. Furthermore, the reovirus fusion proteins fuse numerous types of cells suggesting their broad applicability as fusogens. This could include such primary cell types as dendritic cells, neurons, and stem cells which are difficult to t:ransfect using standard transfection reagents.
Accordingly, the reovirus fusion proteins could be used to promote liposome-cell fusion and the efficient intracellular delivery of DNA or other bioactive compounds into a diversity of cultured cell types, primary cell cultures, tissue explants, or in vivo.
In order to use reovirus fusion proteins for heterokaryon production, the proteins will need to be expressed in a controlled, inducible manner from within cells using standard recombinant DNA approaches. The utility of this approach has already been demonstrated in homologous cell-cell fusion in a non-inducibl.e manner. In a similar fashion, these proteins can promote cell-cell fusion between heterologous cell types in an inc3ucible manner, The development of reovixus fusion proteins for enhanced liposome-cell fusion requires the expression and purification of the functional fusion proteins and their incorporation :into liposome membranes to produce proteoliposome;s. The ~:~14 and p22 proteins can be expressed and purified using standard procedures. Expression can be accomplished employing a variety of expression systems, e.g., baculovirus or yeast eukaryotic expression vectors or from prokaryotic expression 'vectors, depending on expression levels and functional activit~~ of the protein. Various detergent extraction procedures can be used to solubilize the proteins, which can then be purified as detergent-protein complexes using standard protein purification protocols. The proteins are readily soluble in vara.o~us detergents (e. g. 0.8% Triton X100, 0.8% NP40, 0.8% octylglucoside) increasing the diversity of approaches available for functional protein purification. The small size of the reovirus fusion proteins suggests that protein solubilization and purification should be considerably more simple than simil~~r approaches to purify larger, more complex membrane proteins.
The detergent-protein complexes can be mixed with lipids and the detergent: removed by dialysis, chromatography, or extraction according to standard published procedures, similar to methods used to generate influenza HA or Sendai virus F protein-containing virosomes (see Grimaldi, Res.
Virol., 146:289-293 (1995) and Ramani et al., FEBS Lett., 404:164-168 (1.997)). These procedures will result in the production of proteoliposomes, lipid vesicles containing the ARV, NBV, or BRV fusion proteins embedded in the vesicle membrane. Once again, optimal conditions for proteoliposome production can be empirically determined as can the lipid composition and size of 'the proteoliposomes which can affect the efficiency of liposome-cell fusion. Bioactive molecules of interest (e. g., nucleie: acids, proteins or peptides, pharmacological compounds, and the like) can be included during the formation of the proteoliposmes to facilitate packaging of the molecule within the liposomes. The proteoliposomes can be purified by centrifugation and used to deliver bioactive molecules intracellularly, either in cell culture or in vivo, by protein-enhanced fusion of the proteoliposomes with cell membranes.
As acknowledged above, the use of liposomes or proteoliposomes for in;racellular delivery of compounds is known in the art, and development of such methodology is proceeding on several fronts. What is unique with the present system is the use of an atypical, previously unidentified group of nonenveloped virus i:usion proteins isolated from the only known fusogenic poikilot;hermic reoviruses. The unusual structural and. functional properties of this new group of fusion proteiras suggest that these proteins may circumvent many of the problems associated with the current development of protein-mediated membrane fusion.
While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.
EXAMPLES
Example 1: Virus growth and purification Viruses were plaque purified and grown in monkey Vero cells. Virus particles were isolated and concentrated from 3U infected cell lysates by differential centrifugation, as previously described (see Duncan, Virology, 219:179-189. (1996) ) .
Example 2: Synthesis and cloning of cDNA
The ~riral genomic dsRNA segments were isolated from concentrated virus stocks pretreated with RNase and DNase to remove extra-v:irion contaminating cellular nucleic acids.
Virus particles were disrupted using 1~ SDS and the viral dsRNA
isolated by phenol-chloroform extraction and ethanol precipitation. Aliquots of genomic dsRNA (20 ug) were poly-A-tailed using E. coli poly-A polymerase, the tailed RNA was fractionated by agarosE~ gel electrophoresis, and individual genomic segments were isolated using the RNaid protocol (Biolol) according to ~;h.e manufacturers specified procedure.
The tailed S class genome segments were used as templates for reverse t:ranscriptio:n, using Superscript reverse transcriptase (Life Technologies Inc.) and an oligo-dT primer.
Aliquots of the plus and minus strand cDNAs were used as templates for PCR amplification using Vent polymerase (New England Biolabs) and an oligo-dT primer containing a NotI
restriction enzyme site.
The products of the PCR reaction were digested with NotI, size-fractionated on agarose gels, and products corresponding to the full length S genome segments were gel-purified using Geneclean (Bio101). The individual, NotI-digested, double-stranded cDNAs were cloned into the NotI site of pBluescript (Stratagene) and used as templates for sequencing.
Example 3: Sequencing and sequence analysis The cloned cDa~As were sequenced using an automated DNA sequences (Licor) at the NRC/Dalhousie Joint Sequencing Core Facility. All sequences were determined in their entirety from both cDNA strands. The full length cDNA sequences were compiled and analyzed using the GCG sequence analysis software (see Devereaux et al., Nucleic Acids Res., 12:387-395 (1984)).
Example 4: Identification of genome segments encoding the fusion proteins of RRV and AQV
The RRV and AQV cDNA clones were subcloned into the eukaryotic expression vector pcDNA3 (Invitrogen) under the control of the CMV promoter. Plasmid DNA was isolated and purified on Qiagen midi columns (Qiagen) according to the manufacturer '; specific:ations. Plasmid DNA (1 ug) was mixed with Lipofecta~mine (3 u7~;1 (Life Technologies Inc. ) and used to transfect sub-confluent cell monolayers grown in 12 well cluster plates. Transfected cell monolayers were incubated at 37°C for 24-4~ hr before being fixed with methanol and stained using a water-soluble Wright-Giemsa stain (DiffQuik; VWR-Canlab), as previously described (see Duncan et al., Virology, 224:453-464 (1996)). Cell fusion was assessed by light microscopy of stained monolayers and syncytial foci were photographed at 100x magnification. Using this protocol, the S1 genome segment of RR'V and genome segment 7 of AQV were determined to encode t:h~e fusion proteins of these viruses.
Example 5: Identification of p14 and p22 fusion proteins Two unrelated fusion proteins responsible for the cell-cell fusion induced by reptilian reovirus (RRV) and aquareovirus (AQV) have been identified. These proteins are referred to herein as p1.4 (for RRV) and p22 (for AQV) to reflect their approximate predicted molecular weights. The genes encoding p14 and p22 have been cloned and sequenced. The sequence-predicted structural organization of these proteins has been analyzed, and the membrane fusion properties thereof have been directly demonstrated.
p14 of reptilian reovixws Sequence analysis determined that the RRV S1 genome segment is 1501 base pairs long and contains the conserved terminal nucleotide sequences 5'-GTTA...TCATC-3' present in other RRV genome segments, indicating the sequence represents the full-length cD:~TA sequence of the S1 genome segment. This represents the first nucleotide sequence reported for any genome segment of any reptilian reovirus.
Sequence ana:l.ysis revealed the presence of two sequential overlapping ORFs. Based on sequence similarity, the second ORF spanning nucleotides 386-1432 (including the ATG
