WO2000050076A1 - Adenoviral vectors, proteins and methods for their use - Google Patents

Adenoviral vectors, proteins and methods for their use Download PDF

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WO2000050076A1
WO2000050076A1 PCT/US2000/004711 US0004711W WO0050076A1 WO 2000050076 A1 WO2000050076 A1 WO 2000050076A1 US 0004711 W US0004711 W US 0004711W WO 0050076 A1 WO0050076 A1 WO 0050076A1
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amino acid
acid sequence
set forth
nucleotide sequence
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Suresh Kumar Mittal
Neeraj Aggarwal
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Purdue Research Foundation
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86Viral vectors
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/10011Adenoviridae
    • C12N2710/10311Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10322New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011Details
    • C12N2710/10011Adenoviridae
    • C12N2710/10311Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341Use of virus, viral particle or viral elements as a vector
    • C12N2710/10343Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • Adenoviruses are non-enveloped DNA viruses that infect a wide variety of cells and some of them typically cause acute respiratory and ocular infections, as well as hemorrhagic cystitis and infantile gastroenteritis. After adenoviruses infect a cell, their DNA does not become incorporated into the genome of the infected cell, but remains episomal. As adenoviruses infect a wide variety of host cells, and have the ability to replicate in both dividing and non-dividing cells, recombinant adenoviruses are frequently used as viral vectors for gene delivery applications, especially to deliver recombinant vaccines.
  • Porcine adenoviruses usually cause inapparent infections. However, they are associated with enteric, respiratory, kidney, and central nervous system infections in swine [Derbyshire J. B. et al. (1975) J. Comp. Path. 85:437-443; Hirahara T., et al. (1990) Jpn. J. Vet. Sci. 52:407-409; Hirahara T., et al. (1990) Jpn J. Vet Sci. 52:1089-1091]. PAds are classified into five currently known serotypes, PAd-1 , -2, -3, -4, & -5 [Derbyshire J. B. et al. (1975) J. Comp. Path.
  • PAd3 was initially isolated from a fecal sample collected from a healthy pig [Clarke M. C. et al. (1967 Arch. Ges. Virusforsch. 21 :91-97].
  • a cross-hybridization experiment using labeled human adenovirus type 2 (HAd2) DNA as a probe resulted in hybridization with PAd3 DNA fragments mainly in the region corresponding to structural proteins [Benko M. et al. (1990) J. Gen. Virol. 71 :465-469].
  • the genome of PAd3 is approximately 35 Kb in size, restriction maps for a number of enzymes have been reported [Garwes D. J. and Xuan H. (1989) Intervirology.
  • E1 genes are the first set of genes to be expressed.
  • the E1 region includes two transcription units, E1A and E1B, each of which expresses multiple proteins as a result of differential splicing and use of different translation reading frames [Berk A.J. and Sharp P. A. (1978) Cell. 14:695-711].
  • E1 gene products of several HAds have been shown to transactivate a variety of viral and cellular promoters, and transform and immortalize cultured rodent cells [Berk A.J. (1986) Ann. Rev. Genet. 20:45-79; Whyte P. et al. (1988) Nature. 334:124-129; Whyte, P. et al. (1989) Cell.
  • vectors may also be useful for vaccinating immunocompromised hosts since they could not spread in vivo. Titers of E1- deleted vectors in E1 -complementing cells are similar to those of E3- deleted vectors, therefore, the cost of production of either vector system would be similar.
  • adenovirus vectors stimulate an immune response that results in elimination of the cells carrying the vectors. Therefore, one of the drawbacks of many of the currently used adenovirus vectors as gene delivery vehicles is that the transgene is expressed for only a short period of time. This loss in expression can not be overcome with frequent rounds of vaccination, as the host-immune response often leads to neutralization of the virus before it enters target cells. A need therefore exists for adenovirus vectors that exhibit prolonged transgene expression. The present invention addresses this need.
  • nucleic acid molecules that encode the proteins recited above are provided.
  • the nucleic acid molecules may be incorporated into a vector to form a recombinant nucleic acid molecule.
  • recombinant nucleic acid molecules may be introduced into a host cell.
  • adenoviral vectors that include the nucleic acid molecules described herein.
  • the vectors may include a foreign promoter, preferably at least one foreign promoter, that is operably linked to a terminal 5' end of the nucleic acid molecule.
  • the vectors may further include a second nucleic acid molecule of interest, such as one having a nucleotide sequence encoding a desired protein.
  • the vector advantageously may also include a second promoter that is operably linked to a terminal 5' end of the second desired nucleic acid molecule, and may further include an adenoviral origin of replication and an inverted terminal repeat sequence.
  • replication-defective porcine adenoviral vectors are provided that include a porcine adenovirus genome, such as a type 3 genome, having a functional deletion in the E1 region.
  • These vectors may also include a nucleotide sequence encoding a protein of interest inserted into the E1 region.
  • the vector also preferably includes a foreign promoter operably linked to a terminal 5' end of the nucleotide sequence encoding the protein of interest, and may include one or more of the viral nucleotide sequences provided herein, preferably with at least one foreign promoter operably linked to a terminal 5' end of these novel nucleotide sequences.
  • a method includes infecting a host cell with an adenoviral vector having an isolated first nucleic acid molecule encoding one or more, preferably at least one, of the novel proteins as described herein and a second desired nucleic acid molecule that may advantageously encode a protein of interest.
  • Each of the nucleic acid molecules preferably independently include a foreign promoter operably linked to their terminal 5' end.
  • a method of making an adenovirus vector includes introducing into a cell an isolated nucleic acid molecule encoding the proteins described herein and a foreign promoter, wherein the foreign promoter is operably linked to a terminal 5' end of the molecule.
  • a method may include infecting a host cell with an adenoviral vector having a first isolated nucleic acid molecule encoding a protein, such as a viral protein, as described herein, and a second desired nucleic acid molecule encoding a desired protein, and culturing the host cells under conditions effective in achieving expression of the desired protein.
  • the adenoviral vector may further include a foreign promoter, preferably at least one, that is operably linked to a terminal 5' end of the first isolated nucleic acid molecule and another foreign promoter so linked to the second desired nucleotide sequence.
  • the desired protein may then be purified by conventional techniques.
  • a method includes introducing into a host cell an isolated first nucleotide sequence as described herein and a foreign promoter, preferably at least one, that is operably linked to a terminal 5' end of said first nucleotide sequence and culturing under conditions to achieve expression of said protein.
  • a foreign promoter preferably at least one, that is operably linked to a terminal 5' end of said first nucleotide sequence and culturing under conditions to achieve expression of said protein.
  • One object of the invention is to provide proteins and RNA involved in viral replication, especially those expected to function in viral latency, as well as nucleotide sequences encoding these gene products.
  • a further object of the invention is to provide a porcine adenoviral type 3 vector having a functional E1 deletion.
  • a further object of the invention is to provide an adenoviral vector that may be used to increase the duration of transgene expression.
  • Yet another object of the invention is to provide methods of utilizing the nucleotide and amino acid sequences described herein.
  • FIGS. 1A-1 G depict the nucleotide sequence and major open reading frames (ORFs) of the PAd3 genome between map unit (m.u.) 0 and 13.7 determined as discussed in Example 1.
  • the first 144 nucleotides (nt) represent an inverted terminal repeat sequence.
  • the conserved region of replication (bold), the putative packaging signals (underlined), a putative nuclear factor III (NFIII) binding site (bold, italics and underlined), and putative SP1 -binding sites (italics and underlined) are indicated.
  • PAd3 E1A, E1 B-202R, E1 B-411 R, plX ORF, novel ORF 184R on the upper strand and an incomplete IVa2 ORF, and four unique ORFs (97R,162R,163R * and 288R) on the lower-strand are shown in bold.
  • the probable TATA motifs and polyadenylation signals are indicated as bold and underlined, and bold and doubled underlined, respectively.
  • FIG. 2 depicts open reading frames (ORFs) in PAd3 E1 and plX regions of the PAd genome.
  • the left-end PAd3 genome between m.u. 0 and 13.7 was sequenced as described in Example 1 to provide a sequence of 4769 nt.
  • Three frames of ORFs on the upper-strand are indicated as a, b, and c.
  • three frames of ORFs on the lower-strand are indicated as d, e, and f.
  • the direction of ORFs are shown by an open arrow on the left. All ORFs of 50 or more amino acids following methionine are shown.
