The subject matter of this application was made with support from the United States Government under grant MCB9106818 of the National Science Foundation and grant GM22200 of the National Institutes of Health.

FIELD OF THE INVENTION

The present invention relates generally to capsid polypeptides, and more particularly to a viral capsid polypeptide which is capable of inhibiting viral packaging. The viral capsid polypeptide consists of a portion of a viral capsid protein of an RNA virus and includes a multimerization domain of the viral capsid protein.

BACKGROUND OF THE INVENTION

Throughout this application various publications are referenced, many in parenthesis. Full citations for these publications are provided at the end of the Detailed Description. The disclosures of these publications in their entireties are hereby incorporated by reference in this application.

A virus is a genetic element that contains either deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) , that can alternate between an intracellular state and an extracellular state. In either state, viruses are sub icroscopic particles containing nucleic acid surrounded by protein and occasionally containing other components. Viruses can vary widely in size, shape, chemical composition, range of organisms attacked, kinds of cell damage induced, and range of genetic capabilities. Viruses are known to infect animals, plants, bacteria, and fungi .

An analysis of the chemical structure of viruses shows that some contain RNA and others DNA. The DNA molecules of many viruses are double stranded (dsDNA) , but some have single stranded DNA (ssDNA) . While the RNA molecules of most RNA viruses are single stranded (ssRNA) , a few viruses with double stranded RNA (dsRNA) are known. The structure (size and shape) of viruses are exceedingly diverse. The internal structure of viruses, however, generally has the nucleic acid located in the center surrounded by a protein coat called the capsid.

The individual proteins that make up the capsid are called protein subunits, or capsomeres.

Replication of viruses requires that the virus induce a living cell of a host organism to make more of the essential components needed by the virus particle for growth. These components must then be assembled in the proper order and the new virus particles must escape from the cell and infect other cells. The various phases of this replication process can be summarized as: 1) attachment (adsorption) of a virus particle to a sensitive cell; 2) penetration into the cell by the virus or its nucleic acid; 3) replication of the viral nucleic acid; 4) production of capsid protein and other essential viral constituents; 5) assembly of nucleic acid and capsid protein into new virus particles (or packaging of the virus particle) ; and 6) release of mature virus particles from the cell.

With this life cycle (replication process) of a virus in mind, one should be able to attack the progression of viral infection in an animal, plant, bacteria or fungi by interfering with (or inhibiting) any of the stages of the viral life cycle essential to viral replication. Given the severity of many viral infections, in all types of hosts, a need always exists for new methods of inhibiting viral replication as a means of

treating and/or preventing deleterious viral infections. Examination of particular viruses could lead to the discovery of such methods, which in turn could lead to the elucidation of a method applicable to all or nearly all viruses .

Several methods have been explored to interfere with viral replication. Recently, considerable attention has been focused on the capsid proteins (also called coat proteins, cap or gag) . These proteins form one of the major structural components of the virus particles and hence are ideal as possible targets for viral interference. It has been observed that over expression of full-length viral coat proteins results in inhibition of viral replication. This phenomenon is well documented in plant cells. U.S. Patent Nos. 4,970,168 and 5,185,253 to Turner disclose a method for producing transgenic plants that are resistant to Potato virus X and Potato virus Y, by inserting a gene encoding the full length coat protein into the plant's genome. U.S. Patent No. 5,304,730 to Lawson et al . discloses a DNA sequence encoding a potato leafroll virus (PLRV) coat protein that can be used to confer resistance to PLRV. Each of the above patents discloses expression of the full length coat protein of a plant virus in order to confer resistance to that virus. Recently, persistent viral infections have been encountered in yeast cultures of Saccharomyces cerevisiae . This yeast is commonly used in fermentation processes and has gained significant recognition as an expression system for producing high levels of recombinant proteins . As a result, this yeast expression system is currently being used for producing certain vaccines. For example, yeast- derived recombinant hepatitis B vaccines have replaced plasma-derived hepatitis B vaccines in the United States. Two double stranded RNA viruses are known to exist as permanent infections of the yeast Saccharomyces

cerevisiae . These viruses, ScVLl and ScVLa, are totiviruses: that is, dsRNA viruses in which all viral functions are encoded on a single segment of dsRNA. In the literature, LI is synonymous with L-A, and La is synonymous with L-BC (Bruenn, 1988) . Each has two large open reading frames, cap (encoding the viral capsid protein Cap) and pol (encoding the viral RNA-dependent RNA polymerase, or RDRP, Pol) . The two open reading frames overlap and the pol region is translated as a Cap-Pol fusion by translational frameshifting (Diamond et al . , 1989; Tu et al. , 1992; Tzeng et al . , 1992) . Totivirus particles (including ScVLl) are icosohedral, in which 120 copies of Cap are arranged in 12 pentameric units (Cheng et al . , 1994) . ScVLl and ScVLa are related viruses (Bruenn, 1993) that do not package each other's RNA (El- Sherbeini et al . , 1984; Reilly et al . , 1984; Thiele et al. , 1984) .

Due to the important uses of Saccharomyces cerevisiae, it would be beneficial to find ways to confer resistance in this organism to the ScVLl and ScVLa viruses. In this regard, Valle and Wickner have shown that overexpression of both full-length Cap and Cap-Pol proteins interferes with maintenance of ScVLl, which is then lost from the cells (Valle and Wickner, 1993) . Recently, it has also been shown that overproduction of full length La Cap alone interferes with ScVLa replication (Yao et al . , 1995) . However, overproduction of full length LI Cap increases the viral copy number of SvVLl (Wickner et al . , 1991) . Expression of cDNA clones of fragments of viral capsid proteins was shown to be an effective means of conferring resistance to a virus (Lindbo and Dougherty, 1992a) . This resistance, however, appeared to be mediated by RNA-RNA interactions (Lindbo and Dougherty, 1992b) and the production of peptide

fragments encoded by the cDNA fragments of the full length viral capsid protein did not occur.

Since it is more efficient to work with shorter DNA segments, a need continues to exist for new and/or improved methods and means for treating and/or preventing viral infection using shorter DNA segments. Methods and means which inhibit viral packaging can provide one answer to this need.

SUMMARY OF INVENTION

To this end, the subject invention provides a viral capsid polypeptide capable of inhibiting viral packaging, the viral capsid polypeptide consisting of a portion of a viral capsid protein of an RNA virus and including a multimerization domain of the viral capsid protein. The invention further provides an isolated nucleic acid molecule encoding such a viral capsid polypeptide. In one embodiment, the RNA virus is the ScVLl virus of Saccharo yces cerevisiae, and in another embodiment the RNA virus is the ScVLa virus of

Saccharomyces cerevisiae .

The isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the viral capsid polypeptides results in production of the encoded polypeptide which inhibits viral packaging in the host cell.

The invention thus also provides a method of inhibiting viral packaging in a host cell, which comprises introducing the nucleic acid molecules of the subject invention into the cell and allowing the cell to express the nucleic acid molecules. This results in inhibition of viral packaging in the cell. The packaging of yeast, plant, and mammalian viruses can thus be inhibited in cells, such as the yeast, plant, and mammalian cells,

respectively, infected by these viral pathogens. By inhibiting viral packaging, the viral "particles" are not packaged and cannot complete their life cycle, and therefore cannot move on to infect subsequent cells.

BRIEF DESCRIPTION OF THE DRAWINGS These and other features and advantages of this invention will be evident from the following description of preferred embodiments when read in conjunction with the accompanying drawing in which:

FIG. 1 shows the location of the region required for interference m LI and La Cap. Residues without parentheses refer to LI Cap and residues with parentheses refer to La Cap. Alignments were performed with gap (Devereux et al . , 1984) . The region similar to the picornavirus vp3 was described previously (Bruenn et al. , 1989) .

DETAILED DESCRIPTION As used herein, the term "isolated" when used in conjunction with a nucleic acid molecule refers to: 1) a nucleic acid molecule which has been separated from an organism in a substantially purified form (i.e. substantially free of other substances originating from that organism) , or 2) a nucleic acid molecule having the same nucleotide sequence as the molecule in item 1 but not necessarily separated from the organism (i.e. synthesized nucleic acid molecules), or 3) a nucleic acid molecule which is a portion of the molecule of item 1 or a portion of the molecule of item 2. A "portion" refers to part of the entire molecule, that part being, for example, between about 60% and about 70% of the entire molecule, and preferably between about 63% and about 69% of the entire molecule.

As further used herein, the terms "corresponding to" or "having" or "as shown in" or "consisting of" when used in conjunction with a SEQ ID NO for a nucleotide sequence refer to a nucleotide sequence which is substantially the same nucleotide sequence, or derivatives thereof (such as deletion and hybrid variants thereof, splice variants thereof, etc.) . Nucleotide additions, deletions, and/or substitutions, such as those which do not affect the translation of the DNA molecule, are within the scope of a nucleotide sequence corresponding to or having or as shown in or consisting of a particular nucleotide sequence (i.e. the amino acid sequence encoded thereby remains the same) . Such additions, deletions, and/or substitutions can be, for example, the result of point mutations made according to methods known to those skilled in the art. It is also possible to substitute a nucleotide which alters the amino acid sequence encoded thereby, where the amino acid substituted is a conservative substitution or where amino acid homology is conserved. It is also possible to have minor nucleotide additions, deletions, and/or substitutions which do not alter the function of the resulting viral capsid polypeptide. These are also within the scope of a nucleotide sequence corresponding to or having or as shown in or consisting of a particular nucleotide sequence.

Similarly, the term "corresponding to" or "having" or "as shown in" or "consisting of" when used in conjunction with a SEQ ID NO for an amino acid sequence refers to an amino acid sequence which is substantially the same amino acid sequence or derivatives thereof. Amino acid additions, deletions, and/or substitutions which do not negate the ability of the resulting polypeptide to inhibit viral packaging are within the scope of an amino acid sequence corresponding to or having

or as shown in or consisting of a particular amino acid sequence. Such additions, deletions, and/or substitutions can be, for example, the result of point mutations in the DNA encoding the amino acid sequence, such point mutations made according to methods known to those skilled in the art. Substitutions may be conservative substitutions of amino acids. Two amino acid residues are conservative substitutions of one another, for example, where the two residues are of the same type. In this regard, proline, alanine, glycine, serine, and threonine, all of which are neutral, weakly hydrophobic residues, are of the same type. Glutamine, glutamic acid, asparagine, and aspartic acid, all of which are acidic, hydrophilic residues, are of the same type. Another type of residue is the basic, hydrophilic amino acid residue, which includes histidine, lysine, and arginine . Leucine, isoleucine, valine, and methionine, all of which are hydrophobic, aliphatic amino acid residues, form yet another type of residue. Yet another type of residue consists of phenylalanine, tyrosine, and tryptophan, all of which are hydrophobic, aromatic residues. Further descriptions of the concept of conservative substitutions are given by French and Robson 1983, Taylor 1986, and Bordo and Argos 1991.