start codon and exclud:i.n.g the termination codon) encodes the 349 amino acid homolog of the typical reovirus cell attachment protein, termed sigma C" for avian reovirus and sigma 1 for mammalian reovirus (ShaF~ouri et al., J. Gen. Virol., 76:1515-1520) .
The first ORF, spanning nucleotides 25-399, encodes a previously unidentified predicted 125 amino acid protein. The predicted 125 amino acid product of the first ORF had no homolog amongst the known reovirus proteins, and displayed no significant sequence s:~milarity to any proteins contained in the databanks. The fi~_~>t: ORF was subcloned by PCR using specific primers into pc:DNA3. Expression of the first ORF
alone in transfected cells resulted in cell-cell fusion (see Example 8 ) ind.icating t~l~iat RRV encodes a novel 14 Kda fusion protein, termed p14.
p22 of Aquareavirus:_ The AQV genome segment 7 sequence was found to be 1399 base pairs long a~~d. contained the conserved terminal nucleotide sequences 5'-GTTT...TCATC-3' present in other AQV
genome segments, indicating the sequence represents the full-y length cDNA sequence of. genome segment 7.
Sequence anal~~sis revealed a complex arrangement of ORFs on the AQV genome ~;egment 7. The first methionine start codon does not occur until 489 nucleotides from the 5'-end of the gene. This start cc>don is followed by a single ORF
encoding a predicted 2'78 amino acid protein, presumably the previously identified N~~28 protein, the only known gene product of this genome segment I;Subramanian et al., virology, 205:75-81 (1994)). In the 5'-terminal 488 nucleotides preceding this start codon, a.ll 'three reading frames contain small ORFs. The ORF in reading' frame "a"' spans nucleotides 1-267, the ORF in reading frame "b" spans nucleotides 8-610, and the ORF in reading frame "c" spans nucleotides 3-482 (all excluding the termination codon). Each of these ORFs lack a conventional ATG
start codon, but could conceivably be functional using a codon other than met.hionine as a start codon, as occurs, for example, with the C proteins encoded by the P mRNA of Sendai virus (see Curran and Ko7.akofsky, ~sMBO J., 7:245-251 (1988)). These AQV
ORFs can encode 89, 20:1,, or 160 amino acid proteins for reading frames a, b, and c, respectively.
The 5'-terminal 485 nucleotides of the AQV genome segment 7 were' subcloned into pcDNA3 by PCR amplification using specific primers, expressed in tra.nsfected cells, and found to induce cell-cell fusion. This result confirms that the AQV
fusion protein was encoded by one of the three predicted small 30~ ORFs present near the 5'-end of the genome segment.
Of t:he three possible gene products encoded by this region, the potential 201 amino acid product of reading frame b, termed p22, was predicted to be a membrane localized protein, as would be expected for a protein capable of causing membrane fusion (a transmembrane region is predicted to exist in p22, as determined ~m;ing the TMpred and PSORT algorithms;
see Example 7). In order to confirm that p22, encoded by the second ORF, represents the AQV fusion protein, site-directed mutagenesis was used to insert a C to T substitution at position 59. This sub:~t.itution introduces a translation stop site into reading frame b specifically, which would result in the premature termination of the p22 ORF after only 17 codons.
Such a substitution eliminated cell fusion, confirming that the predicted p22 gene product of ORF b represents the AQV fusion protein. Since the p22 ORF lacks the typical methionine start codon, the precise stax:t site of p22 is not presently known but clearly resides in one of the 17 codons that occur upstream of the nucleotide substitution used to engineer a stop codon at position 59.
Example 6: Sequence analysis of p14 The RRV p14 fusion protein is one of the smallest known fusion proteins at only 125 amino acids in length. It shares no significant sequence similarity with any protein in the databases, includirig~ those implicated as membrane fusion proteins such as the enveloped virus fusion proteins, the cellular SNARE protein:, or even the previously identified fusion proteins of avian reovirus and Nelson Bay reovirus (Shmulevitz and Duncan, EMBO J., 19:902-912 (2000), White, Annu. Rev. Physiol., 52:675-697 (1990), Weber et al., Cell, 92:759-772 (1998)). As such, p14 represents a novel membrane fusion protein.
The p14 protein contains a single predicted transmembrane domain, detected using both the TMpred and PSORT
algorithms, located between residues 39-57 (see Figure 4). It also contains ,an N-terminal myristylation consensus sequence (MGXXXS/T) suggesting the protein may be acylated. Sequence analysis also detected a cluster of basic residues on the C-proximal side of the transmembrane domain, and a polyproline motif and N-linked glycosylation consensus sequence (NXS/T) located near the C-terminus of the protein (see Figure 4).
Interestingly, p14 lacks an obvious fusion peptide, a small stretch of hydrophobic amino acids located in the extracellular domain of enveloped virus fusion proteins and involved in insertion of_ a fusion prote~.n into the target membrane. Moreover, the p14 protein lacks any apparent heptad repeats required for the coiled coil rearrangements observed in enveloped virus fusion proteins. The lack of fusion peptide and heptad repeat motifs in p14 suggests that p14 may promote membrane fusion through a mechanism distinct from that proposed for the well described enveloped virus fusion proteins (see White, Ann. Rev. Physi.o:l., 52:675-697 (1990)).
Example 7: SE~quence analysis of p22 The AQV p22 fusion protein shares no obvious sequence homology with either t:he RRV p14 fusion protein, the fusion proteins of avian reovirus or Nelson Bay reovirus, or any other protein in the databases. The p22 protein is approximately 201 amino acids i:n size. Th.e precise size is not known and depends on which of the 17 N-terminal codons present in the p22 ORF
serve as the start codon for this protein. The p22 protein has a predicted transmembrane domain located approximately between residues 43-60. As with p14, p22 contains clusters of basic residues adjacent to the transmembrane domains and p22 also lacks obvious fusion peptide and heptad repeat motifs.
Example 8: T'he p14 and p22 fusion proteins induce cell-cell fusion The fusion-irlducing potential of these reovirus proteins has been directly demonstrated by expressing them in transfected cells in t~.ie absence of any other reovirus proteins; intracellular expression triggers the induction of cell-cell fusion and s~~n.cytium formation characteristic of virus infection by this group of fusogenic reoviruses. Thus, quail or Vero cell monolayers were mock transfected, or transfected with plasm:id. DNA expressing the RRV or AQV fusion proteins. Transfected cells were fixed and the nuclei stained using a Wright-Giemsa ~~tain at 24 hr post infection and the stained monolayers were photographed at 100x magnification.
Transfection of plasmids expressing either RRV p14 or AQV p22 resulted in ext:e:nsive cell fusion and the development of multinucleated syncvytia Cpolykaryons). The appearance of polykaryons was evident. when transfected cells were stained to display the cell nuclei, which clearly showed the clustering of nuclei within large syxicytial cells. Mock transfected cells showed no signs of syn.y~tium formation, indicating that cell fusion was the direct r_e~sult of the expression of the reovirus proteins within transfected cells. These results conclusively demonstrate the membrarae~ fusion-inducing capability of the reovirus fusion protein; of the invention.
Transfection of other reovirus proteins fails to induce cell fusion, indicating that this is a p14- or p22-specific event. zn addi.tion, both proteins induce cell fusion in a variety of cell types of avian or mammalian origin indicating the general utility of these proteins to induce membrane fusion.
While the invention has been described in detail with reference to certain prE:ferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that: which is described and claimed.