  • ORFs that have demonstrated homology with known adenovirus proteins are indicated by hatched boxes and novel ORFs that have homology with EBNA1 , EBNA2 and other transcriptional initiation factors are indicated by bricked boxes.
  • the putative ORF 163R* did not have an initiation codon.
  • FIG. 3 depicts a comparison of the amino acid sequence of four important functional regions in the E1 A proteins of PAd3, PAd4, HAd5, and
  • FIG. 4 depicts a comparison of amino acid sequences of the putative PAd3 E1 B proteins as described in Example 1.
  • B & C homologous regions of the PAd3 E1B-202R, BAd3 E1 B-19k, and HAd5 E1 B-19k;
  • FIG. 5 shows a homology comparison among N-terminal regions of
  • HAd5 plX homologs of three adenoviruses Identical residues are shown in bold. The residue numbers for each of the viruses are shown.
  • FIG. 6 depicts a homology comparison among C-terminal regions of HAd5 IVa2 homologs of three adenoviruses. Identical residues are shown in bold. The residue numbers for each of the viruses are shown.
  • FIGS. 7A-7B show comparisons of the amino acid sequence of the PAd3 five novel ORFs.
  • FIG. 7A, 1 region of homology between PAd3 184R and bovine Na+/Ca+,K+ exchanging protein (PIR S20969 1 );
  • FIG. 7A, 4) region of homology between PAd3 97R and EHV transcriptional control protein 57 (PIR S55652 f );
  • FIG. 7A, 1 region of homology between PAd3 184R and bovine Na+/Ca+,K+ exchanging protein
  • FIG. 7A 5A, 5) region of homology between 162R and equine herpesvirus (EHV) membrane glycoprotein (DBJ D88734 1 ; FIG. 7A, 6) region of homology between 162R and trans-acting transcriptional protein ICP0 of herpes simplex virus, (SP P28284 1 ); FIG. 7B, 7) region of homology between PAd3 288R and EBNA1 ; FIG. 7B, 8) region of homology between PAd3 288R and the transcription factor GATA-4 of mice (SP 0083691*); FIG. 7B, 9) region of homology between PAd3 163R* and EBNA2 (Pir
  • FIG. 8 depicts an SDS-polyacrylamide gel from the purification of PAd3 162R protein expressed in bacteria according to the protocol in Example 2.
  • a plasmid containing PAd3 162R ORF under the control of an inducible bacteriophage T7 promoter was used to transform bacteria expressing T7 RNA polymerase.
  • Bacterial suspension cultures at approximately 0.6 O.D. were treated with 1.5 mM of IPTG to induce the expression of the 162R protein as a his-tag fusion protein.
  • Bacterial cultures were subjected to cellular disruption using a French press and insoluble inclusions containing the fusion protein were isolated and solubilized in a 6M gaunidinium HCI solution.
  • the solution was passed through a 0.2 ⁇ m filter and the filtrate was applied onto a Ni ++ affinity column.
  • the his-tag fusion protein bound to Ni ++ column was eluted following the manufacturer's instructions using a kit (Novagen, Inc.). All protein fractions were separated by SDS-PAGE and then stained with Coomassie blue.
  • the PAd3 162R fusion protein is indicated by an arrow. The molecular weight (MW) markers are shown on the left.
  • FIG. 9 depicts a Western dot-blot analysis of IPTG-induced or uninduced bacterial cell extracts using HRP-conjugated S-tag.
  • DE3 bacteria containing pET30-162R were grown in the presence or absence of IPTG.
  • Total bacterial protein, soluble or insoluble (pellet) protein extracts were analyzed by Western blot using HRP-conjugated S-tag.
  • the PAd3 162R fusion protein is indicated by an arrow.
  • the molecular weight (MW) markers are shown on the left.
  • Novel nucleotide sequences expected to encode proteins involved in controlling viral latency have been identified in porcine adenovirus.
  • the proteins, and/or RNA encoded by these nucleotide sequences may advantageously maintain the adenovirus in a latent state, an adenoviral vector including such sequences, along with a transgene of interest, may not elicit an immune response after being introduced into a host cell. This may increase the duration of expression of the transgene in the cell.
  • other nucleotide sequences are provided that are expected to encode other proteins involved in viral replication, including proteins involved in stimulating DNA synthesis, transactivation of transcription, packing of the full-length genome into virions and shutting down of host cell protein synthesis.
  • nucleic acid molecules are also provided that include the nucleotide sequences encoding the proteins described herein.
  • the nucleic acid molecules may be incorporated into a host cell.
  • methods of expressing a desired protein are also provided, as well as methods of expressing the proteins recited herein.
  • purified proteins are provided that are expected to function in adenoviral replication.
  • the proteins are expected to function in viral latency as more fully discussed in example 1.
  • Amino acid sequences of these proteins, from a porcine adenovirus in one form of the invention, are set forth in SEQ ID NOS:1 , 2, 3, 4, and 5 (97R, 162R, 163R*, 288R and 184R, respectively) and also shown in FIGS. 1A-1 G.
  • the proteins have amino acid sequences from a porcine adenovirus, as set forth in SEQ ID NOS:6, 7, 8, 9, and 10 (E1A, E1 B-202, E1 B-474, plX and IVa2, respectively) and also shown in FIGS. 1A-1G.
  • the polypeptides described herein are substantially pure (i.e., the novel proteins are essentially free, e.g., at least about 95% free, from other proteins with which they naturally occur).
  • porcine adenovirus amino acid sequences it is understood that the invention is not limited to the specific amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10.
  • adenovirus-related protein is used to refer generally to proteins having the features described herein and preferred examples include polypeptides having the amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10. Also included within this definition, and in the scope of the invention, are variants of the polypeptides which function as described herein, especially variants involved in controlling the latent state of adenovirus.
  • adenoviral serotypes commonly express and utilize homologous proteins, which include the insertions, substitutions and/or deletions discussed above, and yet which effectively provide similar function.
  • an amino acid sequence isolated from another serotype may differ to a certain degree from the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, and yet have similar functionality with respect to catalytic and regulatory function. Amino acid sequences comprising such variations are included within the scope of the present invention and are considered substantially or sufficiently similar to a reference amino acid sequence.
  • variants of the proteins described herein are expected to be functionally similar to that set forth in SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10, for example, if they include amino acids which are conserved among a variety of serotypes or if they include non-conserved amino acids which exist at a given location in another serotype that expresses proteins having the functions described herein.
  • Another manner in which similarity may exist between two amino acid sequences is where a given amino acid of one group (such as a non- polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid or a charged polar basic amino acid) is substituted with another amino acid from the same amino acid group.
  • the uncharged polar amino acid serine may commonly be substituted with the uncharged polar amino acid threonine in a polypeptide without substantially altering the functionality of the polypeptide. Whether a given substitution will affect the functionality of the enzyme may be determined without undue experimentation using synthetic techniques and screening assays known in the art.
  • the invention therefore also encompasses polypeptides having amino acid sequences similar to the amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4 and 5 that have at least about 50%, 40%, 60%, 50% and 40% identity thereto, respectively, that preferably function as described herein.
  • the amino acid sequences have at least about 70%, 60%, 70%, 70%, and 60% identity to the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, respectively, more preferably at least about 80% identity to SEQ ID NOS:1 , 2,3, 4 and 5 and most preferably at least about 90% identity to SEQ ID NOS:1 , 2, 3, 4, and 5.
  • the amino acid sequences include those set forth in SEQ ID NO:2 from amino acid 1 to amino acid 38, in SEQ ID NO:3 from amino acid 41 to amino acid 163, and in SEQ ID NO:4 from amino acid 106 to amino acid 146 having at least the percentage identity to these sequences described above.
  • the invention also encompasses amino acid sequences similar to these amino acid sequences having at least about 50%, 40%, 40%, 30% and 90% identity thereto, respectively, that preferably function in viral replication.
  • the amino acid sequences have at least about 60%, 60%, 60%, and 50%, identity to the sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively, and further preferably at least about 80%, 80%, 80% and 70% identity to the sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively.
  • the amino acid sequences encompassed by the invention include amino acid sequences having at least about 90% identity to the sequences set forth in SEQ ID NOS:6, 7, 8 and 9.
  • the amino acid sequences include those set forth in SEQ ID NO:8 from amino acid 1 to amino acid 367, and in SEQ ID NO:9 from amino acid 1 to amino acid 135, and sequences having at least the percentage identity to these sequences described above. Percent identity between two nucleotide sequences or two amino acid sequences may be determined, for example, by comparing sequence information over the entire sequence using the Gap program, version 9, available from Genetics Computer Group (GCG) (Madison, Wisconsin).
