Chemistry: natural resins or derivatives; peptides or proteins; – Proteins – i.e. – more than 100 amino acid residues
Reexamination Certificate
1999-07-27
2001-07-10
Wortman, Donna C. (Department: 1642)
Chemistry: natural resins or derivatives; peptides or proteins;
Proteins, i.e., more than 100 amino acid residues
Reexamination Certificate
active
06258937
ABSTRACT:
BACKGROUND OF THE INVENTION
The field of the invention is cellular receptors for viruses of the family Hepadnaviridae, and nucleic acids encoding the same.
The human hepatitis B virus (HBV) and related animal viruses that infect woodchucks, ground squirrels, Pekin ducks, and herons form a group of hepatotropic DNA viruses in the family Hepadnaviridae. In humans, HBV causes acute and chronic liver disease and hepatocellular carcinoma.
The initial event of infection, interaction between the viral envelope protein and specific cellular receptor(s), is poorly understood. Hepadnaviruses express at least two co-terminal envelope proteins from a single envelope gene by alternative use of in-frame AUG codons. The large envelope protein (pre-S/S protein) of duck hepatitis B virus (DHBV) contains a 161-163 amino acid segment called the pre-S domain, and a carboxylterminal 167 amino acid segment called the S domain. The small envelope protein (S protein alone) is produced by translation from an internal AUG codon. The large envelope protein of HBV is similar, but has pre-S1 and pre-S2 domains in place of the single DHBV pre-S domain. As a result, two pre-S containing proteins are produced: a large envelope protein (preS1+preS2+S) and a middle envelope protein (preS2+S). The large envelope protein is myristylated and phosphorylated (Grgacic et al.,
J. Virol.
68:7344-7350, 1994; Macrae et al.,
Virology
181:359-363, 1991; Persing et al.,
J. Virol.
61:1672-1677, 1987). The large envelope protein mediates infection by DHBV and by hepatitis delta virus (HDV), which borrows the envelope proteins of other hepadnaviruses to enter a hepatocyte (Fernholz et al.,
Virology
197:64-73, 1993; Summers et al.,
J. Virol.
65:1310-1317, 1991; Sureau et al.,
J. Virol.
67:366-372). The pre-S domain is believed to be responsible for binding a cellular receptor. Although several cellular proteins bind the HBV envelope, none have been shown to be the actual receptor (Budkowska et al.,
J. Virol.
67:4316-4322, 1993; Budkowska et al.,
J. Virol.
69:840-848, 1995; Hertogs et al.,
Virology
197:549-557, 1993; Mehdi et al.,
J. Virol.
68:2415-2424, 1994; Neurath et al.,
J. Exp. Med.
176:1561-1569, 1992; Pontisso et al.,
J. Gen. Virol.
73:2041-2045, 1992).
Since no cell culture system is available for the study of HBV, DHBV was developed as a model system. DHBV infection of ducklings and primary duck hepatocytes has been well characterized (Pugh et al.,
Virology
172:564-572, 1989; Tuttleman et al.,
J. Virol.
58:17-25, 1986).
SUMMARY OF THE INVENTION
The invention features a purified nucleic acid that encodes a member of the hepadnavirus family of cellular receptors, or, where the receptor is a complex of two or more polypeptides, a component thereof. By “a member of the hepadnavirus family of cellular receptors” (hereafter a “hepadnavirus receptor”) is meant a protein that binds the pre-S domain of the hepadnavirus large envelope protein so as to mediate or induce entry of a hepadnavirus virion into a host cell. A “hepadnavirus receptor”, as used herein, can be the whole receptor, where the receptor is a monomer, or a subunit of a hepadnavirus receptor that binds the pre-S receptor binding site of the pre-S domain.
The identity of a hepadnavirus receptor that “mediates entry of the hepadnavirus virion into a host cell” can be confirmed using two biological activity assays. First, an antibody preparation specific for a member of the hepadnavirus family of receptor proteins should have the ability to block, inhibit, or reduce hepadnaviral infection of, or entry into, a cell, the virus being capable of infecting the same cell type in the absence of antibody. Either polyclonal or monoclonal receptor-specific antibodies can be used. For the second assay, a cDNA that encodes a member of the hepadnavirus family of receptor proteins is transfected into a cell line. The cell line is one that is ordinarily not a target cell for a hepadnavirus. Transfection of receptor-encoding cDNA sequence into these cells should confer properties on the cell line that enable them to be infected by a hepadnavirus, or enable the virus to bind to the cell surface. By performing these two aforementioned assays, a hepadnavirus receptor is distinguished from a non-receptor hepadnavirus pre-S binding protein.