Summary of Sequences:
SEQ ID N0:1 is a cDNA nucleotide sequence of the S1 genome segment of reptilian reovirus (RRV). The open reading frame encoding p14 is shown.
SEQ ID N0:2 is the deduced amino acid sequence of the p14 protein enc;oded by SEQ ID NO:1.
SEQ ID N0:3 is a cDNA nucleotide sequence of the S1 genome segment of reptilian reovirus (RRV). The open reading frame encoding the sigma C protein is shown.
SEQ 'ID N0:4 is the deduced amino acid sequence of the sigma C protein encoded by SEQ ID N0:1 or 3.
SEQ ID N0:5 i.s a cDNA nucleotide sequence of genome segment 7 of aquareovirus (AQV). 'The apen reading frame encoding p14 as shown.
SEQ ID N0:6 is the deduced amino acid sequence of the p22 protein encoded by SEQ ID N0:5. Note that the initiator codon is one of the first 17 codons; its precise position has not been determined.
SEQ ID N0:7 is a cDNA nucleotide sequence of genome segment 7 of aquareovi.rus (AQV). The open reading frame encoding the NS28 protein is shown.
SEQ ID N0:8 is the deduced amino acid sequence of the NS28 protein encoded by SEQ TD N0:5 or 7.

SEQUENCE LISTING
(1) GENERAL INFOF:MATTON:
( i ) APPLICAI'iT : FUSOGENI:K INC .
(ii) TITLE OF' INVENTION: MEMBRANE FUSION PROTEIN
(iii) NUMBER OF SEQUENCES: 8 (iv) CORRESPONDENCE ADDRESS:
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O (B) STF:EET: P.O. BO:K 2999, STATION D
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(D) STF~TE: ONT
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(F) ZIP: K1P 5Y6 (v) COMPUTEF; READABLE FORM:
(A) MEL)IUM TYPE: Floppy disk (B) COMPUTER: IBM 1?C compatible (C) OPE;RATI:NG SYSTEM: PC-DOS/MS-DOS
(D) SOE'TWARE: AaC:CI (text) ZO (vi) CURRENT APPLICATION DATA:
(A) APPLICATION I~IUMBER:
(B) FILING DATE: 02~-JUNE-2003 (C) CLF,SSIFICATIC)N:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUP4BER:
(B) FILING DATE:
(viii) ATTORNEY'/AGENT INFORMATION:
(A) NAME: SMART tK BIGGAR
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3O (C) REE'ERENCE/DOC'KET NUMBER: 78973-13 (ix) TELECOMMUNICATION TPdFORMATION:
(A) TEhEPHONE: (61:3)-232-2486 (B) TEhEFAK: (61~~ ) --232-8440 (2) INFORMATION F'OR SEQ ID NO.: 1:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1501 (B) TYPE: nucleic acid (C) STRANDEDNES'S:
4O (D) TOPOLOGY:
(ii) MOLECULE TYPE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: reptilian reovirus (ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (25) . . (402) (xi) SEQUENCE DESCRIPTION: :~EQ ID NO.: 1:
GTTATTTTTT TCCTCC~ATGA AGCC .ATG GGG AGT GGA CCC TCT AAT TTC GTC 51 Meet Gly Ser Gly Pro Ser Asn Phe Val ~ 5 AAT CAC GCA CCT C:IGA GAA GCA ATT GTA ACC GGT TTG GAG AAA GGG GCA 99 Asn His Ala Pro C'ly Glu Ala Ile Val Thr Gly Leu Glu Lys Gly Ala GAT AAA GTA GCT G'~GA ACG ATA 7.."CA CAT ACG ATT TGG GAA GTG ATC GCC 147 Asp Lys Val Ala Gly Thr Ile Ser His Thr Ile Trp Glu Val Ile Ala GGA TTA GTA GCC '7.'TG CTG ACr~. 'T'TC TTA GCG TTT GGC TTC TGG TTG TTC 195 Gly Leu Val Ala heu Leu Thr :Phe Leu Ala F~he Gly Phe Trp Leu Phe Lys Tyr Leu Gln hys Arg Arch Glu Arg Arg Arg Gln Leu Thr Glu Phe CAA CGGTAT (.TACGG TAC AGGTTGAGT GAGATCCAG AGA 2 AAA AA'.I' 91 ~AGC