  • GCG Genetics Computer Group
  • nucleic acid molecules originally isolated from porcine adenovirus, are provided that encode the proteins having the amino acid sequences described herein, including those amino acid sequences having the aforementioned percent identities to the respective amino acid sequences.
  • the nucleotide sequences encode proteins that are expected to control viral latency and are set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, as well as in FIGS. 1A-1 G, or similar nucleotide sequences discussed herein.
  • the nucleotide sequences encode other proteins involved in viral replication, and are set forth in SEQ ID NOS:6, 7, 8, 9, and 10 and FIGS. 1A-1 G or similar nucleotide sequences discussed herein.
  • nucleotide sequences that encode a protein that includes an amino acid sequence having at least about 50%, 40%, 60%, 50%, and 40% identity to the amino acid sequence set forth in SEQ ID NOS:1 , 2, 3 , 4 and 5, respectively.
  • amino acid sequences have at least about 70%, 60%, 70%, 70%, and 60% identity to the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, respectively, more preferably at least about 80% identity to SEQ ID NOS:1 , 2, 3, 4 and 5 and most preferably at least about 90% identity to SEQ ID NOS:1 , 2, 3, 4, and 5.
  • the nucleotide sequence encodes a protein having an amino acid sequence set forth in SEQ ID NO:2 from amino acid 1 to amino acid 38, in SEQ ID NO:3 from amino acid 41 to amino acid 163, and in SEQ ID NO:4 from amino acid 106 to amino acid 146 having at least the percentage identity to these sequences described above for SEQ ID NOS:2, 3, and 4.
  • the invention also encompasses nucleotide sequences that encode proteins that include amino acid sequences having at least about 50%, 40%, 40%, 30% and 90% identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8, 9, and 10, respectively, that preferably function in viral replication.
  • the nucleotide sequences encode a protein having an amino acid sequence that has at least about 60%, 60%, 60%, and 50%, identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively, and further preferably at least about 80%, 80%, 80% and 70% identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively.
  • the nucleotide sequences encode a protein having an amino acid sequence having at least about 90% identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8 and 9.
  • the nucleotide sequence encodes a protein having an amino acid sequence set forth in SEQ ID NO:8 from amino acid 1 to amino acid 367, and in SEQ ID NO:9 from amino acid 1 to amino acid 135, and sequences having at least the percent identity to these sequences described above for SEQ ID NOS:8 and 9.
  • RNA nucleotide sequences provided herein are also preferably transcribed to form an RNA nucleotide sequence, such as an mRNA transcript. Therefore, the RNA nucleotide sequences corresponding to the DNA sequences recited herein in SEQ ID NOS: 1 , 2, 3, 4 and 5, as well as in SEQ ID NOS:6, 7, 8, 9, and 10, wherein deoxythymidine ("T") is replaced by uridine (“U”), are also provided herein. RNA nucleotide sequences substantially similar to the transcripts are also encompassed in the invention, as well as RNA transcripts having the percent identity to these sequences as described herein for the DNA sequences.
  • isolated nucleic acid is intended to refer to nucleic acid which is not in its native environment.
  • the nucleic acid is separated from other contaminants that naturally accompany it, such as proteins, lipids and other nucleic acid sequences.
  • the term includes nucleic acid which has been removed or purified from its naturally- occurring environment or clone library, and further includes recombinant or cloned nucleic acid isolates and chemically synthesized nucleic acid.
  • nucleotide sequence is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, including deoxyribonucleic acid and ribonucleic acid, and derivatives thereof.
  • encoding and coding refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme or other protein that has a specific function.
  • the process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e., insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence.
  • base changes i.e., insertions, deletions, substitutions
  • nucleic acid sequences encoding variant amino acid sequences are within the scope of the invention.
  • Modifications to a sequence, such as deletions, insertions, or substitutions in the sequence, which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are expressly contemplated by the present invention.
  • alterations in a nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site are contemplated.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, may also be expected to produce a biologically equivalent product.
  • Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the encoded polypeptide molecule would also not generally be expected to alter the activity of the polypeptide. In some cases, it may in fact be desirable to make mutations in the sequence in order to study the effect of alteration on the biological activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art.
  • the nucleotide sequences have substantial similarity to the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, and, in other preferred embodiments, to SEQ ID NOS:6, 7, 8, 9, and 10, and variants described herein.
  • the term "substantial similarity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence is sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions. This method of determining similarity is well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1 , pp.
  • nucleic acid sequences which are complementary to the sequences specifically shown in the sequence listing.
  • a further requirement of the preferred polynucleotide is that it must encode a polypeptide having similar functionality to the proteins described herein.
  • the nucleotide sequences are deoxyribonucleic acid (DNA) sequences.
  • the nucleotide sequences are RNA sequences, preferably an RNA transcript, such as an mRNA transcript, produced from transcribing the nucleotide sequences provided herein.
  • RNA sequences including those transcribed from SEQ ID NOS:1 , 2, 3, 4, and 5, may also be involved in controlling viral latency.
  • nucleotide sequences having selected percent identities to the nucleotide sequence set forth in SEQ ID NO:1 , 2, 3, 4, and 5 are provided.
  • nucleotide sequences are provided that have at least about 50% identity, preferably at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to the nucleotide sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5.
  • the nucleotide sequences may further have the percent identities mentioned herein to a nucleotide sequence of substantial length within the porcine adenoviral genome.
  • such length may be less than about 100, 200, 500, 600 or 900 nucleotides in SEQ ID NOS:1 , 2, 3, 4, and 5, or may be the length of the entire sequence in these SEQ ID NOS.
  • a further requirement is that the nucleotide sequences described in SEQ ID NOS:1 , 2, 3, 4, and 5 encode proteins or RNA, preferably of adenoviral origin, that function as described herein.
  • nucleotide sequences are provided that have at least about 50% identity, preferably at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to the nucleotide sequences set forth in SEQ ID NOS:6, 7, 8, 9, and 10.
  • the nucleotide sequences may further have the percent identities mentioned herein to a nucleotide sequence of substantial length within the porcine adenoviral genome. For example, such length may be less than about 600, 700 and 1500 nucleotides, for SEQ ID NOS:6, 7, 8, 9 and 10, or may be the length of the entire sequence in these SEQ ID NOS.
  • the nucleotides sequences provided in SEQ ID NOS:6, 7, 8, 9, and 10 must encode proteins or RNA, preferably of adenoviral origin, that function as described herein.
  • the percent identity may be determined, for example, by comparing sequence information using the Gap program described above.
  • the nucleotide sequences include those having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:2 from nucleotide 1 to nucleotide 114, those set forth in SEQ ID NO:3 from nucleotide 121 to nucleotide 489 and those set forth in SEQ ID NO:4 from nucleotide 316 to nucleotide 438. Nucleotide sequences having the selected percent identities to these sequences, as discussed for SEQ ID NOS:2, 3 and 4 above, are also provided, as are nucleotide sequences which are substantially similar to these nucleotide sequences.
  • nucleotide sequences include those having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:8 from nucleotide 1 to nucleotide 1101 and those set forth in SEQ ID NO:9 from nucleotide 1 to nucleotide 405 .
  • a suitable DNA sequence may be obtained by cloning techniques using cDNA libraries. Such cDNA libraries are available commercially or may be constructed using standard methods known in the art. Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of adenoviral serotypes by means of nucleic acid hybridization or polymerase chain reaction (PCR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, nucleotide sequences having substantial similarity thereto, or portions thereof as described herein. Alternately, a suitable sequence may be made by techniques which are well known in the art.
  • nucleic acid sequences encoding the proteins described herein, or acting as templates for the RNA transcripts may be constructed by recombinant DNA technology, for example, by cutting or splicing nucleic acids using restriction enzymes and DNA ligase.
  • nucleic acid sequences may be constructed using chemical synthesis, such as solid-phase phosphoramidate technology. PCR may be used to increase the quantity of nucleic acid produced.
  • the particular nucleic acid sequence is of a length which makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments which may be synthesized and ligated together to form the entire desired sequence by methods known in the art.
  • RNA sequences may also be produced from the DNA templates by methods known to the skilled artisan.
  • recombinant nucleic acid molecules are provided.
  • the nucleic acid molecules include nucleotide sequences encoding the proteins described herein.