As further used herein, the term "corresponding to" or "having" or "as shown in" or "consisting of" when used in conjunction with a SEQ ID NO for a nucleotide or amino acid sequence is intended to cover linear or cyclic versions of the recited sequence (cyclic referring to entirely cyclic versions or versions in which only a portion of the molecule is cyclic, including, for example, a single amino acid cyclic upon itself) , and is intended to cover derivative or modified nucleotide or amino acids within the recited sequence. For example, those skilled in the art will readily understand than an adenine nucleotide could be replaced with a methyladenine, or a

cytosine nucleotide could be replaced with a methylcytosine, if a methyl side chain is desirable. Nucleotide sequences having a given SEQ ID NO are intended to encompass nucleotide sequences containing these and like derivative or modified nucleotides, as well as cyclic variations. As a further example, those skilled in the art will readily understand that an asparagine residue could be replaced with an ethylasparagine if an ethyl side chain is desired, a lysine residue could be replaced with a hydroxylysine if an OH side chain is desired, or a valine residue could be replaced with a methylvaline if a methyl side chain is desired. Amino acid sequences having a given SEQ ID NO are intended to encompass amino acid sequences containing these and like derivative or modified amino acids, as well as cyclic variations. Cyclic, as used herein, also refers to cyclic versions of the derivative or modified nucleotides and amino acids.

With these definitions in mind, the subject invention provides an isolated purified viral capsid polypeptide capable of inhibiting viral packaging. The viral capsid polypeptide consists of a portion of a viral capsid protein of an RNA virus and includes the multimerization domain of the viral capsid protein. Preferably, the viral capsid polypeptide extends from the amino terminal of the viral capsid protein through to the carboxy terminal of the multimerization domain of the viral capsid protein. The multimerization domain of the viral capsid protein may, in one embodiment, have an eight-fold beta barrel structure. A beta barrel structure of a capsid protein refers to the portion of the protein which forms a beta barrel tertiary structure. The amino acid sequence of the capsid protein is the protein's primary structure, and the protein's secondary structure is the three dimensional structure of the protein (for example, an alpha helix, or

a beta-sheet, or a beta-turn) . The individual secondary structural elements of the protein next associate to form the protein's tertiary structure. The tertiary structure reflects the interactions between the amino acid side chains, as well as between amide bonds, of the protein. Two examples of general classifications of tertiary structure include helix bundles and beta barrels. Proteins may also self-assemble into multimeric forms that stabilize their tertiary structure. The orientation of individual monomers as a multimeric protein is referred to as the quaternary structure, and the portion of each protein involved in the formation of such multimers is referred to as a multimerization domain.

RNA viruses which have viral capsid proteins which include a multimerization domain are numerous. These RNA viruses include yeast, plant, and mammalian viruses. For example, there are two viruses of the yeast Saccharomyces cerevisiae which have a viral capsid protein (Cap) which includes a multimerization domain in its quaternary structure. These two viruses are ScVLl and ScVLa. Further descriptions of RNA viruses with multimerization domains, and in particular those with a beta barrel structure, can be found in Getzoff et al . 1986, Chelvanayagam et al . 1992, and Ross ann 1987. To explain the concept and meaning of multimerization domains and beta barrels further, a similarity had been observed between the picornavirus vp3 protein and a region of the cap protein of a Saccharomyces cerevisiae virus (Bruenn et al . 1989) . In the subset of viruses with icosahedral symmetry, there is extensive secondary and tertiary structural homology among the viral capsid proteins. All have one or more capsid proteins with an eight stranded anti-parallel beta-barrel structure (Rossmann, 1987) . In most of the plant viruses, such as tomato bushy stunt virus (TBSV) , Southern bean mosaic

virus (SBMV) , turnip yellow mosaic virus (TYMV) , and brome mosaic virus (BMV) , the capsid is composed of 180 copies of one such protein of about 30 kilodaltons, in which a central S domain of about 190 amino acids adopts the beta- barrel structure. These viruses have a T=3 symmetry. The small plus strand animal viruses, the picornaviruses, such as foot and mouth disease virus (FMDV) , poliovirus (polio) , human rhinovirus (HRV) , encephalomyocarditis virus (EMC) , and Mengovirus (Mengo) have a pseudo T=3 structure. Their icosahedral faces have three different capsid proteins, each of about 250 amino acids: VP1, VP2, and VP3. These are of different primary sequence but essentially have identical protein folding, and replace the three proteins of identical sequence present on each face of the T=3 plant virus capsid. There is no detectable primary sequence similarity between the T=3 plant virus capsid proteins and the picornavirus capsid proteins, but the structural homology between them and the S domain is striking (Rossmann, 1987) . These beta barrel structures, where determined, form a part of the multimerization domain of the capsid protein.

The capsid proteins of the plant and animal RNA viruses for which X-ray crystal structures exist have been compared by a process of three-dimensional mapping. This analysis demonstrates that the proportion of structurally equivalent residues is highest for VP3 versus the plant virus capsid proteins (Rossmann, 1987) . Among the picornaviruses, VP3 also shows the greatest conservation. For instance, the VP3 of Mengovirus is more than 90% structurally identical to the VP3 of HRV14 (Rossmann,

1987) , while they share only about 48% amino acid sequence similarity. Although there is no X-ray structure for the alphavirus Sindbis (SNBV) , high resolution electron microscopy has demonstrated a T=3 symmetry for the nucleocapsid. The single viral nucleocapsid protein, of

264 amino acids, has detectable sequence similarity to the picornavirus VP3.

A multimerization domain and a beta barrel structure, as used herein, are as described above. ScVLl and ScVLa are two examples of RNA viruses whose capsid proteins include a multimerization domain. The nucleotide and amino acid sequences of ScVLl are known (see Diamond et al . 1989) , and are provided herein as SEQ ID NO:l (nucleotide) and SEQ ID NO:2 (amino acid sequence of the cap protein) . The viral capsid polypeptide of the subject invention is encoded by nucleotides 30 to 1421 of SEQ ID NO:l (amino acids 1 to 473 of SEQ ID NO:2) . This viral capsid polypeptide is capable of inhibiting packaging of the ScVLl virus in Saccharomyces cerevisiae, and the sequence of the polypeptide consists of a portion (473 of 680 amino acids or about 70%) of the Cap protein of ScVLl, and includes the multimerization domain (about amino acids 203-448) .

The nucleotide and amino acid sequences of ScVLa are also known (see Park et al . 1996) , and are provided herein as SEQ ID NO:3 (nucleotide) and SEQ ID NO:4 (amino acid sequence of the cap protein) . The viral capsid polypeptide of the subject invention is encoded by nucleotides 24 to 1352 of SEQ ID NO:3 (amino acids 1 to 443 of SEQ ID NO: ) . This viral capsid polypeptide is capable of inhibiting packaging of the ScVLa virus in Saccharomyces cerevisiae, and the sequence of the polypeptide consists of a portion (443 of 697 amino acids or about 64%) of the Cap protein of ScVLa, and includes the multimerization domain (about amino acids 202-442) .

The subject invention further provides an isolated nucleic acid molecule encoding a viral capsid polypeptide capable of inhibiting viral packaging. The viral capsid polypeptide, as described above, consists of a portion of a viral capsid protein of an RNA virus and

includes the multimerization domain of the capsid protein. The nucleic acid molecule is preferably ribonucleic acid (RNA) , and can be genomic or recombinant, biologically isolated or synthetic. One embodiment of the nucleic acid molecule encoding a viral capsid polypeptide of the subject invention has a nucleotide sequence as shown in nucleotides 30 to 1421 of SEQ ID NO:1 (a nucleic acid molecule which encodes amino acids 1 to 473 of SEQ ID NO: 2) . This nucleic acid molecule encodes a viral capsid polypeptide which is capable of inhibiting packaging of the ScVLl virus in Saccharomyces cerevisiae, and the sequence of the polypeptide consists of a portion (473 of 680 amino acids) of the Cap protein of ScVLl, and includes the multimerization domain (about amino acids 203-448) .

Another embodiment of a nucleic acid molecule encoding a viral capsid polypeptide of the subject invention has a nucleotide sequence as shown in nucleotides 24 to 1352 of SEQ ID NO:3 (a nucleic acid molecule which encodes amino acids 1 to 443 of SEQ ID

NO: ) . This nucleic acid molecule encodes a viral capsid polypeptide which is capable of inhibiting packaging of the ScVLa virus in Saccharomyces cerevisiae, and the sequence of the polypeptide consists of a portion (443 of 697 amino acids) of the Cap protein of ScVLa, and includes the multimerization domain (about amino acids 202-442) .

The nucleic acid molecules of the subject invention can be expressed in desirable host cells using conventional techniques. The viral capsid polypeptide encoded thereby can be expressed in any desirable host and can be introduced into the host by introducing the nucleic acid molecules directly into the host cells or by using a vector system to introduce the nucleic acid molecules. If it is desirable to inhibit viral packaging in mammalian tissue culture cells (for example, Hela cells, Cv-l cells,

COS cells) in vitro expression can be used. For other applications of the subject invention, in vivo expression can be used, for example to inhibit viral packaging in plant species and/or yeast species. For example, a plant can be stably transformed with the nucleic acid molecule of the subject invention which renders the plant resistant to a particular virus (by inhibiting packaging of that virus in the transformed plant) . Seeds produced by that transformed plant could then be grown up to result in plants resistant to the virus.