0 Gln LysArgTyr heuArg Asn.;SerTyr ArgheuSer GluIleGln Arg CCT ATATCACAG CACGAA TAC_'GAAGAC CCA2'ACGAG CCACCAAGT CGT 339 Pro IleSerGln FiisGlu Tyx Asp ProTyrGlu ProProSer Arg Glu AGG AAACCACCC CCTCCT CC'I"TATAGC ACATACGTC AACATCGAT AAT 387 Arg LysProPro I?roPro Prc~ Ser ThrTyrVal AsnIleAsp Asn Tyr .L 115 12 GTC TCAGCCATT '.fAGTGATGAGCAA CGGAGGGCCA ATGCTTGGCA

TTATTAAGTT

Val SerAlaIle TTTGCTGACGGAGGAACATCAGGA(~CCGATGTAGATGAGTTGATACGTCGCATGGCGGCA502 TTAGAGGTATCGTTA(3TAGAGATAi?.GGCGAGACCTGAC'GGTTCTAGATGGGGATGTAGCC562 TCTGTAATCCGTAGACTACAGGACC~C'TGAGGACGCGATAACGGCATTGTCCAACGCGATG622 ATAGCGGCTTTGGAGACGGCGGTGF.,C'TCAGAACACGAAGGACATAGATAGTGTGCGTAGC742 AGAGATGTGTCGACACAGGGACTG'I'C:AATCACTGATTTGCAGGCGCGAGTAGCTAAATTA862 GAAAGGGAAGCTGAACCGACGTCG'I'TCGAATGGCCACTGAGAAAAGATGCGAAGAGTGGA922 TTGCTATCATTGAAC'.CGGGATCCT'I'G~;~TTCTTAGAAACGACTGAAATATTTGGACTCTCA982 TGGGCGCAGTCTGGA(3TTGAGATGGG.AGCCACAACTGGACAAGGAGAATGGCATACACAA1042 AGTGGTGATTACTTG'.CACACCGTGAGCCTTAACTTTAAATTCTACAGATACAGGTCTATG1102 GGAGCCTTTTCACTC'CCAACCGGGAA'rGCGTTGCTGAACGGCCCAAAGGTGGAGCTACGT1162 TCGTCCACCACGAGA'TTTCCGTTGACGTTCGTGACACGAATAACGGTAGGAGGAAGTGAA1282 TATACCATGCCAATTACGGTGACAAT.ACGACGAATTAGTGGTGTGGATACAATCGTGCTA1342 ACTCCAGCGGATTTGCCAGGCGCCAC.AAGCTATCCATGTTATCTGAGGGGGGAGTCGATA1402 GGTGAAGCACGATGGGACGTGCGAGG.AAAGCTATTCATC 1501 (2) INFORMATION FOR SEQ ID NO.: 2:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 7.25 (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TYPE: polypeptide (vi) ORIGINAL SOURCE:
(A) ORGANISM: reptiliaal :reovirus (xi) SEQUENCE DESCRIPTION: SEQ ID NO.: 2:
Met Gly Ser Gly hro Ser Asn ~Phe Val Asn His Ala Pro Gly Glu Ala Ile Val Thr Gly heu Glu Lys Gly Ala Asp Lys Val Ala Gly Thr Ile Ser His Thr Ile Trp Glu Val :Ile Ala Gly Leu Val Ala Leu Leu Thr Phe Leu Ala Phe Gly Phe Trp Leu Phe Lys Tyr Leu Gln Lys Arg Arg Glu Arg Arg Arg Gln Leu Thr c;lu Phe Gln L~ys Arg Tyr Leu Arg Asn Ser Tyr Arg Leu Ser Glu Ile G1n Arg Pro Ile Ser Gln His Glu Tyr f!5 90 95 Glu Asp Pro Tyr Glu Pro Pro Ser Arg Arg Lys Pro Pro Pro Pro Pro Tyr Ser Thr Tyr Val Asn Ile Asp Asn Val Ser Ala Ile 115 :120 125 (2) INFORMATION FOR SEQ ID NO.: 3:
(i) SEQUENCE CHARACTERISTIC:3 4O (A) LENGTH: 7.501 (B) TYPE: nucle~.ic acid (C) STRANDEDNE:~S:
(D) TOPOLOGY:
(ii) MOLECULE TS'PE: DNA
(vi) ORIGINAL SOURCE:
(A) ORGANISM: reptilian :reovirus (ix) FEATURE
(A) NAME/KEY: CDS
(B) LOCATION: (386)..(14:35) 5O (xi) SEQUENCE DFsSCRIPTION: SEQ ID NO.: 3:
GTTATTTTTT TCCTCCiATGA AGCCATGGGG AGTGGACC'CT CTAATTTCGT CAATCACGCA 60 CCTGGAGAAG CAATTC3TAAC CGGT'.I'TGGAG AAAGGGGC'AG ATAAAGTAGC TGGAACGATA 12 0 TCACATACGA TTTGGC3AAGT GATC(3CCGGA TTAGTAGCCT TGCTGACATT CTTAGCGTTT 180 GGCTTCTGGT TGTTCAAGTA TCTCC'AAAAG AGAAGAGAAA GAAGGAGACA ACTCACTGAG 240 TTCCAAAAAC GGTAT(:TACG GAATAGCTAC AGGTTGAGTG AGATCCAGAG ACCTATATCA 300 TATAGCACAT ACGTC31ACAT CGATA :ATG TCT CAG C'.CA TTT AGT GAT GAG CAA 412 Met Ser Gln Pro Phe Ser Asp Glu Gln Arg Arg Ala Ile :Cle Lys Leu Cys Leu Ala Phe Ala Asp Gly Gly Thr 'IO TCA GGA GCC GAT (3TA GAT GAG TTG ATA CGT CGC ATG GCG GCA TTA GAG 508 Ser Gly Ala Asp Val Asp Glu Leu Ile Arg Arg Met Ala Ala Leu Glu :30 35 40 Val Ser Leu Val (31u Ile Arc_( Arg Asp Leu Thr Val Leu Asp Gly Asp Val Ala Ser Val :Ile Arg Arg Leu G1n Asp Ala Glu Asp Ala Ile Thr Ala Leu Ser Asn :Ala Met Gln Val Val Gln Ser His Ile Glu Glu Ile Val Thr Gln Val .Arg Lys Gl.n Val Glu Gln Ile Ala Ala Leu Glu Thr C~ GCG GTG ACT CAG AAC ACG AAG GAC ATA GAT AGT GTG CGT AGC ACG GTA 748 Ala Val Thr Gln .Asn Thr Lys Asp Ile Asp Ser Val Arg Ser Thr Val Thr Asp Leu Gly Ser 'Leu Va:l Ser Ala Glu Lys Val Arg Leu Asp Gly Val Ala Arg Asp Val Ser Thr Gln Gly