  • the nucleotide sequences have substantial similarity or identity, both as defined above, to the nucleotide sequences set forth in either SEQ ID NOS:1 , 2, 3, 4, and 5 or SEQ ID NOS:6, 7, 8, 9, and 10, or the selected sequences within these sequences as described above, or combinations thereof.
  • the proteins produced have the amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, or variants thereof as described above.
  • Recombinant nucleic acid molecules may be constructed by incorporating the desired nucleotide sequence within a vector according to methods well known to the skilled artisan and as described for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982) and Current Protocols in Molecular Biology, John Wiley and Sons, edited by Ausubel et al. (1988).
  • vectors are known that may be used in the invention.
  • various plasmid and phage vectors are known that are ideally suited for use in the invention.
  • pET30 and ⁇ GT11 may be used in the invention.
  • the desired recombinant vector may be constructed by ligating DNA linker sequences to the 5' and 3' ends of the desired nucleotide insert, cleaving the insert with a restriction enzyme that specifically recognizes sequences present in the linker sequences and the desired vector, cleaving the vector with the same restriction enzyme, mixing the cleaved vector with the cleaved insert and using DNA ligase to incorporate the insert into the vector as known in the art.
  • Preferred vectors are plasmid vectors.
  • the plasmid vectors include at least one of the nucleotide sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, and preferably an adenoviral origin of replication.
  • a plasmid vector includes at least one nucleotide sequence selected from the nucleotide sequences set forth in SEQ ID NOS:3 and 4. Such vectors may advantageously be used to introduce the nucleotide sequences into a cell for production of adenoviral vectors as described below.
  • the vectors may include other nucleotide sequences, such as those encoding selectable markers, including those for antibiotic resistance or color selection.
  • the vectors also preferably include a transcriptional regulatory nucleotide sequence, such as a promoter.
  • the desired nucleic acid insert is preferably operably linked to the promoter.
  • a nucleic acid is "operably linked" to a another nucleic acid sequence, such as a promoter sequence, when it is placed in a specific functional relationship with the other nucleic acid sequence.
  • the functional relationship between a promoter and a desired nucleic acid insert typically involves the nucleic acid and the promoter sequences being contiguous such that transcription of the nucleic acid sequence will be facilitated.
  • Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1 ) result in the introduction of a frame-shift- mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region.
  • the promoter element is generally upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence.
  • a wide variety of promoters are known in the art, including cell- specific promoters, inducible promoters, and constitutive promoters.
  • the promoters may further be selected such that they require activation by activating elements known in the art, so that production of the gene products, including the protein encoded by the nucleic acid sequence insert as well as the RNA transcript, may be regulated as desired.
  • the promoter is a foreign promoter.
  • a "foreign promoter" is defined herein as a promoter other than the natural, or native, promoter involved in transcribing a particular region of DNA.
  • the vectors may further include other regulatory elements, such as enhancer sequences, which cooperate with the promoter to achieve transcription of the nucleic acid insert coding sequence.
  • the vectors may include another nucleotide sequence insert that encodes a protein that may aid in purification of the desired protein encoded by the desired nucleotide sequence.
  • the additional nucleotide sequence is positioned in the vector such that a fusion, or chimeric, protein is obtained.
  • a protein described herein may be produced having at its C-terminal end several histidine molecules joined to the protein. Therefore, the additional nucleotide sequence may include, for example, the nucleotide sequence encoding multiple histidines.
  • the protein may be isolated on a nickel chromatography column, which will bind the histidine in the protein being purified. After purification procedures known to the skilled artisan, the additional amino acid sequence may be cleaved with an appropriate enzyme, such as a protease. The desired protein may then be isolated from the other proteins, or fragments thereof, by methods known in the art.
  • the preferred recombinant vectors may be used to transform, or introduce nucleic acid into, a host cell, and thus may include a nucleic acid having a nucleotide sequence, for example, that encodes a protein of interest.
  • Such methods include, for example, those described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), as well as Current Protocols in Molecular Biology, John Wiley and Sons, edited by Ausubel et al. (1988).
  • the host cell may produce the desired protein, RNA, or variants thereof as described above.
  • a host cell that includes the preferred recombinant vectors described above.
  • the recombinant vectors are introduced into a host cell to advantageously form an adenoviral vector as described below.
  • the adenoviral vector may then be used to introduce a desired nucleotide sequence into a cell.
  • host cells may be used in the invention, including prokaryotic and eukaryotic host cells, eukaryotic host cells are preferred. Further preferred are animal host cells, including swine, bovine, murine and avian host cells. Human host cells are most preferred.
  • the host cells may include cell lines that transiently or stably produce the adenoviral vectors herein described.
  • a further aspect of the invention provides recombinant adenoviral vectors.
  • adenoviral vectors are preferred vectors for introducing into a host cell a nucleotide sequence encoding a protein of interest.
  • an adenoviral vector may include the nucleotide sequences described herein, especially the nucleotide sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, that is preferably replication-defective so that it will not reproduce when introduced into a host cell.
  • the adenoviral vector may include at least one of the nucleotide sequences described herein, such as those set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, preferably a nucleotide sequence encoding a desired protein product and typically a promoter, preferably at least one, as described above.
  • a recombinant adenoviral vector preferably a porcine adenovirus type 3 vector
  • an adenoviral genome having a functional deletion in its E1 region is provided that includes an adenoviral genome having a functional deletion in its E1 region.
  • functional deletion is meant a deletion that results in a replication- defective adenovirus.
  • various portions of the E1 region may be deleted, including, for example, portions of the E1 A and E1 B genes.
  • the entire E1 region may be deleted.
  • the deleted portions of the E1 region, or other applicable region of the genome are advantageously replaced with a nucleotide sequence of interest.
  • the nucleotide sequence preferably includes a gene that encodes a protein of interest.
  • a promoter sequence may also be operably linked to a terminal 5' end of the nucleotide sequence encoding a protein of interest, as discussed above.
  • the vector having the functional E1 deletion in its genome may further include at least one of the introduced nucleotide sequences described herein, especially those set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, and preferably includes at least one promoter sequence operably linked to a terminal 5' end of these nucleotide sequences.
  • adenoviral vectors are well known in the art, including direct cloning and homologous recombination methods.
  • methods for producing such vectors may be found in the following references: Yeh, P. and Perricaudet, M. (1997) FASEB J. 11 :615-623; Wang, Q. et al. (1997) Gene Ther. 4:393-400; Gilgenkrantz, H. et al. (1995) Hum. Gene Ther. 6:1265-1274; and U.S. Patent Nos. 5,518,913; 5,585,362 and 5,698,202.
  • a method typically includes introducing into a cell a transcriptional regulatory nucleotide sequence and an isolated nucleic acid molecule as described herein, such as SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10.
  • a method typically involves making a functional deletion in the E1 region of the adenoviral genome and may also include replacing the deleted region of the genome with a nucleotide sequence of interest as described above.
  • a method includes introducing into a host cell a nucleic acid molecule encoding a desired protein and an isolated nucleic acid molecule having a nucleotide sequence having the percent identity or similarity as described herein to the nucleotide sequence selected from SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10, and combinations thereof.
  • Each of the nucleic acid molecules independently preferably include a foreign promoter, further preferably at least one foreign promoter, operably linked to their terminal 5' end.
  • the nucleotide sequences are preferably carried by an adenoviral vector as described above.
  • the desired nucleic acid molecule is introduced by infecting the cell with an adenoviral vector as described herein.
  • the adenoviral vector may be constructed as described herein.
  • the adenoviral vector may include an adenoviral genome with a functional deletion in its E1 region which is replaced by the nucleotide sequence of interest, and may also include one or more of the viral nucleotide sequences described herein.
  • the vector may include one or more of the nucleotide sequences described herein, especially those selected from SEQ ID NOS:1 , 2, 3, 4, and 5, an inverted terminal repeat (ITR) sequence, an adenoviral origin of replication and the nucleotide sequence, such as a transgene, of interest.
  • ITR inverted terminal repeat
  • the cells may be cultured under conditions effective to achieve production of the RNA transcript, and/or expression of the desired protein, depending on whether RNA or protein is desired.
  • methods of expressing a desired protein include introducing into a host cell an isolated nucleic acid molecule that includes a first nucleotide sequence described herein, especially those set forth in SEQ ID NOS:1 , 2, 3, 4, or 5, or combinations thereof, and further preferably includes at least one promoter sequence, such as a foreign promoter sequence, operably linked to a terminal 5' end of the isolated nucleotide sequence.