Techniques for introducing the nucleic acid molecules into the host cells may involve the use of expression vectors which comprise the nucleic acid molecules. These expression vectors (such as plasmids and viruses; viruses including bacteriophage) can then be used to introduce the nucleic acid molecules into suitable host cells .

Various methods are known in the art for introducing nucleic acid molecules into host cells. One method is microinjection, in which nucleic acid is injected directly into the nucleus of cells through fine glass needles (RNA is injected directly into the cytoplasm of cells) . Alternatively, nucleic acid can be incubated with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE, for diethylaminoethyl) has been coupled. The nucleic acid sticks to the DEAE-dextran via its negatively charged phosphate groups. These large nucleic acid-containing particles stick in turn to the surfaces of cells, which are thought to take them in by a process known as endocytosis. Some of the nucleic acid evades destruction in the cytoplasm of the cell and escapes to the nucleus, where it can be transcribed into RNA like any other gene in the cell. In another method, cells efficiently take in nucleic acid in the form of a precipitate with calcium

phosphate. In electroporation, cells are placed in a solution containing nucleic acid and subjected to a brief electrical pulse that causes holes to open transiently in their membranes. Nucleic acid enters through the holes directly into the cytoplasm, bypassing the endocytotic vesicles through which they pass in the DEAE-dextran and calcium phosphate procedures (passage through these vesicles may sometimes destroy or damage nucleic acid) . Nucleic acid can also be incorporated into artificial lipid vesicles, liposomes, which fuse with the cell membrane, delivering their contents directly into the cytoplasm. In an even more direct approach, used primarily with plant cells and tissues, nucleic acid is absorbed to the surface of tungsten microprojectiles and fired into cells with a device resembling a shotgun.

Several of these methods, microinjection, electroporation, and liposome fusion, have been adapted to introduce proteins into cells. For review, see Mannino and Gould-Fogerite 1988, Shigekawa and Dower 1988, Capecchi 1980, and Klein et al . 1987.

Further methods for introducing nucleic acid molecules into cells involve the use of viral vectors. Since viral growth depends on the ability to get the viral genome into cells, viruses have devised clever and efficient methods for doing it. One such virus widely used for protein production is an insect virus, baculovirus. Baculovirus attracted the attention of researchers because during infection, it produces one of its structural proteins (the coat protein) to spectacular levels. If a foreign gene were to be substituted for this viral gene, it too ought to be produced at high level. Baculovirus, like vaccinia, is very large, and therefore foreign genes must be placed in the viral genome by recombination. To express a foreign gene in baculovirus, the gene of interest is cloned in place of the viral coat

protein gene in a plasmid carrying a small portion of the viral genome. The recombinant plasmid is cotransfected into insect cells with wild-type baculovirus DNA. At a low frequency, the plasmid and viral DNAs recombine through homologous sequences, resulting in the insertion of the foreign gene into the viral genome. Virus plaques develop, and the plaques containing recombinant virus look different because they lack the coat protein. The plaques with recombinant virus are picked and expanded. This virus stock is then used to infect a fresh culture of insect cells, resulting in high expression of the foreign protein. In the subject invention, the viral capsid polypeptide for inhibiting viral packaging can be expressed in a cell by introducing the nucleic acid encoding the viral capsid polypeptide by use of another viral vector. For a review of baculovirus vectors, see Miller (1989) . Various viral vectors have also been used to transform mammalian cells, such as bacteriophage, vaccinia virus, adenovirus, and retrovirus . As indicated, some of these methods of transforming a cell require the use of an intermediate plasmid vector. U.S. Patent No. 4,237,224 to Cohen and Boyer describes the production of expression systems in the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated in unicellular cultures including procaryotic organisms and eucaryotic cells grown in tissue culture. The DNA sequences are cloned into the plasmid vector using standard cloning procedures known in the art, as described by Sambrook et al . (1989) .

The viral capsid polypeptide encoded by the introduced nucleic acid molecule will inhibit viral packaging in the host cell, thereby protecting the host cell from infection by the virus.

Various modifications of the nucleic acid and amino acid sequences disclosed herein are covered by the subject invention. These varied sequences still encode a functional viral capsid polypeptide (capable of inhibiting viral packaging) . The invention thus further provides an isolated nucleic acid molecule encoding a viral capsid polypeptide, the nucleic acid molecule encoding a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, the second amino acid sequence selected from the group consisting of amino acids 1 to 473 of SEQ ID NO:2 and amino acids 1 to 443 of SEQ ID N0:4.

As indicated above, levels of the viral capsid polypeptide according to the subject invention can be expressed in a cell by introducing the nucleic acid molecule encoding the viral capsid polypeptide into the cell and by expressing the viral capsid polypeptide encoded thereby. For in vivo expression of viral capsid polypeptide, various gene therapy techniques can be utilized to get the nucleic acid molecule into the desired cell. In plants, for example, various plant specific promoters can be used as well as tissue specific promoters which permit expression of the viral capsid polypeptide in only those tissues that the promoter is "specific" for. In certain circumstances, it may be desirable to administer the viral capsid polypeptide to a subject, in which case the polypeptide is provided in a composition with a suitable carrier. The compositions may be made up in any suitable form appropriate for the desired use; e.g., oral, parenteral, or topical administration. Suitable dosage forms for oral use include tablets, dispersible powders, granules, capsules, suspensions, syrups, and elixirs. Inert diluents and carriers for tablets include, for example, calcium carbonate, sodium carbonate, lactose, and talc. Tablets may also contain

granulating and disintegrating agents such as starch and alginic acid, binding agents such as starch, gelatin, and acacia, and lubricating agents such as magnesium stearate, stearic acid, and talc. Tablets may be uncoated or may be coated by known techniques to delay disintegration and absorption. Inert diluents and carriers which may be used in capsules include, for example, calcium carbonate, calcium phosphate, and kaolin. Suspensions, syrups, and elixirs may contain conventional excipients, for example, methyl cellulose, tragacanth, sodium alginate; wetting agents, such as lecithin and polyoxyethylene stearate; and preservatives, e.g., ethyl-p-hydroxybenzoate.

Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions, and the like. They may also be manufactured in the form of sterile solid compositions which can be dissolved or suspended in sterile injectable medium immediately before use. They may contain suspending or dispersing agents known in the art. Examples of parenteral administration are intraventricular, intracerebral, intramuscular, intravenous, intraperitoneal, rectal, and subcutaneous administration.

In accordance with the above description of the subject invention, it should be readily apparent to those skilled in the art that it is possible to interfere with the replication of a number of RNA viruses by production of viral capsid polypeptides (such polypeptides being less than the entire capsid protein, and, in one preferred embodiment, being an amino terminal fragment of the entire capsid protein) . Interference can occur by a number of mechanisms. The Saccharomyces cerevisiae double-stranded RNA viruses ScVLl and ScVLa, which exist as permanent persistent infections of their host cells, can be cured very efficiently by production of N-terminal fragments of

their capsid proteins. These totiviruses produce only two proteins: a capsid protein (Cap) and a Cap-Pol fusion protein with RNA-dependent RNA polymerase activity. Three types of interference can be detected: interference due to overproduction of both Cap and Cap-Pol, interference due to overproduction of Cap (and consequent distortion of the Cap to Cap-Pol ratio) , and interference due to negative complementation by N-terminal fragments of Cap. Some N-terminal fragments of Cap appear to be incorporated into viral particles, but only in the presence of a complete Cap protein. Incorporation of N-terminal fragments of Cap can result in the formation of defective particles.

MATERIALS AND METHODS

Vectors and strains

The vectors used for expression of cDNAs were pGl, with a yeast glucose-6-phosphate dehydrogenase promoter (Schena et al . , 1991) , or pG4 (see below) . The original source of the full-size LI cDNA was the NIH. All other cDNA clones were derived by applicants . The assays for curing of ScVLa were in strain T120 {MATa makl O trpl ura3 La Ll - 0 Ml - 0) and those for curing of ScVLl were in strain T141-3 (MATa trpl l eu2 his Gal ^* LI Ml La - 0 ) . T120 and T141 were both derived from a cross between BJ2169 (RPMI) and M179 (Yeast Genetics Stock Center) . T141-3 was derived from T141 by curing of ScVLa with pGLal (see below) followed by loss of the plasmid by growth on nonselective media. Strain T141-4, used for expression of both Cap and truncated Cap proteins, was derived from T141-3 by curing of ScVLl with pGLld3 (see below) and curing of the plasmid by growth on nonselective media.

Transformation

Transformation of yeast was by the lithium acetate-polyethylene glycol method (Elbe, 1992) , using selection for TRP1 or LEU2. Strains were always grown on selective media, to retain plasmids, unless otherwise stated.

Mutagenesis

Site-directed mutagenesis was of selected restriction fragments in pGEM7Zf ⁺ , by the method of Kunkel (Kunkel, 1985) . Every mutagenized fragment was completely sequenced before being returned to expression vector constructs for testing. Deletions were made by excising restriction fragments in pGEM7Zf ⁺ and returning the truncated restriction fragments to expression vector constructs by using restriction sites in the multiple cloning sequence. Deletion mutants were made by incorporating restriction fragments of La or Ll cDNA clones containing their entire Cap genes, after blunt- ending with DNAP I Klenow fragment, into blunt-ended restriction sites in pGl or its derivative pG4. Plasmid pG4 was constructed by replacing the Ba ΑΪ -Sal l region of pGl with the sequence having SEQ ID NO:5 : GATCTATAAAGATGTCGGATCCCCCGGGGGTACCGTCGAC, providing an AUG in the vector sequence (underlined) . The La deletion constructs had the following bases from La (from SEQ ID NO:3) : pGLa4 , bases 16-2724; pGLal, bases 16-2360; pGLaldl, bases 16-1536; pGLald2, bases 16-1480; pGLald3 , bases 16-1349; pGLald , bases 16-1304; pGLald5, bases 16- 1279; pGLaldδ, bases 16-1224; pGLald7, bases 58-1349; pGLaldδ, bases 315-1349. The Ll deletion constructs had the following bases from Ll (from SEQ ID N0:l) : pGLl, bases 1-4571; pGLldl, bases 1-2095; pGLld2 , bases 1-1835; pGLld3, bases 1-1455; pGLld4, bases 1-1332; pGLlld5, bases 1-1326. The nonsense mutants of La had bases 16-2360 of

La (of SEQ ID NO:3) ; the nonsense mutants of Ll had bases 1-2095 of Ll (of SEQ ID NO:l) . The nonsense mutants of Ll were pGLalm3 (K506O) , pGLalm4 (14430) , pGLalm5 (S4190) , and pGLalmδ (E110) , where 0 indicates ocher, and K506O indicates a change of a lysine at residue 506 to ocher. The nonsense mutants of Ll were pGLlmlδ (G501O) , pGLlml4 (Y4850) , pGLlmll (F4740) , pGLlml2 (E4350) , and pGLlml3 (Q10O) . The La sequence is in GenBank Accession No. U01060 and the Ll sequence is in GenBank Accession No. M28353 (Diamond et al . , 1989) .