Leu Ser Ile Thr Asp Leu Gln Ala Arg Val Ala Lys Leu Glu Arg Glu Ala Glu Pro Thr Ser Phe Glu 4~i TGG CCA CTG AGA AAA GAT GCG AAG AGT GGA 'TTG CTA TCA TTG AAC TGG 940 Trp Pro Leu Arg Lys Asp Ala Lys Ser Gly Leu Leu Ser Leu Asn Trp 5O GAT CCT TGG TTC TTA GAA ACG ACT GAA ATA TTT GGA CTC TC;A TGG GCG 988 Asp Pro Trp Phe Leu Glu Thr Thr Glu Ile 'Phe Gly Leu Ser Trp Ala 5:i Gln Ser Gly Val Glu Met Gly Ala Thr Thr Gly Gln Gly Glu Trp His Thr Gln Ser Gly Asp Tyr Leu Tyr Thr Val Ser Leu Asn Phe Lys Phe TAC AGA TAC AGG 7.'CT ATG GGA GCC TTT TCA CTC TCA ACC GGG AAT GCG 1132 Tyr Arg Tyr Arg Ser Met Gly .Ala Phe Ser Leu Ser Thr Gly Asn Ala TTG CTG AAC GGC (:CA AAG GTGI GAG CTA CGT ATA CCA TAT ACC ACA GGG 1180 Leu Leu Asn Gly I?ro Lys Va:l Glu Leu Arg Ile Pro Tyr Thr Thr Gly 250 255 e:60 265 GGG ACT GGC CTA GAA GGA TC'I' GAC CTA CAA AAC ATG ACG CCA TCG TCC 1228 0 Gly Thr Gly Leu Cslu Gly Sex :Asp Leu Gln Asn Met Thr Pro Ser Ser :'.70 275 280 ACC ACG AGA TTT C:CG TTG ACCs~ 'TTC GTG ACA CGA ATA ACG GTA GGA GGA 12 7 6 Thr Thr Arg Phe Pro Leu Thr :Phe Val Thr Arg Ile Thr Val Gly Gly AGT GAA TAT ACC ATG CCA ATT':~1CG GTG ACA F,TA CGA CGA ATT AGT GGT 1324 Ser Glu Tyr Thr Met Pro Ile 'Thr Val Thr I:le Arg Arg Ile Ser Gly GTG GAT ACA ATC C~TG CTA AC'C' CCA GCG GAT' TTG CCA GGC GCC ACA AGC 1372 Val Asp Thr Ile TTal Leu Thz :Pro Ala Asp Leu Pro Gly Ala Thr Ser TAT CCA TGT TAT C:TG AGG GGG GAG TCG ATA TTT TAC TAC ATG AGG GCT 1420 Tyr Pro Cys Tyr heu Arg Gly G1u Ser Ile Phe Tyr Tyr Met Arg Ala AGG CAG ATG ACG 7.'GA TTGCGTGA~AG AGGGACTCTC CCCGTAAGGT GAAGCACGAT 1475 Arg Gln Met Thr GGGACGTGCG AGGAAAGCTA TTCAT'C 1501 (2) INFORMATION FOR SEQ ID NO.: 4:
(i) SEQUENCE CFiARACTERISTIClS
(A) LENGTH: .l49 (B) TYPE: amino acid (C) STRANDEDNESS:
(D) TOPOLOGY:
(ii) MOLECULE TS!PE: polypept:ide (vi) ORIGINAL SOURCE:
(A) ORGANISM: reptilian :reovirus (xi) SEQUENCE DESCRIPTION: ;SEQ ID NO.: 4:
Met Ser Gln Pro I?he Ser AsI: Glu G1n Arg Arg Ala Ile Ile Lys Leu Cys Leu Ala Phe Ala Asp Gly G1y Thr Ser Gly Ala Asp Val Asp Glu Leu Ile Arg Arg Met Ala Ala.:Leu Glu Val Ser Leu Va1 Glu Ile Arg 35 ~40 45 Arg Asp Leu Thr Val Leu As~ma:ly Asp Val Ala Ser Val Ile Arg Arg Leu Gln Asp Ala Glu Asp Als. Ile Thr Ala I,eu Ser Asn Ala Met Gln Val VaI Gln Ser Iiis Ile Ghu Glu Ile Val Thr Gln Val Arg Lys Gln Val Glu Gln Ile Ala Ala Leu Glu Thr Ala val Thr Gln Asn Thr Lys 1 C~
Asp Ile Asp Ser 'Val Arg Ser. Thr Val Thr Asp Leu Gly Ser Leu Val Ser Ala Glu Lys 'Val Arg Leu Asp Gly Val Ala Arg Asp Val Ser Thr Gln Gly Leu Ser Ile Thr Asp Leu Gln Ala Arg Val Ala Lys Leu Glu 1~ 145 150 155 160 Arg Glu Ala Glu Pro Thr Ser Phe Glu Trp Pro Leu Arg Lys Asp Ala 2() Lys Ser Gly Leu Leu Ser Leu Asn Trp Asp Pro Trp Phe Leu Glu Thr Thr Glu Ile Phe Gly Leu Se:r Trp Ala Gln Ser Gly Val Glu Met Gly 2~i Ala Thr Thr Gly Gln Gly Glu Trp His Thr Gln Ser Gly Asp Tyr Leu Tyr Thr Val Ser Leu Asn Phe Lys Phe Tyr Arg Tyr Arg Ser Met Gly 3~) 225 230 235 240 Ala Phe Ser Leu Ser Thr Gly Asn Ala Leu Leu Asn Gly Pro Lys Val 37 Glu Leu Arg Ile Pro Tyr Thr Thr Gly Gly Thr G:ly Leu G:Lu Gly Ser Asp Leu Gln Asn Met Thr Pro Ser Ser Thr Thr Arg Phe Pro Leu Thr 275 ?.80 285 41l Phe Val Thr Arg Ile Thr Val Gly Gly Ser Glu Tyr Thr Met Pro Ile Thr Val Thr Ile Arg Arg Ile Ser Gly Val Asp Thr Ile Val Leu Thr 4.5 305 310 315 320 Pro Ala Asp Leu Pro Gly Aaa Thr Ser Tyr Pro Cys Tyr Leu Arg Gly 5~ Glu Ser Ile Phe Tyr Tyr Met Arg Ala Arg Gln Met Thr (2) INFORMATION FOR SEQ Ii) NO. : 5:
55 (i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 1399 (B) TYPE: nucleic acid (C) STRANDEDNESS:
(D) TOPOLOGY.
6O ( i i ) MOLECULE 'TYPE : DNA
(vi) ORIGINAL SOURCE:

(A) ORGANISM: aquareov:i.rus (ix) FEATURE
(A) NAME/KEY: misc_feat.ure (B) LOCATION: (8). (610) (C) OTHER INFORMATION: Initiator codon unknown; coding sequence begins at a position wit:
hin the first 17 codon (xi) SEQUENCE DESCRIPTION: S:EQ ID NO.: 5:
gttttag tca atc atc ctg gg~~ aat acc atc t:ca aac acc gtt cag tac 49 Ser Ile :Cle Leu G1.~ .Asn Thr Ile Ser Asn Thr Val Gln Tyr acg gta ctg cag atc gac aga tct tgc tgt atc aaa acc agc ctc acc 97 Thr Val Leu Gln :Cle Asp Arc~~ Ser Cys Cys Ile Lys Thr Ser Leu Thr gcc act tcc gaa gcc act tcc~ tgg gcc atc c:cc cct ctc gca atc tgt 145 Ala Thr Ser Glu Ala Thr SeewTrp Ala Ile Pro Pro Leu Ala Ile Cys tgc tgc tgt tgc atc tgc tgt .acc ggc gga cta tat ctc gtt cat tct 193 Cys Cys Cys Cys :Cle Cys Cyc,'Thr Gly Gly Leu Tyr Leu Val His Ser gga cgt ttt cca ggc ctc ag4: cga agg ttg gac gtg ctc gga ggt tcg 241 Gly Arg Phe Pro Gly Leu Sex .~3.rg Arg Leu Asp Val Leu Gly Gly Ser ggg tca acc cca aaa cac tcc~ ctg cgt agc cac cgg cac cca aag cca 289 Gly Ser Thr Pro Lys His Ser :Leu Arg Ser His Arg His Pro Lys Pro cgt gta cat cgc c~tt agc ttc .agt gat tct agt gac tct agt gat atc 337 Arg Val His Arg Val Ser Phe Ser Asp Ser Ser Asp Ser Ser Asp Ile 95 100 1.05 110 tct gat ctg gaa t:tg cct cgg cac ggg tct cat cct ctg gcg cat tca 385 Ser Asp Leu Glu l~eu Pro Arg :His Gly Ser His Pro Leu Ala His Ser :C15 120 125 ttc agg cct gaa c3tc gat cgc~ cac cgc cct cgt ccc tca acg caa gtc 433 Phe Arg Pro Glu Val Asp Arg :His Arg Pro Arg Pro Ser Thr Gln Val cag cag aca tct i:tc atc ccg ctc gta cca ctc agg tcc gga tca agt 481 Gln Gln Thr Ser 1?he Ile Pro :Leu Val Pro Leu Arg Ser Gly Ser Ser tta gac gat ggg atc gta cgc tct caa ccc t.ca cgg gat tcg cgg ccc 529 Leu Asp Asp Gly :Cle Val Arg Ser Gln Pro Ser Arg Asp Ser Arg Pro 160 16~~ 170 cac gag caa ttt gag gat tgg ctt caa caa gca cat ctc cta cga cca 577 His Glu Gln Phe c3lu Asp Trp Leu Gln Gln Ala His Leu Leu Arg Pro 175 180 1.85 190 gga cga gtt tcc gga tct acc aac ccc ttc acc tgaccacatt cccgactggg 630 Gly Arg Val Ser Gly Ser Thz- .Asn Pro Phe Thr :L95 200 tgacggatca tgacaagttc aacggtcatc ccctccccct cgtctacgat ggacgtctga 690 cacccatcac gggtcctcac catcttt:ggg agcctgacag ttatgtagag tggcagacct 750 gggggtgcct ccgacccttc tctcctt;t:ca gcgtttggcc accaacggta ccgaactggt 810 tcagccgtaa ggtcctccac gtcttcagca acatgtcccc gtacgcctgc gctgctgaga 870 agagtcccaa tccccttccc tactggcgtt tgaatgatca gggtcgtgac tggagcgtat 930 tctgggactt aatttggcga tgtgctcaga cacgtggtgc tcgcatctgt tttgcgaaga 990 cccccttcat ccagacgatg ctacgcc:tga ctgacgatca gctgtcccgt cttccatccg 1050 ctgaggatcc aatcagtctc ttaaacatcg caggatggga cgcccttctt ctcaacggtc 1110 ttccccctaa cctggtgcga gcatt.gatga ggtcccctcc aaacccagag gtcgttgagc 1170 tggatctgct cgtctcctgg ttcgatc~t.cg tgattcgtat tccctatgac gtgcaacacc 1230 ccctaggcct tggtttcagc cctgatc:aat tttggactca tccgttcgtc gtcctgtgct 1290 acctgcgctg gcgtttgttg ggagqtc3acg actaggatgg cgtccgcgac agttgaggcc 1350 tgggcctcgg ggatttagtc ccctgtc:gcc agcgtgactg ctattcatc 1399 (2) INFORMATION F'OR SEQ ID NO.: 6:
(i) SEQUENCE CHARACTERISTTCS
(A) LENGTH: 201 (B) TYPE: amino acid (C) STRANDEDNES~S:
(D) TOPOLOGY:
( ii ) MOLECULE T~i.'PE : polypept: ide (vi) ORIGINAL SOURCE:
(A) ORGANISM: aquareovirus (ix) FEATURE
(A) NAME/KEY: misc_featu~re (B) LOCATION: (8). (610) (C) OTHER INFORMATION: Initiator codon unknown; coding sequence begins at a position within the fwrst 17 codon (xi) SEQUENCE DESCRTPTION SEQ ID NO.: 6:
Ser Ile Ile Leu Gly Asn Thr :Ile Ser Asn Thr Val Gln Tyr Thr Val Leu Gln Ile Asp Arg Ser Cys Cys Ile Lys Thr Ser Leu Thr Ala Thr Ser Glu Ala Thr Ser Trp Ala :Ile Pro Pro Leu Ala Ile Cys Cys Cys 35 ~40 45 Cys Cys Ile Cys C:ys Thr Gly Gly Leu Tyr Leu val His Ser Gly Arg Phe Pro Gly Leu Ser Arg Arg :Leu Asp Val Leu Gly Gly Ser Gly Ser Thr Pro Lys His Ser Leu Arg Ser His Arg His Pro Lys Pro Arg Val His Arg Val Ser 1?he Ser Asp Ser Ser Asp Ser Ser Asp Ile Ser Asp loo los llo Leu Glu Leu Pro Arg His Gly ~~er His Pro Leu Ala His Ser Phe Arg 115 1.20 125 Pro Glu Val Asp Arg His Arg F°ro Arg Pra Ser Thr Gln Va1 Gln Gln Thr Ser Phe Ile Pro Leu Val Pro Leu Arg Ser G1y Ser Ser Leu Asp Asp Gly Ile Val Arg Ser Gln. Pro Ser Arg Asp Ser Arg Pro His Glu Gln Phe Glu Asp Trp Leu Gln Gl.n A1a His Leu Leu Arg Pro Gly Arg Val Ser Gly Ser Thr Asn Pro F>he Thr 19 5 2 C~ 0 (2) INFORMATION FOR SEQ ID 7:
NO.:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 1398 (B) TYPE: nucleic acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: DNA