  • a second nucleotide sequence encoding a desired protein is also introduced into the host cell.
  • the protein may typically be one that provides some benefit to the host cell, including replacing the function of a non-functional protein, or one of decreased function, in the host cell.
  • the nucleotide sequence encoding the desired protein preferably includes a promoter sequence operably linked to a terminal 5' end of the nucleotide sequence.
  • the nucleotide sequences are preferably introduced into the host cell by first incorporating the sequences into an adenoviral vector.
  • the desired cells may be infected with the adenoviral vector according to known procedures in the art so that the desired nucleotide sequence may be introduced into the cell.
  • the host cells are then cultured under conditions, well known to the skilled artisan, effective to achieve expression of the desired polypeptide.
  • the desired polypeptide may then be purified using conventional techniques.
  • the transgene may be expressed by utilizing a promoter as described herein while the virus is in a latent state, and, when it is desired that the virus be cleared from the system, the virus is then returned to a non-latent state by regulating production of the RNA and/or proteins described herein. This provides a method of sequential expression of the transgene according to the need.
  • a method includes introducing into a host cell an isolated nucleotide sequence as described herein, preferably having at least one foreign promoter operably linked to its terminal 5' end. The cells may then be cultured under conditions to achieve expression of the protein.
  • EXAMPLE 1 SEQUENCE ANALYSIS OF PORCINE ADENOVIRUS Cell Culture and Virus
  • MEM Eagles minimum essential medium
  • PAd3 kindly supplied by Dr. W. Mengeling, USDA, Agricultural Research Service, National Animal Disease Center, Ames, IA, was grown in ST cells and purified by cesium chloride density-gradient centrifugation [Graham F. L. and Prevec L. (1991 ) in Murray E. J. (ed): Methods in Molecular Biology: Gene Transfer and Expression Protocols, Clifton, Humana Press, Vol. 7, pp 109-128].
  • DNA was extracted from purified virions following a technique described previously [Graham F. L. and Prevec L. (1991) in Murray E. J. (ed): Methods in Molecular Biology: Gene Transfer and Expression Protocols, Clifton, Humana Press, Vol. 7, pp 109-128].
  • the terminal protein attached to PAd3 DNA was removed by sodium hydroxide treatment.
  • PAd3 DNA was digested with BamHI, separated on an agarose gel by electrophoresis and a 4.8 kb fragment containing the left-end of PAd3 genome between m.u. 0 and 13.7 was purified and inserted at the Hincll-BamHI site of pUC18 to obtain pPAd-E1.
  • a large scale purification of pPAd-E1 was performed by alkaline lysis and cesium chloride purification [Sambrook J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press].
  • pPAd-E1 was sequenced using universal and reverse primers and then the entire sequence on both strands was obtained by primer walking following the dideoxy nucleotide chain termination technique [Sanger F. et al (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467] using an Applied Biosystems 373A automated sequencer. All sequence analyses were done using the GCG sequence analysis program.
  • ORFs homologous to HAd5 E1A, E1 B-19k, E1 B-55k, plX, plVa2 and five novel ORFs were identified in the PAd3 genome (Fig. 1A, 1 B and 2).
  • Our PAd3 sequence yielded approximately 41 % and 99% homology at the nucleotide level with the E1 regions of PAd4 [Kleiboeker S. B. (1995) Virus Res. 31 :17-25] and recently published sequence of PAd3 [Reddy P. S. et al. (1998) Virus Res. 58:97-106], respectively.
  • the inverted terminal repeat (ITR) sequence from nt 1-144 was identical to the published sequence of PAd3 ITR [Reddy P. S. (1995) Virology 212:237-239].
  • the PAd3 ITR also contains a 10-nt (8-17 nt) sequence that may be important for virus DNA replication [Shinagawa M. et al (1987) Gene 55:85-93]. It has been demonstrated that there are seven A-repeat packaging domains located within nt 194-380 of the Had5 genome which are involved in viral DNA packaging [Grable M. and Hearing P. (1990) J. Virol. 64:2047-2056; Schmid S. I. and Hearing P. (1997) J. Virol.
  • PAd3 at least has two TTTTG domains that are usually a part of A-repeat packaging domains and are located at nt 37-41 and 265-269.
  • the consensus nuclear factor III (NFIII)- binding site [Hatfield L. and Hearing P. (1993) J. Virol. 67:3931-3939] is located at nt 130-140.
  • Four GC-rich regions similar to the SP-I binding site were present at nt 68-75, 77-82, 198-204, and 213-219.
  • E1A Region Within the 288R E1A protein of HAd, binding sites for a number of cellular proteins including p300, retinoblastoma protein (Rb), cyclin A, an associated protein kinase pSS"" 2 and other unassigned proteins have been defined [Whyte P. et al. (1988) Nature 334:124-129; Whyte P. et al. (1989) Cell 56:67-75; Lundblad J. R. et al. (1995) Nature 374:85-88; Arany Z. et al. (1995) Nature 374:81-84].
  • the predicted PAd3 E1A ORF contains 170 amino acid residues (R) with an initiation codon at nt 533 followed by a stop codon at nt 1046 (Fig. 1A and 2).
  • This ORF showed approximately 27%, 26%, and 41% amino acid identity and approximately 34%, 35%, and 45% amino acid similarity with E1A of BAd3, HAd5 and PAd4, respectively.
  • a probable intron of 100 bp with its splice donor and splice acceptor sites at nt 1044, and 1145, respectively may result in continuation of E1A ORF until a stop codon appears at 1221.
  • the resultant putative PAd3 E1 A is expected to be of 195R.
  • the putative E1A 'TATA' box and the polyadenylation signal are located at nt 449 and 1287, respectively.
  • the conserved region 3 (CR3) of the 288R E1A of HAd5 includes the activation region, the metal binding region, and the promoter binding region [Lillie J. W. and Green M. R. (1989) Nature 338:39-44].
  • the predicted activation, metal binding, and the promoter binding regions of PAd3 E1A showed homology with similar E1A sequences of HAd5, BAd3, and PAd4 (Fig. 3A-C). There was a complete conservation of the metal binding (C-X 2 -C-X 13 -C-X 2 -C) motif of PAd3 E1A.
  • the putative PAd3 E1A region between amino acids 112 and 126 had sufficient homology with the Rb binding region [Dyson N. et al. (1990) J. Virol. 64:1353-1356] of E1A of HAd5 and BAd3 (Fig. 3D).
  • the putative PAd4 E1A [Kleiboeker S. B. (1995) Virus Res. 31 :17-25] did not seem to contain either the promoter binding domain or the Rb binding domain.
  • the E1B transcription unit of HAd5 consists of two major messages to produce 19k and 55k proteins. Both of these proteins independently inhibit p53-mediated apoptosis by different mechanisms [Wold W. S. M. et al. (1994) Trends Microbiol. 2:437-443]. Coexpression of both E1A and E1B (19k and or 55k) is required for high frequency transformation of cultured rodent cells [Wold W. S. M. et al. (1994) Trends Microbiol. 2:437-443].
  • the putative first PAd3 E1B ORF begins at nt 1462 to the termination codon at nt 2068.
  • This ORF defines a polypeptide of 202R that had approximately 23%, and 26% amino acid identity and approximately 31%, and 39% amino acid similarity with BAd3 E1 B-19k (Fig. 4A) and HAd5 E1B-19k, respectively.
  • the two other regions of HAd5 E1 B-19k that are conserved in PAd3 E1B-202R are also shown (Fig. 4B & C). It was surprising to note that the published PAd4 E1 region sequence [Kleiboeker S. B. (1995) Virus Res. 31 :17-25] did not contain an ORF similar to PAd3 E1 B-202R.
  • the putative second PAd3 E1 B ORF starts at nt 1830 and terminates at nt 3255.
  • This ORF defines a polypeptide of 475R that had approximately 32%, 28% and 30% amino acid identity and approximately 43%, 37%, and 36% amino acid similarity with E1 B-55k homologs of BAd3, HAd5 and PAd4, respectively (Fig. 4D).
  • the putative E1 B TATA motif and the polyadenylation signal are located at nt 1330 and 3340, respectively.
  • the HAd5 plX protein is a minor structural protein that is essential for the packaging of full-length genomes [Ghosh-Choudhury G. et al. (1987) EMBO J 6:1733-1739].
  • An ORF starting at nt 3395 and terminating at nt 3992 represents a protein of 198R.