RNA extraction and Northern hybridization

Crude RNA (including DNA) was prepared from whole cells by phenol extraction (Bruenn and Kane, 1978) . These preparations have primarily dsRNA and the smaller single-stranded RNAs of the cell. For Northern analysis, total RNA was extracted with phenol and glass beads . Northern transfers of native RNA and hybridization to T7 or SP6 RNA transcripts of cDNAs were performed as described (Huan et al . , 1991) . Probes were made from pGEMLl-4 (bases 1-521 of Ll [SEQ ID N0:1] in pGEM7Zf ⁺ ) or pGEMLal-6 (bases 16-487 of La [SEQ ID NO:3] in the same vector) .

Western transfers

ScV particles were prepared by differential centrifugation and CsCl equilibrium gradient centrifugation as described (Shen and Bruenn, 1993) . SDS- polyacrylamide gel electrophoresis (SDS-PAGE) and Western transfers were performed as previously described and probed with polyclonal antibodies to the ScVLl Cap (Diamond et al . , 1989) . Crude protein extracts of cells were prepared by mechanical lysis as described (Sambrook et al . , 1989) using 100 μl of buffer for a 10-ml culture at OD ₅₅₀ = 1.

Killer assays

Detection of the ScVMl phenotype (production of kl killer toxin) was performed as described (Huan et al . , 1991) .

EXAMPLE I

Overproduction of Cap fragments interferes with ScVLa

An initial observation was that overproduction of La Cap from a cDNA expression vector resulted in curing of ScVLa (Yao et al . , 1995) . This is in contrast to what had been reported for ScVLl (Valle and Wickner, 1993) . In accordance with the subject invention, several deletion mutants lacking C-terminal portions of the La Cap were prepared and tested for their ability to cure ScVLa.

Unexpectedly, all of these (pGLaldl-pGLald3) were at least as efficient at curing ScVLa as constructs containing the full-size La cap reading frame (see Table 1) .

More particularly, the dsRNAs from a series of transformants expressing portions of the La Cap protein in a strain (T120) that originally had ScVLa (but no ScVLl) were elaborated on a 1% agarose gel . The control (vector alone, pGl) , the two nonsense mutants pGLalm5 and m6, and the deletion pGLad5 did not interfere with ScVLa but the rest of the expression vector constructs did interfere.

The transformant colonies isolated after transformation with the expression plasmid were all cured of ScVLa after their initial 20 or so generations of growth to form a visible colony. Since the assay for the presence of ScVLa requires an RNA extraction, only 10 clones of each construct were tested. None retained ScVLa, while the control, with vector alone, always retained ScVLa. Previous constructs lacking small portions of the C-terminus of the La Cap did not interfere efficiently (Yao et al . , 1995), but these had C-terminal

substitutions from the Ll Cap, which apparently affect the La Cap interaction with wild-type La Cap monomers.

Several more constructs were made, in which increasingly larger deletions were made from the C- terminus of the La Cap, in order to determine which portion of the gene was required for interference. These experiments (Table 1) delimited the required region to the N-terminal 425 amino acids of La Cap, which is a protein of 697 amino acids (see SEQ ID NO:4) . Removal of even a few amino acids from the N-terminus was fatal (Table 1) . This interpretation of the data assumes that all the noninterfering constructs were properly transcribed, and this was verified by Northerns of nondenatured RNA.

Specifically, a 1.5% nondenaturing agarose gel of total RNA from transformants was denatured and transferred to nitrocellulose and probed with an La minus-strand probe. Those transformants not cured by the expression vectors (pGl, pGLalm5 and m6, and pGLald5) still had La dsRNA. All other transformants were missing La, and all the transformants that should express La RNA

(including those that did not interfere with ScVLa) had several bands of La plus-strand transcript from the expression vector. The multiple bands were the result of alternate secondary structures of the RNAs, since a denaturing gel shows only one band. Note that progressively larger deletions (dl-d5) had smaller transcripts. Every construct was shown to be well- transcribed. The multiple bands of La transcript from the expression vector were the result of alternate secondary structures of the RNA, as shown by Northerns with denatured RNA.

In many cases, interference with viral replication can occur by production of interfering, untranslated viral RNAs (Fitchen and Beachy, 1993) . For instance, this will work with ScVLl, by overproduction of

its packaging signal (Huan et al . , 1991; Shen and Bruenn, 1993) . However, the region of La responsible for the interference observed here does not include its putative packaging signal. Nevertheless, another series of constructs were made that terminate La Cap synthesis with nonsense mutations, preserving the rest of the sequence intact. As expected, any mutant with a nonsense codon preceding the C-terminus of the essential region was incapable of interference, while mutants with nonsense codons C-terminal to the essential region were functional (Table 1) . All of these mutants are normally transcribed as well. This is strong evidence that N-terminal fragments of the La Cap protein are responsible for the observed interference. Experiments in which the presence of ScVLa is tested immediately after transformation with cDNA expression vectors demonstrate that interference with ScVLa (mirroring the situation with ScVLl) is much more effective with the N-terminal fragments of Cap than with the full-size Cap. As expected, La Cap or its N-terminal fragments do not interfere with ScVLl, and Ll Cap or its N-terminal fragments do not interfere with ScVLa.

EXAMPLE II

Overproduction of Cap fragments interferes with ScVLl

Given the sensitivity of ScVLa to overproduction of N-terminal fragments of its Cap protein, as well as the complete protein, it was surprising that ScVLl was not sensitive to overproduction of its Cap protein. A number of deletion constructs of ScVLl cap were prepared and overproduction of N-terminal fragments of the Ll Cap tested for their effect on ScVLl. Again, the N-terminal fragments interfered with ScVLl (Table 1) .

Specifically, the dsRNAs from a series of transformants expressing portions of the Ll Cap protein in a strain (T141-3) that originally had ScVLl (but no ScVLa) were elaborated on a 1% agarose gel. Note that the constructs that did interfere with ScVLl (Lld2 and Lld3) also eliminated ScVMl . The region responsible was mapped by deletions to the N-terminal 476 amino acids of Ll Cap, a protein of 680 amino acids (see SEQ ID NO:2) (Table 1) . In this case, the initial screening assay for interference is the absence of the killer phenotype, which depends on ScVMl, whose dsRNA encodes a killer toxin, and whose replication depends on ScVLl. This provides a more facile assay, so more clones can be screened rapidly. The absence of ScVLl and ScVMl in the cured clones was verified by RNA extraction.

Dependence on the N-terminal fragments of Cap, rather than its RNA, was verified by making nonsense mutants of Ll cap (Table 1) . However, none of the nonsense mutants of Ll cap (except when combined with deletions; see below) successfully interfered with ScVLl. This appears to be partially due to low steady-state mRNA levels in these expression constructs. Those Ll nonsense mutants that should interfere with viral particle assembly (Llmll, Llml4, Llml5) did not have significant steady-state levels of transcript, while the deletions (Lldl-Lld5) and the nonsense mutants that should not interfere (Llml2 and Llml3) all had reasonable levels of mRNA. The mutants Llmll, Llml4, and Llml5 did have reduced levels of Ll dsRNA present, as though interference was occurring but was not very efficient. As above, several bands of transcript were visible, and the deletions had progressively smaller mRNAs . The low levels of mRNA in those constructs that should interfere with ScVLl (Llmll, Llml4 , and Llml5) were correlated with a reduced amount of Ll dsRNA, as though interference was

still occurring but was inefficient. However, the two nonsense mutants that should not interfere with ScVLl (Llml2 and Llml3; as deduced from the results with the deletion mutants) did make levels of mRNA as high or higher than those of the deletion constructs that interfere (Lld2 and Lld3) , so one may still conclude that interference requires Cap, not cap mRNA.

All of the deletion mutants and nonsense mutants (with the exception of Llml3 , which produces a Cap of only nine amino acids) produced fragments of Ll Cap of the expected sizes, detectable by polyclonal antibodies. Most of the deletion and nonsense mutants of Ll cap were tested for the presence of the expected Ll Cap N-terminal fragment. Crude protein from transformants was run on a 15% SDS-PAGE, transferred to nitrocellulose, probed with anti-Ll Cap polyclonal antibody, and antigen-antibody complexes were visualized with bacterial alkaline phosphatase-conjugated protein A. Ll was the control Cap from wild-type ScVLl particles and M was prestained protein markers. Although there appeared to be somewhat lower levels of the Llml5, Llml4, and Llmll proteins than the equivalent (Lld3) deletion protein, consistent with their inability to interfere, there was not a good correlation between RNA level and protein level. Another construct (pGLlmlld3) , with the mil mutation combined with a deletion of the region of the mRNA encoding amino acids 579-680 of SEQ ID NO:2, interfered as well as d2 (Table 1) . Hence the unexpected failure of the nonsense mutants Llmll, Llml4 , and Llml5 to interfere was the result of some complex interaction between the 3' portion of the Ll cap mRNA and the nonsense codon, presumably during translation. Both the nonsense mutants and the deletion mutants clearly implicated the amino-ter inal portion of Ll Cap, amino acids 1-473 of SEQ ID NO: 2, in interference.

EXAMPLE III

Mechanism of interference

The mechanism of interference by overproduction of both Cap and Cap-Pol is thought to be titration of a cellular factor required for viral replication (Valle and Wickner, 1993) . Drastic alterations in the ratio of Cap to Cap-Pol are also known to interfere with viral replication (Dinman and Wickner, 1992) . Consequently, interference by overproduction of Cap is probably due to distortion of the ratio of Cap to Cap-Pol and is efficient only if the amount of protein synthesized by virtue of the presence of an expression vector is close to that normally produced by the virus: in other words, when the viral copy number is low.