(vi) ORIGINAL SOURCE:

(A) ORGANISM: aquareovi.rus (ix) FEATURE

(A) NAME/KEY: CDS

(B) LOCATION: (488) . . (1.3'<'4) (xi) SEQUENCE DESCRIPTION: ID NO.:
SEQ 7:

GTTTTAGTCA ATCATC'CTGG GGAATACCATCTCAAACACCGTTCAGTACA CGGTACTGCA60 GATCGACAGA TCTTGCTGTA TCAAAACC~AGCCTCACCGCCACTTCCGAAG CCACTTCCTG120 GGCCATCCCC CCTCTC'GCAA TCTGTTGCTGCTGTTGCATCTGCTGTACCG GCGGACTATA180 TCTCGTTCAT TCTGGP,CGTT TTCCAGGCCTCAGCCGAAGGTTGGACGTGC TCGGAGGTTC240 GGGGTCAACC CCAAAP,CACT CGCTGCGTAGCCACGGCACCCAAAGCCACG TGTACATCGC300 f GTTAGCTTCA GTGATT'CTAG TGACTC'.CAGTGATATCTCTGATCTGGAATT GCCTCGGCAC360 TCAACGCAAG TCCAGC.'AGAC ATCTTTCATCCCGCTCGTACCACTCAGCTC CGGATCAAGT480 CTC CAC

Met Gly Ser Tyr Ala :Leu Asn Pro Gly Ile Arg Gly Pro His ACG AGC AAT TTG AGG ATT GGt:AAC AAG ATC TCC TAC GAC CAG 577 'rTC CAC

Thr Ser Asn Leu Arg Tle Gly Asn Lys Ile Ser Tyr Asp Gln Phe His .ACC CCT

Asp Glu Phe Pro Asp heu Pro Pro Ser Asp His Ile Pro Asp 'Thr Pro a5 40 45 TGG GTG ACG GAT C:AT GAC AAG 'rTC AAC GGT C:AT CCC CTC CCC CTC GTC 673 Trp Val Thr Asp His Asp Lys :Phe Asn Gly His Pro Leu Pro Leu Val TAC GAT GGA CGT C:TG ACA CCC .ATC ACG GGT CCT CAC CAT CTT TGG GAG 721 Tyr Asp Gly Arg heu Thr Prr_~ I.le Thr Gly Pro His His Leu Trp Glu O CCT GAC AGT TAT GTA GAG TGG ~~AG ACC TGG GGG TGC CTC CGA CCC TTC 769 Pro Asp Ser Tyr Val Glu Trp G:ln Thr Trp Gly Cys Leu Arg Pro Phe Ser Pro Phe Ser Val Trp Prc~ Pro Thr Val F'ro Asn Trp Phe Ser Arg AAG GTC CTC CAC GTC TTC AGC' ;?SAC ATG TCC C'CG TAC GCC TGC GCT GCT 865 Lys Val Leu His Val Phe Se:~ ;4sn Met Ser Pro Tyr Ala Cys Ala Ala 7.15 120 125 GAG AAG AGT CCC AAT CCC CT'I' CCC TAC TGG C'GT TTG AAT GAT CAG GGT 913 Glu Lys Ser Pro Asn Pro Leu.:Pro Tyr Trp Arg Leu Asn Asp Gln Gly CGT GAC TGG AGC C~TA TTC TGG GAC TTA ATT TGG CGA TGT GCT CAG ACA 961 Arg Asp Trp Ser Val Phe Trp ;Asp Leu Ile Trp Arg Cys Ala Gln Thr 3O CGT GGT GCT CGC ATC TGT TT'I' GCG AAG ACC C'CC TTC ATC CAG ACG ATG 10 0 9 Arg Gly Ala Arg Ile Cys Phe ;Ala Lys Thr Pro Phe Ile Gln Thr Met CTA CGC CTG ACT GAC GAT CAG'~CTG TCC CGT CTT CCA TCC GCT GAG GAT 1057 Leu Arg Leu Thr Asp Asp G1n :Leu Ser Arg Leu Pro Ser Ala Glu Asp CCA ATC AGT CTC 7.'TA AAC ATC GCA GGA TGG GAC GCC CTT CTT CTC AAC 1105 Pro Ile Ser Leu heu Asn Ile ;Ala Gly Trp Asp Ala Leu Leu Leu Asn GGT CTT CCC CCT AAC CTG GTC: CGA GCA TTG A,TG AGG TCC CCT CCA AAC 1153 Gly Leu Pro Pro Asn Leu Val :4rg Ala Leu Met Arg Ser Pro Pro Asn CCA GAG GTC GTT GAG CTG GA'I'CTG CTC GTC TCC TGG TTC GAT GTC GTG 1201 Pro Glu Val Val C~lu L~eu Asr~:Leu Leu Val Ser Trp Phe Asp Val Val Ile Arg Ile Pro Tyr Asp Val G1n His Pro Leu Gly Leu Gly Phe Ser 240 24!~ 250 CCT GAT CAA TTT 7.'GG ACT CA'.I' CCG TTC GTC GTC CTG TGC TAC CTG CGC 1297 Pro Asp Gln Phe .'rp Thr His Pro Phe Val Val Leu Cys Tyr Leu Arg TGG CGT TTG TTG GGA GGT GAC!GAC TAG GATGGCGTCC GCGACAGTTG 1344 Trp Arg Leu Leu Gly Gly Asp .Asp AGGCCTGGGC CTCGGGGATT TAGTCCC'CTG TCGCCAGCGT GACTGCTATT CATC 1398 (2) INFORMATION
FOR
SEQ
ID
NO.:
8:

(i) SEQUENCE CHARACTERISTICS

(A) LENGTH: 278 (B) TYPE: amino acid (C) STRANDEDNESS:

(D) TOPOLOGY:

(ii) MOLECULE TYPE: polype:pt:ide (vi) ORIGINAL SGURCE:

(A) ORGANISM: aquareovi.rus (xi) SEQUENCE DESCRIPTION: SEQ ID
NO.: 8:

Met Gly Ser Tyr Ala Leu Asn Pro His Gly Ile Arg Gly Pro Thr Ser Asn Leu Arg Ile Cily Phe Asn 7~ys His Ile Ser Tyr Asp Gln Asp Glu Phe Pro Asp Leu Pro Thr Pra Ser Pro Asp His Ile Pro Asp Trp Val 35 ~40 45 Thr Asp His Asp hys Phe Asn G:ly His Pro Leu Pro Leu Val Tyr Asp Gly Arg Leu Thr Pro Ile Thr Gly Pro His His Leu Trp Glu Pro Asp Ser Tyr Val Glu '.~rp Gln Thr Trp Gly Cys Leu Arg Pro Phe Ser Pro Phe Ser Val Trp '.Pro Pro Thr Val Pro Asn Trp Phe Ser Arg Lys Val Leu His Val Phe Ser Asn Met Ser Pro Tyr Ala Cys Ala Ala G1u Lys 40' Ser Pro Asn Pro Leu Pro Tyr Trp Arg Leu Asn Asp Gln Gly Arg Asp Trp Ser Val Phe Trp Asp Leu Ile Trp Arg Cys Ala Gln Thr Arg Gly Ala Arg Ile Cys Phe Ala Lys Thr Pro Phe Ile Gln Thr Met Leu Arg Leu Thr Asp Asp Gln Leu Se:r Arg Leu Pro Ser Ala Glu Asp Pro Ile Ser Leu Leu Asn Ile Ala Gly Trp Asp Ala Leu Leu Leu Asn Gly Leu Pro Pro Asn Leu Val Arg Ala Leu Met Arg Ser Pro Pro Asn Pro Glu 210 27.5 220 Val Val Glu Leu Asp Leu Leu Val 5er Trp Phe Asp Val Val Ile Arg Ile Pro Tyr Asp Val GIn His Pro Leu Gly Leu Gly Phe Ser Pro Asp 2!E5 250 255 Gln Phe Trp Thr H:is Pro Phe val val Leu Cys Tyr Leu Arg Trp Arg ~J 260 265 270 Leu Leu Gly Gly Asp Asp

Claims (25)

CLAIMS:

1. An isolated protein:
(a) which is a membrane fusion protein;
(b) whose amino acid sequence is free of fusion peptide motif I; wherein the fusion peptide motif I:
(i) is an amino acid sequence 17 to 28 residues long;
(ii) has a hydrophobicity value from 0.6 to 0.7; and (iii) has a content of alanine plus glycine from 29 to 43%;
(c) whose amino acid sequence is free of fusion peptide motif II; wherein the fusion peptide motif II:
(i) is an amino acid sequence 16 to 28 residues long;
(ii) has a hydrophobicity value from 0.3 to 0.4;
(iii) has a content of alanine plus glycine from 29 to 43%; and (iv) contains a heptad repeat;
(d) which comprises an amino acid sequence which has at least 33% identity overall to SEQ ID NO:6; and (e) which comprises a transmembrane domain whose amino acid sequence has at least 60% amino acid sequence identity to the sequence 5' LAICCCCCICCTGGLYLV 3' of SEQ ID NO:6.

2. The protein of claim 1 which is encoded by the genome of a reovirus selected from the genus consisting of Orthoreovirus and Aquareovirus,

3. The protein of claim 1 which is encoded by the genome of a reovirus which naturally infects a poikilothermic host.

4. The protein of any one of claims 1 to 3 which has an isoelectric point equal to or greater than 7.

5. The protein of any one of claims 1 to 4 further comprising a positive cluster; wherein the positive cluster consists of at least three positively charged amino acid residues within a contiguous sequence of at mast 25 residues, wherein the contiguous sequence is within at most 100 residues flanking the transmembrane domain at the C-terminal side.

6. The protein of any one of claims 1 to 5 further comprising a fatty acylation sequence.

7. An isolated protein comprising SEQ ID NO:6.

8. An isolated polynucleotide encoding the protein according to any one of claims 1 to 8.

9. An isolated polynucleotide comprising SEQ ID NO:5, or degenerate variations thereof which encode the same amino acid sequences, or splice variant nucleotide sequences thereof.

10. The isolated polynucleotide according to claim 8 or 9 operatively associated with a promoter.

11. The polynucleotide of claim 10 wherein the promoter is inducible.

12. A cell expressing the isolated protein according to any one of claims 1 to 7.

13. A call expressing the polynucleotide according to any one of claims 8 to 11.

14. A liposome containing the protein according to any one of claims 1 to 7.

15. A liposome containing the polynucleotide according to any one of claims 8 to 11.

16. An antibody against the protein according to any one of claims 1 to 7.

17. A method to promote fusion between two or more membranes, comprising contacting the membranes to be fused with an effective amount of the protein according to any one of claims 1 to 7.

18. The method according to claim 17 wherein the membranes are selected from the group consisting of cell membranes, liposome membranes and proteoliposome membranes.

19. The method according to claim 17 wherein the membranes are membranes of cells, and wherein heterokaryons are produced.

20. The method according to claim 17 wherein the membranes are of an immortalized cell and a primary B cell or T
cell, for producing a hybridoma cell.

21. The method according to claim 20 wherein the hybridoma cell produces a substance selected from: monoclonal antibodies, cytokines and immune modulators.

22. The method according to claim 21 wherein the hybridoma cells produce monoclonal antibodies, and wherein the membranes are of an immortalized cell and an antibody-synthesizing cell.

23. The method according to claim 22 wherein said immortalized cell is a human or mouse B cell myeloma cell or a T cell myeloma cell, and wherein said antibody-synthesizing cell is an isolated spleen cell from an immunized mammal.

24. The method according to claim 17, wherein the membranes are selected from the group consisting of: liposome membranes, liposome membrane and cell membrane, and proteoliposome membrane and cell membrane.

25. The method according to claim 24 wherein the liposome or proteoliposome contains a bioactive drug.