  • This putative PAd3 plX showed 22%, and 23% amino acid identity and 30%, and 30% amino acid similarity with plX of BAd3 and HAd5, respectively.
  • the N-terminal portion of plX of both HAd5 and BAd3 showed extensive homology with the predicted PAd3 plX (Fig. 5).
  • the putative PAd3 plX TATA motif and the polyadenylation signal are present at nt 3339 and 4085, respectively.
  • the sequence information for the putative PAd4 plX ORF is not available.
  • the HAd IVa2 message transcribed from the lower-strand is involved in adenovirus replication [Swaminathan S. and Thimmapaya B. (1995) in Doerfler W. and Bohm P. (ed) The Molecular Repertoire of Adenoviruses III: Biology and Pathogenesis. Curr Topics Microbiol. Immunol., Berlin, Spring- Verlag, Vol 1999/111, pp 177-194].
  • the lower-strand of PAd3 genome between m.u. 13.7 and 1 1.7 contains an incomplete ORF representing an approximately 50% C-terminal portion of the predicted PAd3 IVa2 protein (SEQ ID NO:10 and FIG. 2).
  • the C-terminal of putative 288R protein demonstrated 44% identity with herpesvirus papio Epstein- Barr virus nuclear antigen 1 (EBNA1 ) (Fig. 7B, 7.) [Yates J. L. et al. (1996) Virology 222:1-13] and 36% identity with the GATA-4 transcription factor of mice (Fig. 7B, 8.) [Arceci R. J. et al. (1993) Mol. Cell Biol. 13:2235-46].
  • Herpesvirus papio infects baboons and is similar to Epstein-Barr virus (EBV) of human [Heller M. and Kieff E. (1981) J. Virol. 37:821-826].
  • EBNA1 is involved in maintaining the EBV genome during a latent phase by providing a stable and regulated replication [Yates J. L. et al. (1984) Proc. Natl. Acad .Sci. USA 81 :3806- 3810; Yates J. L. et al. (1985) Nature 313:812-815].
  • GATA-4 is a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally-derived tissues and heart and is involved in gene expression in these tissues [Arceci R. J. et al. (1993) Mol. Cell. Biol. 13:2235-46].
  • the 163R * predicted protein contains a proline- and alanine-rich region (83-179 residues) that showed 54% identity with the EBV nuclear antigen (FIG. 7B- 9) (EBNA2) [Sample J. et al. (1986) Proc. Natl. Acad. Sci. USA 83:5096- 6000] and 40% identity with a human transcription initiation factor II (TFII) (Fig. 7B, 10.) [Tanese N. Et al. (1996) Proc. Natl. Acad. Sci. USA 93:13611-13616].
  • EBNA2 is responsible for transforming B lymphocytes [Jones M. D. et al.
  • these novel ORFs showed homology either with EBNA antigens or other cellular and viral transcription factors, these putative proteins appear to be stimulating or inhibiting viral replication.
  • PAd3 162R and 97R putative proteins may be trans-activating adenovirus replication similar to trans-activating immediate early proteins of herpesviruses. These predicted proteins may play a role in initial viral replication, virus latent phase or virus replication during reactivation. Some of these PAd3 putative proteins may function as a complex with other viral or cellular protein/s.
  • a 709 bp Hindlll fragment containing the ORF of 162R was isolated from pPAd-E1 and cloned into the Hindlll site of pUC18.
  • the resultant plasmid was digested with BsmBI and cohesive ends were filled in using

Abstract

Purified proteins that function in viral replication, especially in controlling the viral latent state, are provided. Nucleotide sequences encoding these proteins are also provided. Both DNA sequences, and the RNA transcripts are encompassed by the invention. The invention also provides recombinant vectors including the nucleotide sequence encoding the novel proteins described herein. In a preferred form of the invention, a plasmid is provided that includes the nucleotide squence encoding the novel proteins described herein, along with an adenoviral origin of replication. Further provided are host cells that include the recombinant vectors described herein, adenoviral vectors and methods of expressing viral proteins and other desired proteins utilizing the nucleotide sequences, recombinant vectors and host cells described herein.

Description

ADENOVIRAL VECTORS, PROTEINS AND METHODS FOR THEIR USE
CROSS-REFERENCE TO RELATED APPLICATIONS
The present application claims the benefit of U.S. Provisional Patent Application Serial Number 60/121 ,647, filed on February 24, 1999 which is hereby incorporated by reference in its entirety.
This invention was made with government support under Public Health Service grant number GM55168-01 awarded by the National
Institutes of Health/National Institute of General Medical Sciences. The government has certain rights in the invention.
BACKGROUND OF THE INVENTION Adenoviruses are non-enveloped DNA viruses that infect a wide variety of cells and some of them typically cause acute respiratory and ocular infections, as well as hemorrhagic cystitis and infantile gastroenteritis. After adenoviruses infect a cell, their DNA does not become incorporated into the genome of the infected cell, but remains episomal. As adenoviruses infect a wide variety of host cells, and have the ability to replicate in both dividing and non-dividing cells, recombinant adenoviruses are frequently used as viral vectors for gene delivery applications, especially to deliver recombinant vaccines.
Porcine adenoviruses (PAds) usually cause inapparent infections. However, they are associated with enteric, respiratory, kidney, and central nervous system infections in swine [Derbyshire J. B. et al. (1975) J. Comp. Path. 85:437-443; Hirahara T., et al. (1990) Jpn. J. Vet. Sci. 52:407-409; Hirahara T., et al. (1990) Jpn J. Vet Sci. 52:1089-1091]. PAds are classified into five currently known serotypes, PAd-1 , -2, -3, -4, & -5 [Derbyshire J. B. et al. (1975) J. Comp. Path. 85:437-443; Hirahara T., et al. (1990) Jpn. J. Vet. Sci. 52:1089-1091]. Oronasal inoculation of pigs or colostrum-deprived piglets with PAd-1 , -2, or -3 did not result in clinical signs or gross lesions, [Derbyshire J. B. et al. (1975) J. Comp. Path. 85:437-443; Sharpe H. B. A. and Jesset D. M. (1967) J. Comp. Pathol. 77:45-50; Tuboly, T. et al. (1993) Res. Vet. Sci. 54:345-350] suggesting that these serotypes usually lead to subclinical infections. PAd3 was initially isolated from a fecal sample collected from a healthy pig [Clarke M. C. et al. (1967 Arch. Ges. Virusforsch. 21 :91-97]. A cross-hybridization experiment using labeled human adenovirus type 2 (HAd2) DNA as a probe resulted in hybridization with PAd3 DNA fragments mainly in the region corresponding to structural proteins [Benko M. et al. (1990) J. Gen. Virol. 71 :465-469]. The genome of PAd3 is approximately 35 Kb in size, restriction maps for a number of enzymes have been reported [Garwes D. J. and Xuan H. (1989) Intervirology. 30:234-236; Reddy P. S. et al. (1993) Intervirology 36:161 -168 ] and the early region 3 (E3) has been sequenced [Reddy P. S. et al. (1995) Virus Res. 36:97-106].
Following adenovirus infection, the E1 genes are the first set of genes to be expressed. The E1 region includes two transcription units, E1A and E1B, each of which expresses multiple proteins as a result of differential splicing and use of different translation reading frames [Berk A.J. and Sharp P. A. (1978) Cell. 14:695-711]. E1 gene products of several HAds have been shown to transactivate a variety of viral and cellular promoters, and transform and immortalize cultured rodent cells [Berk A.J. (1986) Ann. Rev. Genet. 20:45-79; Whyte P. et al. (1988) Nature. 334:124-129; Whyte, P. et al. (1989) Cell. 56:67-75; Lillie J.W. and Green M. R. (1989) Nature. 338:39-44; Lundblad J. R. et al. (1995) Nature. 374:85-88; Arany Z. et al. (1995) Nature. 374:81-84].
Recent studies involving HAd E1 insertion vectors as recombinant vaccine delivery vehicles have demonstrated that they may be better than HAd E3 insertion vectors [Eloit M. and Adam M. (1995) J. Gen. Virol. 76:1583-1589; Fooks A. R. et al. (1995) Virology 210:456-465; Mittal S. K. et al. (1996) Virology 222:299-309; Xiang Z. Q. et al. (1996) Virology 219:220- 227]. Since E1 insertion vectors do not actively replicate in their natural hosts, they should be safer as virus excretion from vaccinated animals would not occur. These vectors may also be useful for vaccinating immunocompromised hosts since they could not spread in vivo. Titers of E1- deleted vectors in E1 -complementing cells are similar to those of E3- deleted vectors, therefore, the cost of production of either vector system would be similar.