However, interference by N-terminal fragments of Cap is insensitive to viral copy number and must therefore take place by a third mechanism. One possibility is negative complementation: the incorporation of one or more copies of a partially functional Cap monomer (an N-terminal fragment) in a nascent viral particle may disrupt the particle. Since there are 118 copies of Cap (and two of Cap-Pol) per particle, if incorporation of a single fragment disrupted a particle, production of N-terminal fragments of Cap would be very efficient in interfering with ScV packaging. N-terminal fragments of the Cap protein of another totivirus are known to be incorporated into aberrant particles (Cadd et al . , 1994) . Viral particle preparations were made from a strain without any resident ScV particles (no ScVLa or ScVLl) after introduction of expression vectors producing only the interfering N-terminal fragments of La. Expression of the full-size La Cap resulted in efficient production of empty ScVLa particles, but expression of

interfering N-terminal fragments of Cap produced no detectable particles. This result was confirmed by SDS-PAGE of fractions from the gradients.

The same was true of strains producing only the N-terminal fragments of the Ll Cap. However, when two plasmids were introduced -- one making the Ll Cap and Cap-Pol proteins and a second making an N-terminal fragment of Ll Cap (Lld2) -- the result was the production of empty particles that appeared to have both the fragment and the full-size Cap.

The same particles that apparently had both the full-size Cap and the deletion product were seen when the helper vector produced Ll Cap but not Ll Cap-Pol. This is consistent with the negative complementation model for interference, in which a Cap-Cap interaction is implicated. Only the larger N-terminal fragments of Cap are detected in heteromultimeric viral particles, however. For instance, Lld3 and Lald3 Cap do not appear in viral particles in similar experiments.

EXAMPLE IV

Three types of interference were observed with the ScV life cycle due to production of viral proteins from cDNA expression vectors. First, overproduction of both Cap and Cap-Pol, as has been observed previously (Valle and Wickner, 1993) , will eliminate ScVLl. This was postulated to involve interaction with cellular proteins. Second, overproduction of Cap alone will cure ScVLa, but not ScVLl. This appears to be due to distortion of the ratio of Cap to Cap-Pol, which is critical (Ribas and Wickner, 1992) and affects ScVLa but not ScVLl because the copy number of the former is much lower and it is consequently much more sensitive to production of Cap from cDNA clones. Overproduction of Ll Cap is also known to

elevate the number of ScVLl particles per cell (Wickner et al . , 1991) , making it more difficult to distort the ratio of Cap to Cap-Pol by overexpression of Cap. ScVLa copy number may not be derepressed by overproduction of La Cap. Another totivirus, LRV, can also be cured by overexpression of its capsid polypeptide (Widmer, 1995) . Third, and most interesting, is the highly efficient elimination of either ScVLl or ScVLa by overproduction of N-terminal fragments of their capsid polypeptides. This is a unique method of viral interference .

Interference with ScVLa and ScVLl by synthesis of fragments of Cap is highly efficient. This interaction might be with either a cellular or viral gene product. If interaction were with some cellular component, this interaction would also be expected to take place with wild-type Cap, which it does not, since curing by wild-type Ll Cap is not detectable and is less efficient with La Cap. Hence, some abnormal interaction must be taking place with some component of the viral particle. There are only three components in the viral particle: Cap, Cap-Pol, and the viral RNA. These experiments implicate Cap in the fatal interaction.

Production of a complete Cap protein from a nuclear promoter results in formation of empty viral particles. However, fragments of Cap lacking the

C-terminus of the protein are not detectably incorporated into viral particles except in the presence of equivalent quantities of wild-type Cap. They appear to be partially crippled in multimer formation. Some N-terminal fragments of Cap appear to be capable of some but not all of the interactions required for particle formation. Recent cryoelectron microscopy has demonstrated that ScVLl (as well as another fungal dsRNA virus) does have 120 copies of Cap arranged in 12 pentamers (Cheng et al . , 1994) . The fragments would form heteropentons incapable of correct

assembly into particles (e.g., Lld3), or heteromultimeric particles incapable of packaging or replicating the viral RNA (e.g. , Lld2) .

The region of Cap required for interference includes the region of Ll and La Cap with 37.7% amino acid identity, the only major region of sequence similarity between the two, and its C-terminal border is close to the C-terminal limit of the region with some similarity to the picornavirus vp3 (Bruenn et al . , 1989) , as shown in Fig. 1. The C-terminal border of the La Cap required for interference maps almost precisely to the end of the region with similarity to vp3 (425-442 compared to 443) . This is consistent with the existence of a similar protein fold (an eightfold beta barrel) in ScV responsible for multimerization.

This very efficient method of interference with viral replication may be useful in other viral systems. Specifically, it may be readily applicable to making transgenic plants resistant to plant viruses. Many of these are known to have single capsid polypeptides with an eightfold beta barrel structure (Rossmann, 1987) that may also be susceptible to interference by N-terminal capsid polypeptide fragments.

Although preferred embodiments have been depicted and described in detail herein, it will be apparent to those skilled in the relevant art that various modifications, additions, substitutions and the like can be made without departing from the spirit of the invention and these are therefore considered to be within the scope of the invention as defined in the claims which follow.

TABLE 1 Curing of ScVLa and ScVLl

Deletions Nonsense mutants

Construct aa La cap Curing (%) Construct aa La cap Curing (%)

pGLa4 1-697 100 pGLalm3 1-505 100 pGLal 1-697 100 pGLalπvl 1-442 100 pGLaldl 1-502 100 pGLalm5 1-418 <10 pGLald2 1-476 100 pGLalm6 1-10 <10 pGLald3 1-443 100 pGLald4 1-428 50 pGLald5 1-418 <10 pGLald6 1-401 <10 pGLald7 6-443 <10 pGLaldδ 98-443 <10

Construct aa Ll cap Curing (%) Construct aa Ll cap Curing (%)

pGLl ^a 1-680 100 pGLlmlδ 1-500 <5 pGLldl 1-680 <5 pGLlml4 1-484 <5 pGLld2 1-603 100 pGLlmll 1-473 <5 pGLld3 1-476 80-100 pGLlml2 • 1-434 <5 pGLld4 1-435 <5 pGLlml3 1-9 <5 pGLld5 1-433 <5 pGLlmlld3 1-473 100

Complete Ll cDNA, producing both Cap and Cap-Pol.

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SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: The Research Foundation of State University of New York

(ii) TITLE OF INVENTION: CAPSID POLYPEPTIDES AND USE TO

INHIBIT VIRAL PACKAGING

(iii) NUMBER OF SEQUENCES: 5

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Nixon, Hargrave, Devans & Doyle LLP

(B) STREET: Clinton Square, P.O. Box 1051

(C) CITY: Rochester

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(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

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(D) SOFTWARE: Patentln Release #1.0, Version #1.30

(vi) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: U.S. Serial No. 08/674,351

(B) FILING DATE: July 2, 1996

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Goldamn, Michael L.

(B) REGISTRATION NUMBER: 30,727

(C) REFERENCE/DOCKET NUMBER: 19226/741

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: (716) 263-1304

(B) TELEFAX: (716) 263-1600

(2) INFORMATION FOR SEQ ID NO:1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 4580 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 :