By “purified nucleic acid” is meant a nucleic acid that is not immediately contiguous with both of the coding sequences with which it is immediately contiguous (i.e., one at the 5′ and one at the 3′ end) in the naturally-occurring genome of the organism from which the nucleic acid of the invention is derived. The term encompasses deoxyribonucleic acid (DNA), for example, a cDNA or a genomic DNA fragment produced by the polymerase chain reaction (PCR), or produced by restriction endonuclease treatment. The cDNA or genomic DNA fragment can be incorporated into a vector, integrated into the genome of the same or a different species than the organism from which it was originally derived, linked to an additional coding sequence to form a hybrid gene encoding a chimeric polypeptide, or independent of any other nucleic acid sequences. The term also encompasses ribonucleic acid (RNA). The nucleic acid may be double-stranded or single-stranded, sense or antisense.
Examples of purified nucleic acids of the invention include those which encode amino acid sequences substantially the same as those shown in
FIGS. 18 and 19
; and those having sequences that are either identical to, or hybridize under conditions of high or moderate stringency to, the 2.5 kb p170 cDNA included in the ATCC deposit Ep170pUC, designated No. 69869. High stringency conditions are herein defined as the following: hybridizing with 50% deionized formamide, 800 mM NaCl; 20 mM Pipes, pH 6.5, 0.5% SDS, 100 &mgr;g/ml denatured, sonicated salmon sperm DNA at 42° C. for 12-20 hours, washing with 30 mM NaCl/3.0 mM sodium citrate (0.2×SSC)/0.1% SDS at 55° C., while moderate stringency conditions are as follows: hybridizing with 50% deionized formamide, 800 mM NaCl; 20 mM Pipes, pH 6.5, 0.5% SDS, 100 &mgr;g/ml denatured, sonicated salmon sperm DNA at 42° C. for 12-20 hours, washing with 75 mM NaCl/7.5 mM sodium citrate (0.5×SSC)/0.1% SDS at 55° C. Such hybridization conditions are useful in a method of identifying a nucleic acid sequence encoding a hepadnavirus receptor polypeptide. The method involves providing a genomic or cDNA library; contacting the library with a nucleic acid that encodes a portion of a hepadnavirus receptor, e.g., the duck p170 receptor, under conditions permitting hybridization between the nucleic acid and a homologous nucleotide sequence in the library; and identifying a clone from the library which hybridizes to the nucleic acid, hybridization being indicative of the presence in the clone of a nucleotide sequence homologous to a hepadnavirus receptor-encoding nucleotide sequence.
The invention also includes fragments of a purified nucleic acid that encodes a member of the hepadnavirus family of receptors. Examples include a nucleic acid of at least 20 nucleotides in length, or at least 30 or 50 nucleotides in length, that includes a strand which hybridizes under high stringency conditions to either the sense or antisense strand of a nucleic acid encoding part or all of a naturally occurring hepadnavirus receptor polypeptide. A nucleic acid fragment is useful, e.g., as a probe for identifying additional members of the hepadnavirus family of receptors, or for administering a portion of a hepadnavirus receptor sequence to a cell, e.g., a cell in a patient, using gene therapy techniques. Such portions of a hepadnavirus can include the peptides 1-4 shown in FIG.
14
A.
The invention also includes vectors (e.g., plasmids, phage, or viral nucleic acid) or cells (prokaryotic or eukaryotic) which contain nucleic acids encoding any of the various hepadnavirus receptors of the invention. The vector can be any vector suitable for maintaining or making multiple copies of a nucleic acid of the invention, or can be one t
Li Jisu
Tong Shuping
Wands Jack R.
Brumback Brenda G.
Fish & Richardson P.C.
The General Hospital Corporation
Wortman Donna C.
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