However, many of these adenovirus vectors stimulate an immune response that results in elimination of the cells carrying the vectors. Therefore, one of the drawbacks of many of the currently used adenovirus vectors as gene delivery vehicles is that the transgene is expressed for only a short period of time. This loss in expression can not be overcome with frequent rounds of vaccination, as the host-immune response often leads to neutralization of the virus before it enters target cells. A need therefore exists for adenovirus vectors that exhibit prolonged transgene expression. The present invention addresses this need.
SUMMARY OF THE INVENTION
Open reading frames in nucleotide sequences have been discovered in porcine adenovirus that are expected to encode proteins involved in controlling viral replication, especially in controlling the viral latent phase. Accordingly, the present invention provides these purified proteins.
In yet another aspect of the invention, isolated nucleic acid molecules that encode the proteins recited above are provided. The nucleic acid molecules may be incorporated into a vector to form a recombinant nucleic acid molecule. Moreover, such recombinant nucleic acid molecules may be introduced into a host cell.
Further provided are adenoviral vectors that include the nucleic acid molecules described herein. In one form of the invention, the vectors may include a foreign promoter, preferably at least one foreign promoter, that is operably linked to a terminal 5' end of the nucleic acid molecule. The vectors may further include a second nucleic acid molecule of interest, such as one having a nucleotide sequence encoding a desired protein. The vector advantageously may also include a second promoter that is operably linked to a terminal 5' end of the second desired nucleic acid molecule, and may further include an adenoviral origin of replication and an inverted terminal repeat sequence. In other forms of the invention, replication-defective porcine adenoviral vectors, especially type 3 vectors, are provided that include a porcine adenovirus genome, such as a type 3 genome, having a functional deletion in the E1 region. These vectors may also include a nucleotide sequence encoding a protein of interest inserted into the E1 region. The vector also preferably includes a foreign promoter operably linked to a terminal 5' end of the nucleotide sequence encoding the protein of interest, and may include one or more of the viral nucleotide sequences provided herein, preferably with at least one foreign promoter operably linked to a terminal 5' end of these novel nucleotide sequences. In another aspect of the invention, methods of introducing a desired nucleic acid into a cell are provided. In one form of the invention, a method includes infecting a host cell with an adenoviral vector having an isolated first nucleic acid molecule encoding one or more, preferably at least one, of the novel proteins as described herein and a second desired nucleic acid molecule that may advantageously encode a protein of interest. Each of the nucleic acid molecules preferably independently include a foreign promoter operably linked to their terminal 5' end.
In a further aspect of the invention, a method of making an adenovirus vector is provided. In one form, the method includes introducing into a cell an isolated nucleic acid molecule encoding the proteins described herein and a foreign promoter, wherein the foreign promoter is operably linked to a terminal 5' end of the molecule.
In other aspects of the invention, methods of expressing a desired protein are provided. A method may include infecting a host cell with an adenoviral vector having a first isolated nucleic acid molecule encoding a protein, such as a viral protein, as described herein, and a second desired nucleic acid molecule encoding a desired protein, and culturing the host cells under conditions effective in achieving expression of the desired protein. The adenoviral vector may further include a foreign promoter, preferably at least one, that is operably linked to a terminal 5' end of the first isolated nucleic acid molecule and another foreign promoter so linked to the second desired nucleotide sequence. The desired protein may then be purified by conventional techniques. In another aspect of the invention, methods of expressing viral proteins are provided. In one embodiment, a method includes introducing into a host cell an isolated first nucleotide sequence as described herein and a foreign promoter, preferably at least one, that is operably linked to a terminal 5' end of said first nucleotide sequence and culturing under conditions to achieve expression of said protein. One object of the invention is to provide proteins and RNA involved in viral replication, especially those expected to function in viral latency, as well as nucleotide sequences encoding these gene products.
A further object of the invention is to provide a porcine adenoviral type 3 vector having a functional E1 deletion.
A further object of the invention is to provide an adenoviral vector that may be used to increase the duration of transgene expression.
Yet another object of the invention is to provide methods of utilizing the nucleotide and amino acid sequences described herein. These and other objects and advantages of the present invention will be apparent from the descriptions herein.
BRIEF DESCRIPTION OF THE FIGURES
FIGS. 1A-1 G depict the nucleotide sequence and major open reading frames (ORFs) of the PAd3 genome between map unit (m.u.) 0 and 13.7 determined as discussed in Example 1. The first 144 nucleotides (nt) represent an inverted terminal repeat sequence. The conserved region of replication (bold), the putative packaging signals (underlined), a putative nuclear factor III (NFIII) binding site (bold, italics and underlined), and putative SP1 -binding sites (italics and underlined) are indicated. PAd3 E1A, E1 B-202R, E1 B-411 R, plX ORF, novel ORF 184R on the upper strand and an incomplete IVa2 ORF, and four unique ORFs (97R,162R,163R* and 288R) on the lower-strand are shown in bold. The probable TATA motifs and polyadenylation signals are indicated as bold and underlined, and bold and doubled underlined, respectively.
FIG. 2 depicts open reading frames (ORFs) in PAd3 E1 and plX regions of the PAd genome. The left-end PAd3 genome between m.u. 0 and 13.7 was sequenced as described in Example 1 to provide a sequence of 4769 nt. Three frames of ORFs on the upper-strand are indicated as a, b, and c. Similarly, three frames of ORFs on the lower-strand are indicated as d, e, and f. The direction of ORFs are shown by an open arrow on the left. All ORFs of 50 or more amino acids following methionine are shown. ORFs that have demonstrated homology with known adenovirus proteins are indicated by hatched boxes and novel ORFs that have homology with EBNA1 , EBNA2 and other transcriptional initiation factors are indicated by bricked boxes. The putative ORF 163R* did not have an initiation codon.
FIG. 3 depicts a comparison of the amino acid sequence of four important functional regions in the E1 A proteins of PAd3, PAd4, HAd5, and
BAd3 showing the areas of homology. Residues that are identical in all four E1A proteins are in bold. The residue numbers for each of the viruses are shown. FIG. 4 depicts a comparison of amino acid sequences of the putative PAd3 E1 B proteins as described in Example 1. A) homology between the BAd3 E1 B-19k and the PAd3 E1 B-202R; B & C) homologous regions of the PAd3 E1B-202R, BAd3 E1 B-19k, and HAd5 E1 B-19k; and D) homology comparison among C-terminal regions of HAd5 E1 B-55k homologs of four adenoviruses are presented. Identical residues are shown in bold. The residue numbers for each of the viral proteins are shown.
FIG. 5 shows a homology comparison among N-terminal regions of
HAd5 plX homologs of three adenoviruses. Identical residues are shown in bold. The residue numbers for each of the viruses are shown.
FIG. 6 depicts a homology comparison among C-terminal regions of HAd5 IVa2 homologs of three adenoviruses. Identical residues are shown in bold. The residue numbers for each of the viruses are shown.
FIGS. 7A-7B show comparisons of the amino acid sequence of the PAd3 five novel ORFs. FIG. 7A, 1 ) region of homology between PAd3 184R and bovine Na+/Ca+,K+ exchanging protein (PIR S209691); FIG. 7A, 2) region of homology between PAd3 184R and BICP4 (Gl L14321+); FIG. 7A, 3) region of homology between PAd3 97R and EHV membrane glycoprotein (DBJ 088734*); FIG. 7A, 4) region of homology between PAd3 97R and EHV transcriptional control protein 57 (PIR S55652f); FIG. 7A, 5) region of homology between 162R and equine herpesvirus (EHV) membrane glycoprotein (DBJ D887341 ; FIG. 7A, 6) region of homology between 162R and trans-acting transcriptional protein ICP0 of herpes simplex virus, (SP P282841); FIG. 7B, 7) region of homology between PAd3 288R and EBNA1 ; FIG. 7B, 8) region of homology between PAd3 288R and the transcription factor GATA-4 of mice (SP 0083691*); FIG. 7B, 9) region of homology between PAd3 163R* and EBNA2 (Pir | 1542447*); and FIG. 7B, 10) region of homology between PAd3 163R* and the human transcription initiation factor II (TFII) 130k subunit (SP 10002681*) are presented. Identical residues are shown in bold. The residue numbers for each of the proteins are shown. *Protein sequence database number.