GAAAAATTTT TAAATTCATA TAACTCCCCA TGCTAAGATT TGTTACTAAA AACTCTCAAG 60

ATAAATCGTC TGATCTATTC TCTATTTGTT CTGATCGCGG AACTTTTGTT GCTCATAATA 120

GAGTTCGGAC TGATTTCAAG TTTGACAACT TAGTATTTAA CCGAGTTTAT GGTGTTTCTC 180

AAAAATTTAC GTTAGTCGGT AACCCGACAG TCTGCTTTAA TGAAGGCAGT TCTTACCTAG 240

AAGGTATTGC TAAAAAGTAC CTAACTTTGG ATGGAGGACT TGCCATTGAC AATGTCCTCA 300

ATGAGTTGAG ATCCACTTGC GGTATCCCAG GTAATGCTGT TGCGTCTCAT GCGTATAATA 360

TTACATCCTG GCGTTGGTAT GACAATCACG TGGCACTGTT GATGAACATG TTGCGTGCTT 420

ACCACTTACA AGTATTGACC GAACAGGGCC AATATAGCGC TGGAGATATC CCTATGTACC 480

ATGATGGACA TGTCAAAATC AAGCTACCAG TGACTATCGA TGACACGGCA GGCCCAACAC 540

AATTCGCTTG GCCTAGTGAC AGGTCTACTG ATTCGTATCC TGATTGGGCA CAGTTTTCTG 600

AATCATTTCC ATCAATCGAC GTCCCGTACC TAGATGTTAG GCCATTGACC GTAACGGAAG 660

TCAATTTCGT GCTTATGATG ATGAGTAAGT GGCATAGACG TACTAACTTA GCGATAGACT 720

ACGAGGCACC CCAACTAGCT GATAAGTTCG CTTACCGCCA TGCGCTTACT GTTCAAGACG 780

CTGACGAGTG GATAGAAGGC GATAGAACTG ATGACCAGTT CCGCCCCCCC TCGTCTAAAG 840

TAATGTTATC GGCACTTCGT AAGTACGTGA ACCGTAACAG GCTGTACAAT CAGTTTTACA 900

CTGCAGCACA ACTGTTAGCT CAAATTATGA TGAAACCTGT CCCTAACTGC GCTGAGGGCT 960

ACGCTTGGCT GATGCATGAC GCATTGGTCA ATATACCAAA ATTTGGGTCT ATTCGAGGAA 1020

GGTACCCTTT TTTGTTATCA GGTGATGCAG CGTTGATTCA GGCTACAGCC CTAGAAGACT 1080

GGTCTGCTAT CATGGCGAAA CCCGAGCTGG TGTTCACTTA CGCGATGCAG GTGTCAGTAG 1140

CGTTAAACAC CGGACTATAC TTACGTCGCG TTAAGAAAAC AGGCTTCGGC ACAACTATAG 1200

ATGACAGCTA TGAAGATGGA GCGTTTTTGC AACCGGAGAC GTTCGTTCAG GCCGCACTAG 1260

CATGTTGTAC CGGACAAGAT GCGCCCCTAA ATGGGATGTC AGATGTGTAT GTCACTTATC 1320

CAGATCTTCT AGAATTTGAT GCTGTTACAC AAGTACCCAT CACGGTCATT GAGCCCGCTG 1380

GCTATAACAT TGTTGATGAT CATTTAGTGG TTGTGGGTGT ACCTGTGGCA TGTTCACCAT 1440

ACATGATATT TCCAGTAGCT GCGTTTGATA CTGCAAATCC TTACTGTGGG AATTTTGTCA 1500

TTAAGGCTGC TAACAAGTAT CTCCGTAAGG GTGCCGTGTA TGATAAACTC GAAGCATGGA 1560

AGTTGGCCTG GGCACTGAGG GTAGCCGGGT ATGACACTCA CTTCAAAGTG CATGGCGATA 1620

CACACGGCTT AACTAAGTTC TATGCTGACA ACAGTGACAC ATGGACACAC ATACCTGAAT 1680

TTGTCACTGA CGGTGACGTG ATGGAAGTAT TCGTTACTGC CATCGAACGC AGAGCTAGAC 1740

ATTTCGTTGA ACTACCTAGA CTGAATTCAC CAGCATTCTT CAGATCTGTA GAAGTCAGCA 1800

CCACTATATA TGATACTCAT GTGCAGGCTG GTGCGCATTC AGTGTATCAT GCTAGACGAA 1860

TCAATCTTGA TTATGTTAAG CCTGTTTCGA CCGGCATTCA GGTGATCAAT GCGGGCGAAC 1920

TTAAGAACTA CTGGGGTAGT GTGCGTCGTA CTCAGCAGGG TTTAGGAGTG GTAGGTCTTA 1980

CGATGCCAGC TGTAATGCCT ACCGGAGAAC CTACAGCTGG CGCTGCCCAC GAAGAGTTGA 2040

TAGAACAGGC GGACAATGTT TTAGTAGAGT AAACGTAATC GAACCCTCAC ACGGACCCCG 2100

CCCTACAAGG TACATACTGC AGGAACCAGG TACGTACCCT GCGTGGATTA GATTCAGGAA 2160

CAGAGTACAA GCTGTATCGC GTCAGAAAGC CACTCACTTC TTGTTTGACA TCGTACCTGC 2220

CGCAGTAATT AGTGATTTTA CTACGTCTGA CACGTCTTCG TTTGCATACA AATCGCACAC 2280

CTACGCTGCA AATGTAACAG CATTGAGGTT CAGTGACACT TATGCCTTGT ACGTACAGAC 2340

TGATACCAAT ATGACAATTT TAAGCCCAGC GGCGCGTCGC CAGGCTTCTG CGACGTACTC 2400

ACAGGTGGCA GGGTTTTGTT ATAACACACC TACCGTTATG GATTCGCTAG CGAATATCTT 2460

GGACGTAGAC CGCAATATAC GACCCAAACA CTTCAAGGGT TTACGGCTAT ACACCAGCTC 2520

TAAGGTCACT GCTCAACATC ATACTCACTT GCGGCCAGAC GAGCTAGTGG AAGCGGCCGC 2580

AAAGGTCTCG CCTAGACGTA AATACTACCT AATGTGTGTA GTTGAGCTGC TCGCGAACTT 2640

ACAAGTAGAT CTTGAAGCAG CAGTAGCTAC TATTCTAGCA TATGTCCTGA CACTAAGTGA 2700

AAAATTTGTA CCAATTTTCT TGGATTCTAG AGCAATATGG GTCGGTGAGC CTGGGCCTGA 2760

TGCTCTGACT GCACGTCTCA AGGCCAGTAG TGGGCAGATC AAGAGCATAC ACACGGCTGA 2820

TTACGAACCA CTCACTGAAC TATTCGAGTT AGCAGTATTG ATGAACCGAG GTGTTGGCCA 2880

TGTCTCTTGG CAAGCTGAAA AGGATCATCG CTTGAATCCC GACGTGGCTG TAGTTGATCA 2940

AGCACGGCTA TATTCGTGTG TGCGCGACAT GTTCGAAGGA TCAAAGCAGA CGTATAAATA 3000

TCCCTTTATG ACGTGGGATG ACTACACTGC AAACAGATGG GAGTGGGTTC CAGGTGGCAG 3060

TGTCCACTCT CAATACGAAG AAGACAACGA TTATATCTAT CCTGGTCAGT ATACTAGGAA 3120

CAAGTTCATA ACTGTTAACA AAATGCCCAA ACACAAAATA TCTAGAATGA TAGCATCACC 3180

GCCTGAGGTA CGAGCTTGGA CGTCGACGAA GTACGAATGG GGCAAGCAAC GTGCTATCTA 3240

CGGGACGGAT CTACGAAGTA CACTGATAAC TAACTTTGCA ATGTTCAGGT GCGAGGATGT 3300

TCTCACTCAC AAGTTCCCAG TAGGCGACCA GGCAGAGGCA GCAAAGGTGC ACAAACGGGT 3360

GAACATGATG CTGGACGGTG CCTCTAGTTT CTGCTTCGAT TATGATGACT TCAATTCTCA 3420

GCATTCGATA GCTAGTATGT ATACGGTTTT GTGCGCTTTC AGGGACACAT TTAGTCGCAA 3480

CATGTCTGAT GAACAAGCAG AGGCGATGAA CTGGGTGTGT GAGTCCGTCA GACACATGTG 3540

GGTACTAGAT CCTGATACCA AGGAGTGGTA CAGACTACAA GGTACATTAC TGTCAGGATG 3600

GCGGTTAACC ACATTTATGA ATACTGTGCT AAACTGGGCG TATATGAAAT TAGCTGGCGT 3660

ATTTGATCTG GATGACGTTC AAGACTCGGT ACACAACGGT GATGATGTTA TGATTAGTCT 3720

CAACCGCGTG AGCACAGCAG TGAGAATAAT GGACGCTATG CACCGGATAA ATGCGCGAGC 3780

ACAGCCGGCG AAGTGTAACT TGTTTTCGAT AAGTGAATTT CTGAGGGTAG AACACGGTAT 3840

GAGCGGAGGC GATGGTCTTG GGGCTCAGTA CTTAAGTAGG TCTTGTGCTA CTCTTGTACA 3900

CAGTAGGATT GAGTCTAACG AACCACTGTC AGTAGTACGA GTTATGGAAG CAGACCAGGC 3960

TAGATTGCGC GACCTGGCAA ACAGAACGCG GGTACAATCT GCGGTAACAG CGATAAAAGA 4020

ACAACTCGAC AAACGTGTCA CTAAGATATT CGGAGTTGGT GATGACGTTG TGCGCGACAT 4080

ACACACAGCT CACAGGGTGT GTGGCGGTAT CTCGACTGAT ACCTGGGCAC CGGTTGAAAC 4140

TAAGATAATA ACAGACAATG AAGCATATGA AATACCATAC GAAATAGATG ATCCATCATT 4200

TTGGCCAGGG GTAAATGATT ATGCTTATAA AGTCTGGAAA AATTTCGGAG AACGACTCGA 4260

ATTTAATAAA ATTAAAGATG CCGTAGCTAG AGGGAGTAGG AGCACTATAG CTCTGAAACG 4320

TAAGGCTAGG ATAACATCTG AGAAGAATGA ATTCGCTAAC AAGTCGGAAT GGGAAAGGAC 4380

AATGTACAAA GCCTATAAGG GTTTGGCAGT CTCATACTAT GCTAACCTGA GCAAATTCAT 4440

GAGTATACCA CCAATGGCGA ACATTGAATT TGGGCAGGCT AGATATGCTA TGCAAGCAGC 4500

CCTTGATAGT TCTGATCCAC TCCGGGCATT ACAGGTCATA CTGTAGTTGC CAAAAAGATA 4560

ATGGGAATTA CCCATATGCA 4580

(2) INFORMATION FOR SEQ ID NO:2 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 680 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

Met Leu Arg Phe Val Thr Lys Asn Ser Gin Asp Lys Ser Ser Asp Leu 1 5 10 15

Phe Ser He Cys Ser Asp Arg Gly Thr Phe Val Ala His Asn Arg Val 20 25 30

Arg Thr Asp Phe Lys Phe Asp Asn Leu Val Phe Asn Arg Val Tyr Gly 35 40 45

Val Ser Gin Lys Phe Thr Leu Val Gly Asn Pro Thr Val Cys Phe Asn 50 55 60

Glu Gly Ser Ser Tyr Leu Glu Gly He Ala Lys Lys Tyr Leu Thr Leu 65 70 75 80

Asp Gly Gly Leu Ala He Asp Asn Val -Leu Asn Glu Leu Arg Ser Thr 85 90 95

Cys Gly He Pro Gly Asn Ala Val Ala Ser His Ala Tyr Asn He Thr 100 105 110

Ser Trp Arg Trp Tyr Asp Asn His Val Ala Leu Leu Met Asn Met Leu 115 120 125

Arg Ala Tyr His Leu Gin Val Leu Thr Glu Gin Gly Gin Tyr Ser Ala 130 135 140

Gly Asp He Pro Met Tyr His Asp Gly His Val Lys He Lys Leu Pro 145 150 155 160

Val Thr He Asp Asp Thr Ala Gly Pro Thr Gin Phe Ala Trp Pro Ser 165 170 175

Asp Arg Ser Thr Asp Ser Tyr Pro Asp Trp Ala Gin Phe Ser Glu Ser 180 185 190

Phe Pro Ser He Asp Val Pro Tyr Leu Asp Val Arg Pro Leu Thr Val 195 200 205

Thr Glu Val Asn Phe Val Leu Met Met Met Ser Lys Trp His Arg Arg 210 215 220

Thr Asn Leu Ala He Asp Tyr Glu Ala Pro Gin Leu Ala Asp Lys Phe 225 230 235 240

Ala Tyr Arg His Ala Leu Thr Val Gin Asp Ala Asp Glu Trp He Glu 245 250 255

Gly Asp Arg Thr Asp Asp Gin Phe Arg Pro Pro Ser Ser Lys Val Met 260 265 270

Leu Ser Ala Leu Arg Lys Tyr Val Asn Arg Asn Arg Leu Tyr Asn Gin 275 280 285

Phe Tyr Thr Ala Ala Gin Leu Leu Ala Gin He Met Met Lys Pro Val 290 295 300

Pro Asn Cys Ala Glu Gly Tyr Ala Trp Leu Met His Asp Ala Leu Val 305 310 315 320

Asn He Pro Lys Phe Gly Ser He Arg Gly Arg Tyr Pro Phe Leu Leu 325 330 335

Ser Gly Asp Ala Ala Leu He Gin Ala Thr Ala Leu Glu Asp Trp Ser 340 345 350

Ala He Met Ala Lys Pro Glu Leu Val Phe Thr Tyr Ala Met Gin Val 355 360 365

Ser Val Ala Leu Asn Thr Gly Leu Tyr Leu Arg Arg Val Lys Lys Thr 370 375 380

Gly Phe Gly Thr Thr He Asp Asp Ser Tyr Glu Asp Gly Ala Phe Leu 385 390 395 400

Gin Pro Glu Thr Phe Val Gin Ala Ala Leu Ala Cys Cys Thr Gly Gin 405 410 415

Asp Ala Pro Leu Asn Gly Met Ser Asp Val Tyr Val Thr Tyr Pro Asp 420 425 430

Leu Leu Glu Phe Asp Ala Val Thr Gin Val Pro He Thr Val He Glu 435 440 445

Pro Ala Gly Tyr Asn He Val Asp Asp His Leu Val Val Val Gly Val 450 455 460

Pro Val Ala Cys Ser Pro Tyr Met He Phe Pro Val Ala Ala Phe Asp 465 470 475 480

Thr Ala Asn Pro Tyr Cys Gly Asn Phe Val He Lys Ala Ala Asn Lys 485 490 495

Tyr Leu Arg Lys Gly Ala Val Tyr Asp Lys Leu Glu Ala Trp Lys Leu 500 505 510

Ala Trp Ala Leu Arg Val Ala Gly Tyr Asp Thr His Phe Lys Val His 515 520 525

Gly Asp Thr His Gly Leu Thr Lys Phe Tyr Ala Asp Asn Ser Asp Thr 530 535 540

Trp Thr His He Pro Glu Phe Val Thr Asp Gly Asp Val Met Glu Val 545 550 555 560

Phe Val Thr Ala He Glu Arg Arg Ala Arg His Phe Val Glu Leu Pro 565 570 575

Arg Leu Asn Ser Pro Ala Phe Phe Arg Ser Val Glu Val Ser Thr Thr 580 585 590

He Tyr Asp Thr His Val Gin Ala Gly Ala His Ser Val Tyr His Ala 595 600 605

Arg Arg He Asn Leu Asp Tyr Val Lys Pro Val Ser Thr Gly He Gin 610 615 620

Val He Asn Ala Gly Glu Leu Lys Asn Tyr Trp Gly Ser Val Arg Arg 625 630 635 640

Thr Gin Gin Gly Leu Gly Val Val Gly Leu Thr Met Pro Ala Val Met 645 650 655

Pro Thr Gly Glu Pro Thr Ala Gly Ala Ala His Glu Glu Leu He Glu 660 665 670

Gin Ala Asp Asn Val Leu Val Glu 675 680

( 2 ) INFORMATION FOR SEQ ID NO : 3 :

( i ) SEQUENCE CHARACTERISTICS :

(A) LENGTH : 4615 base pairs

(B ) TYPE : nucleic acid

( C) STRANDEDNESS : single

(D) TOPOLOGY : l inear

(ii) MOLECULE TYPE: cDNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

GAATTTTTCG GTGAACCGGA ATTATGTCGT CTCTGTTAAA TTCATTACTA CCAGAATATT 60

TTAAACCTAA AACTAATTTG AATATCAACT CTTCTAGGGT CCAATATGGC TTTAATGCTC 120

GCATTGATAT GCAGTATGAA GACGATAGTG GGACTAGAAA AGGCTCAAGA CCCAATGCAT 180

TTATGTCTAA CACAGTTGCT TTTATAGGAA ACTATGAAGG TATTATTGTT GATGACATTC 240

CGATATTGGA TGGTCTTAGG GCCGACATTT TTGATACTCA TGGTGACTTA GACATGGGCC 300

TCGTTGAGGA TGCATTGTCT AAGAGTACCA TGATTAGAAG GAATGTACCA ACTTATACTG 360

CTTACGCGAG TGAATTACTG TATAAGAGAA ACCTTACATC TCTATTTTAC AATATGCTCC 420

GTTTATACTA CATTAAAAAA TGGGGCAGTA TTAAGTATGA AAAAGATGCC ATCTTTTATG 480

ATAATGGCCA CGCCTGTCTT TTAAACAGGC AATTGTTTCC AAAGTCTCGT GATGCTTCTT 540

TGGAATCAAG CCTCTCTTTG CCTGAGGCTG AAATTGCAAT GCTTGATCCT GGCCTGGAAT 600

TTCCAGAAGA GGATGTGCCT GCAATTTTAT GGCACGGCAG AGTGTCATCC AGAGCAACGT 660

GTATCTTAGG GCAAGCTTGC TCAGAGTTCG CGCCTCTGGC CCCCTTTTCG ATTGCGCATT 720

ATTCACCACA ATTGACGAGA AAACTATTTG TCAATGCGCC CGCTGGGATT GAGCCTAGCT 780

CCGGGCGGTA TACTCACGAG GATGTAAAAG ATGCGATTAC GATCCTTGTG TCTGCAAACC 840

AGGCTTATAC TGACTTTGAA GCAGCATACT TGATGCTTGC TCAAACGTTG GTCTCACCTG 900

TACCACGCAC TGCCGAAGCA AGTGCATGGT TCATCAATGC TGGCATGGTC AATATGCCAA 960

CTTTGTCATG TGCAAATGGT TATTATCCAG CACTGACCAA TGTCAATCCT TACCACCGGC 1020

TAGACACATG GAAAGATACG TTAAATCATT GGGTGGCTTA TCCCGACATG CTGTTTTACC 1080

ATTCAGTGGC AATGATTGAG AGCTGCTATG TTGAACTCGG GAATGTGGCT CGTGTGTCAG 1140

ACAGTGATGC AATAAACAAA TACACTTTCA CTGAGCTATC AGTGCAAGGA CGGCCTGTTA 1200

TGAATCGAGG AATTATTGTA GATCTGACAC TTGTGGCAAT GCGTACTGGT AGGGAGATCT 1260

CACTACCTTA CCCGGTCAGC TGTGGCCTGA CCCGTACAGA CGCGTTATTG CAAGGTACTG 1320

AGATTCACGT TCCAGTTGTT GTCAAAGATA TCGACATGCC CCAGTATTAC AACGCGATTG 1380

ATAAGGATGT TATTGAGGGG CAGGAAACTG TGATTAAAGT GAAACAGCTG CCACCAGCTA 1440

TGTATCCAAT TTATACTTAC GGGATCAACA CTACTGAATT CTATTCTGAC CATTTTGAAG 1500

ACCAGGTACA AGTTGAAATG GCACCAATCG ATAATGGAAA AGCAGTTTTT AACGATGCAA 1560

GAAAGTTTTC GAAATTTATG TCCATAATGC GCATGATGGG GAATGATGTT ACTGCTACTG 1620

ATTTAGTTAC AGGTAGAAAA GTGTCGAATT GGGCCGACAA CTCATCAGGG CGTTTCTTGT 1680

ACACGGATGT GAAGTATGAA GGACAAACTG CCTTTTTGGT TGATATGGAT ACTGTCAAGG 1740

CGAGAGACCA CTGTTGGGTG TCAATTGTTG ATCCTAATGG TACAATGAAC TTGTCATATA 1800

AGATGACCAA TTTTAGAGCA GCAATGTTTT CTAGAAACAA GCCCTTGTAT ATGACAGGGG 1860

GGTCAGTCAG GACCATAGCT ACTGGCAATT ATCGAGATGC TGCTGAAAGA TTACGTGCAA 1920

TGGATGAAAC GCTCAGATTA AAACCTTTTA AGATTACTGA GAAGTTGGAT TTTCGTGTAG 1980

CAGCTTACGC GATACCAAGT TTGTCGGGCA GCAATATGCC ATCCTTACAC CATCAGGAAC 2040

AACTACAGAT ATCAGAAGTG GACGCGGAAC CAATCAATCC TATAGGAGAG GACGAACTTC 2100

CACCGGATAT AGAATAGGTG TCGAAGACGA TGAGGACTTA GATATTGGTA CGGTCAAATA 2160

CATTGTGCCA TTGTATTTGA ACGGTGATAA TGTGGCACAA AATTGTTTAG AAGCAACACA 2220

CGTGCTTATC AAAGCTTGTA GTATTGCGAA CCGGATTGTA GATGACGGAG AGGGTCACTG 2280

TTTCACACAG CAAGGGCTGG CGCAGCAGTG GATCTTCCAT AGGGGGGAGA TGATATTTGT 2340

GAAGGCGGTA CGCATTGGTC AACTCAATGC ATATTATGTA GACTATAAGA ACGTCACAAA 2400

TTATAGTCTT AAAACCGCTG CTCAAGTAGG AGCGACGATA TCAAATAACT TACGCCACGG 2460

ATTTGTTGAC AATCAACAAG ACGCATACAC GCGCTTGGTT GCCAACTACT CTGATACGCG 2520

GAAGTGGATA CGTGACAATT TTACATATAA TTATAATATG GAGAAAGAAA AGTATAGGAT 2580

AACCCAATAC CACCATACAC ATGTGAGGTT GAAAGATTTG TTTCCATCCA GGAAAATAGT 2640

TAAACTAGAG GGATATGAAG CCTTGTTGGC AATGATGCTA GACAGGTTTA ACAACATAGA 2700

GTCAACACAT GTAACTTTCT TCACATATTT AAGAGCACTA CCTGACCGTG AAAAAGAAGT 2760

CTTTATTAGC TTAGTCTTAA ACTATAATGG CCTTGGCAGA GAGTGGTTGA AGTCTGAAGG 2820

TGTTAGGGCT AAACAAGCAC AAGGTACTGT GAAATACGAT ATGAGTAAAC TATTTGAACT 2880

GAATGTACTA GAGAACGGAG TTGACGAAGA AGTTGACTGG GAGAAAGAGA AACGCAACAG 2940

GTCAGATATC AAGACTGTTA ACATAAGTTA TGCAAAAGTT CTCGAACATT GTAGAGAGCT 3000

ATTCATCATG GCGAGGGCCG AAGGGAAACG GCCAATGAGG ATGAAATGGC AAGAGTACTG 3060

GAGGCAGAGA GCAGTTATCA TGCCAGGTGG ATCGGTCCAC AGTCAACATC CAGTCGAACA 3120

GGACGTGATT AGAGTATTAC CCAGAGAAAT CAGAAGTAAG AAGGGGGTGG CAAGTGTCAT 3180

GCCATACAAA GAACAGAAGT ATTTCACGTC CAGAAGGCCG GAAATACACG CTTACACTTC 3240

AACGAAATAC GAGTGGGGAA AAGTGAGGGC ACTATATGGG TGTGATTTTT CATCACATAC 3300

AATGGCTGAT TTTGGATTGT TACAATGCGA GGATACATTC CCGGGCTTTG TACCAACAGG 3360

GTCTTACGCC AATGAGGATT ATGTCAGGAC CAGAATTGCT GGGACTCACT CATTGATCCC 3420

TTTCTGTTAC GATTTCGATG ATTTCAACAG CCAACATTCA AAGGAAGCCA TGCAAGCAGT 3480

GATTGATGCA TGGATATCTG TCTATCACGA TAAGTTAACA GATGACCAGA TAGAGGCGGC 3540

AAAGTGGACA CGAAACTCGG TAGATAGAAT GGTCGCTCAC CAACCTAACA CTGGTGAGAC 3600

TTATGATGTT AAAGGGACAC TGTTTAGTGG CTGGCGATTA ACAACATTTT TCAATACGGC 3660

GTTGAACTAT TGCTACCTGG CTAATGCAGG TATAAACTCA CTAGTGCCAA CGAGTCTCCA 3720

TAATGGTGAT GATGTTTTTG CAGGGATAAG GACAATAGCT GACGGTATTT CTTTGATCAA 3780

AAACGCCGCA GCCACGGGAG TTCGCGCTAA TACAACTAAA ATGAACATTG GTACGATAGC 3840

AGAGTTTTTG AGAGTTGATA TGCGTGCAAA AAATAGTACT GGCAGTCAGT ATTTAACAAG 3900

AGGGATTGCT ACCTTCACGC ACAGTAGGGT TGAGTCTGAT GCACCACTGA CATTGCGCAA 3960

TCTAGTATCT GCTTACAAAA CCAGATATGA CGAGATTTTA GCTCGTGGCG CAAGCATCGA 4020

TAACATGAAG CCACTCTATC GTAAGCAATT ATTTTTTGCT AGAAAGTTGT TCAATGTCGA 4080

GAAGGACATT GTTGACAATC TGATAACGAT GGACATATCA TGTGGCGGTT TGCAAGAAAA 4140

GGGTAGGGTA TCAGAGATGG TGTTACAGGA GGTTGACATT GAGAATATAG ATAGTTATAG 4200

GAAGACAAGG ATGATCGCCA AACTGATTGA CAAGGGGGTT GGCGATTATA CTGCATTCCT 4260

GAAAACTAAC TTTTCCGAGA TAGCTGATGC TATCACAAGA GAGACACGTG TAGAGTCAGT 4320

GACCAAGGCT TATAATGTTA AGAAGAAAAC GGTCGTACGC GCGTTTAGGG ACCTAAGCGC 4380

AGCATATCAT GAAAGAGCGG TGAGACATGC TTGGAAGGGG ATGAGTGGAC TACACATAGT 4440

CAACAGGATT CGTATGGGAG TGAGCAACTT AGTAATGGTT GTTAGCAAAA TCAATCCTGC 4500

AAAAGCTAAT GTGCTAGCCA AATCAGGAGA TCCTACAAAA TGGCTTGCAG TCCTTACATG 4560

ATATACAGGC AACCACATAA GACCTGAGAA CAAAGAGTAC ATACGATACT ACGCA 4615

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 697 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS :

(D) TOPOLOGY: linear

( ii ) MOLECULE TYPE : protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

Met Ser Ser Leu Leu Asn Ser Leu Leu Pro Glu Tyr Phe Lys Pro Lys 1 5 10 15

Thr Asn Leu Asn He Asn Ser Ser Arg Val Gin Tyr Gly Phe Asn Ala 20 25 30

Arg He Asp Met Gin Tyr Glu Asp Asp Ser Gly Thr Arg Lys Gly Ser 35 40 45

Arg Pro Asn Ala Phe Met Ser Asn Thr Val Ala Phe He Gly Asn Tyr 50 55 60

Glu Gly He He Val Asp Asp He Pro He Leu Asp Gly Leu Arg Ala 65 70 75 80

Asp He Phe Asp Thr His Gly Asp Leu Asp Met Gly Leu Val Glu Asp 85 90 95

Ala Leu Ser Lys Ser Thr Met He Arg Arg Asn Val Pro Thr Tyr Thr 100 105 110

Ala Tyr Ala Ser Glu Leu Leu Tyr Lys Arg Asn Leu Thr Ser Leu Phe 115 120 125

Tyr Asn Met Leu Arg Leu Tyr Tyr He Lys Lys Trp Gly Ser He Lys 130 135 140

Tyr Glu Lys Asp Ala He Phe Tyr Asp Asn Gly His Ala Cys Leu Leu 145 150 155 160

Asn Arg Gin Leu Phe Pro Lys Ser Arg Asp Ala Ser Leu Glu Ser Ser 165 170 175

Leu Ser Leu Pro Glu Ala Glu He Ala Met Leu Asp Pro Gly Leu Glu 180 185 190

Phe Pro Glu Glu Asp Val Pro Ala He Leu Trp His Gly Arg Val Ser 195 200 205

Ser Arg Ala Thr Cys He Leu Gly Gin Ala Cys Ser Glu Phe Ala Pro 210 215 220

Leu Ala Pro Phe Ser He Ala His Tyr Ser Pro Gin Leu Thr Arg Lys 225 230 235 240

Leu Phe Val Asn Ala Pro Ala Gly He Glu Pro Ser Ser Gly Arg Tyr 245 250 255

Thr His Glu Asp Val Lys Asp Ala He Thr He Leu Val Ser Ala Asn 260 265 270

Gin Ala Tyr Thr Asp Phe Glu Ala Ala Tyr Leu Met Leu Ala Gin Thr 275 280 285

Leu Val Ser Pro Val Pro Arg Thr Ala Glu Ala Ser Ala Trp Phe He 290 295 300

Asn Ala Gly Met Val Asn Met Pro Thr Leu Ser Cys Ala Asn Gly Tyr 305 310 315 320

Tyr Pro Ala Leu Thr Asn Val Asn Pro Tyr His Arg Leu Asp Thr Trp 325 330 335

Lys Asp Thr Leu Asn His Trp Val Ala Tyr Pro Asp Met Leu Phe Tyr

340 345 350

His Ser Val Ala Met He Glu Ser Cys Tyr Val Glu Leu Gly Asn Val 355 360 365

Ala Arg Val Ser Asp Ser Asp Ala He Asn Lys Tyr Thr Phe Thr Glu 370 375 380

Leu Ser Val Gin Gly Arg Pro Val Met Asn Arg Gly He He Val Asp 385 390 395 400

Leu Thr Leu Val Ala Met Arg Thr Gly Arg Glu He Ser Leu Pro Tyr 405 410 415

Pro Val Ser Cys Gly Leu Thr Arg Thr Asp Ala Leu Leu Gin Gly Thr 420 425 430

Glu He His Val Pro Val Val Val Lys Asp He Asp Met Pro Gin Tyr 435 440 445

Tyr Asn Ala He Asp Lys Asp Val He Glu Gly Gin Glu Thr Val He 450 455 460

Lys Val Lys Gin Leu Pro Pro Ala Met Tyr Pro He Tyr Thr Tyr Gly 465 470 475 480

He Asn Thr Thr Glu Phe Tyr Ser Asp His Phe Glu Asp Gin Val Gin 485 490 495

Val Glu Met Ala Pro He Asp Asn Gly Lys Ala Val Phe Asn Asp Ala 500 505 510

Arg Lys Phe Ser Lys Phe Met Ser He Met Arg Met Met Gly Asn Asp 515 520 525

Val Thr Ala Thr Asp Leu Val Thr Gly Arg Lys Val Ser Asn Trp Ala 530 535 540

Asp Asn Ser Ser Gly Arg Phe Leu Tyr Thr Asp Val Lys Tyr Glu Gly 545 550 555 560

Gin Thr Ala Phe Leu Val Asp Met Asp Thr Val Lys Ala Arg Asp His 565 570 575

Cys Trp Val Ser He Val Asp Pro Asn Gly Thr Met Asn Leu Ser Tyr 580 585 590

Lys Met Thr Asn Phe Arg Ala Ala Met Phe Ser Arg Asn Lys Pro Leu 595 600 605

Tyr Met Thr Gly Gly Ser Val Arg Thr He Ala Thr Gly Asn Tyr Arg 610 615 620

Asp Ala Ala Glu Arg Leu Arg Ala Met Asp Glu Thr Leu Arg Leu Lys 625 630 635 640

Pro Phe Lys He Thr Glu Lys Leu Asp Phe Arg Val Ala Ala Tyr Ala 645 650 655

He Pro Ser Leu Ser Gly Ser Asn Met Pro Ser Leu His His Gin Glu 660 665 670

Gin Leu Gin He Ser Glu Val Asp Ala Glu Pro He Asn Pro He Gly 675 680 685

Glu Asp Glu Leu Pro Pro Asp He Glu 690 695

(2) INFORMATION FOR SEQ ID NO: 5

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 40 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA

( xi ) SEQUENCE DESCRIPTION : SEQ ID NO : 5 : GATCTATAAA GATGTCGGAT CCCCCGGGGG TACCGTCGAC 40