FIG. 8 depicts an SDS-polyacrylamide gel from the purification of PAd3 162R protein expressed in bacteria according to the protocol in Example 2. A plasmid containing PAd3 162R ORF under the control of an inducible bacteriophage T7 promoter was used to transform bacteria expressing T7 RNA polymerase. Bacterial suspension cultures at approximately 0.6 O.D. were treated with 1.5 mM of IPTG to induce the expression of the 162R protein as a his-tag fusion protein. Bacterial cultures were subjected to cellular disruption using a French press and insoluble inclusions containing the fusion protein were isolated and solubilized in a 6M gaunidinium HCI solution. The solution was passed through a 0.2 μm filter and the filtrate was applied onto a Ni++ affinity column. The his-tag fusion protein bound to Ni++ column was eluted following the manufacturer's instructions using a kit (Novagen, Inc.). All protein fractions were separated by SDS-PAGE and then stained with Coomassie blue. The PAd3 162R fusion protein is indicated by an arrow. The molecular weight (MW) markers are shown on the left.
FIG. 9 depicts a Western dot-blot analysis of IPTG-induced or uninduced bacterial cell extracts using HRP-conjugated S-tag. DE3 bacteria containing pET30-162R were grown in the presence or absence of IPTG. Total bacterial protein, soluble or insoluble (pellet) protein extracts were analyzed by Western blot using HRP-conjugated S-tag. The PAd3 162R fusion protein is indicated by an arrow. The molecular weight (MW) markers are shown on the left. DESCRIPTION OF THE PREFERRED EMBODIMENTS
For the purposes of promoting an understanding of the principles of the invention, reference will now be made to preferred embodiments and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications of the invention, and such further applications of the principles of the invention as illustrated herein, being contemplated as would normally occur to one skilled in the art to which the invention relates.
Novel nucleotide sequences expected to encode proteins involved in controlling viral latency have been identified in porcine adenovirus. As the proteins, and/or RNA encoded by these nucleotide sequences, may advantageously maintain the adenovirus in a latent state, an adenoviral vector including such sequences, along with a transgene of interest, may not elicit an immune response after being introduced into a host cell. This may increase the duration of expression of the transgene in the cell. In yet other embodiments, other nucleotide sequences are provided that are expected to encode other proteins involved in viral replication, including proteins involved in stimulating DNA synthesis, transactivation of transcription, packing of the full-length genome into virions and shutting down of host cell protein synthesis. Recombinant nucleic acid molecules are also provided that include the nucleotide sequences encoding the proteins described herein. The nucleic acid molecules may be incorporated into a host cell. In another aspect of the invention, methods of expressing a desired protein are also provided, as well as methods of expressing the proteins recited herein.
In a first aspect of the invention, purified proteins are provided that are expected to function in adenoviral replication. In one preferred embodiment, the proteins are expected to function in viral latency as more fully discussed in example 1. Amino acid sequences of these proteins, from a porcine adenovirus in one form of the invention, are set forth in SEQ ID NOS:1 , 2, 3, 4, and 5 (97R, 162R, 163R*, 288R and 184R, respectively) and also shown in FIGS. 1A-1 G.
In yet another embodiment, the proteins have amino acid sequences from a porcine adenovirus, as set forth in SEQ ID NOS:6, 7, 8, 9, and 10 (E1A, E1 B-202, E1 B-474, plX and IVa2, respectively) and also shown in FIGS. 1A-1G. The polypeptides described herein are substantially pure (i.e., the novel proteins are essentially free, e.g., at least about 95% free, from other proteins with which they naturally occur). Although the invention is described with reference to porcine adenovirus amino acid sequences, it is understood that the invention is not limited to the specific amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10. Skilled artisans will recognize that, through the process of mutation and/or evolution, polypeptides of different lengths and having differing constituents, e.g., with amino acid insertions, substitutions, deletions, and the like, may arise that are related to, or sufficiently similar to, a sequence set forth herein by virtue of amino acid sequence homology and advantageous functionality as described herein. The term "adenovirus-related protein" is used to refer generally to proteins having the features described herein and preferred examples include polypeptides having the amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10. Also included within this definition, and in the scope of the invention, are variants of the polypeptides which function as described herein, especially variants involved in controlling the latent state of adenovirus.
It is well known a wide variety of adenoviral serotypes commonly express and utilize homologous proteins, which include the insertions, substitutions and/or deletions discussed above, and yet which effectively provide similar function. For example, an amino acid sequence isolated from another serotype may differ to a certain degree from the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, and yet have similar functionality with respect to catalytic and regulatory function. Amino acid sequences comprising such variations are included within the scope of the present invention and are considered substantially or sufficiently similar to a reference amino acid sequence. Although not being limited by theory, it is believed that the identity between amino acid sequences that is necessary to maintain proper functionality is related to maintenance of the tertiary structure of the polypeptide such that specific interactive sequences will be properly located and will have the desired activity. Although it is not intended that the present invention be limited by any theory by which it achieves its advantageous result, it is contemplated that a polypeptide including these interactive sequences in proper spatial context will have good activity, even where alterations exist in other portions thereof.
In this regard, variants of the proteins described herein are expected to be functionally similar to that set forth in SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10, for example, if they include amino acids which are conserved among a variety of serotypes or if they include non-conserved amino acids which exist at a given location in another serotype that expresses proteins having the functions described herein. Another manner in which similarity may exist between two amino acid sequences is where a given amino acid of one group (such as a non- polar amino acid, an uncharged polar amino acid, a charged polar acidic amino acid or a charged polar basic amino acid) is substituted with another amino acid from the same amino acid group. For example, it is known that the uncharged polar amino acid serine may commonly be substituted with the uncharged polar amino acid threonine in a polypeptide without substantially altering the functionality of the polypeptide. Whether a given substitution will affect the functionality of the enzyme may be determined without undue experimentation using synthetic techniques and screening assays known in the art. The invention therefore also encompasses polypeptides having amino acid sequences similar to the amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4 and 5 that have at least about 50%, 40%, 60%, 50% and 40% identity thereto, respectively, that preferably function as described herein. Preferably, the amino acid sequences have at least about 70%, 60%, 70%, 70%, and 60% identity to the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, respectively, more preferably at least about 80% identity to SEQ ID NOS:1 , 2,3, 4 and 5 and most preferably at least about 90% identity to SEQ ID NOS:1 , 2, 3, 4, and 5. In one specific form of the invention, the amino acid sequences include those set forth in SEQ ID NO:2 from amino acid 1 to amino acid 38, in SEQ ID NO:3 from amino acid 41 to amino acid 163, and in SEQ ID NO:4 from amino acid 106 to amino acid 146 having at least the percentage identity to these sequences described above.
Similarly, with respect to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8, 9, and 10, the invention also encompasses amino acid sequences similar to these amino acid sequences having at least about 50%, 40%, 40%, 30% and 90% identity thereto, respectively, that preferably function in viral replication. Preferably, the amino acid sequences have at least about 60%, 60%, 60%, and 50%, identity to the sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively, and further preferably at least about 80%, 80%, 80% and 70% identity to the sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively. In most preferred embodiments, the amino acid sequences encompassed by the invention include amino acid sequences having at least about 90% identity to the sequences set forth in SEQ ID NOS:6, 7, 8 and 9. In another specific form of the invention, the amino acid sequences include those set forth in SEQ ID NO:8 from amino acid 1 to amino acid 367, and in SEQ ID NO:9 from amino acid 1 to amino acid 135, and sequences having at least the percentage identity to these sequences described above. Percent identity between two nucleotide sequences or two amino acid sequences may be determined, for example, by comparing sequence information over the entire sequence using the Gap program, version 9, available from Genetics Computer Group (GCG) (Madison, Wisconsin). Default parameters are preferably used, with the exception that end gaps are penalized using the end weight option of the program. In another aspect of the invention, isolated nucleic acid molecules, originally isolated from porcine adenovirus, are provided that encode the proteins having the amino acid sequences described herein, including those amino acid sequences having the aforementioned percent identities to the respective amino acid sequences. In one embodiment, the nucleotide sequences encode proteins that are expected to control viral latency and are set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, as well as in FIGS. 1A-1 G, or similar nucleotide sequences discussed herein. In other embodiments, the nucleotide sequences encode other proteins involved in viral replication, and are set forth in SEQ ID NOS:6, 7, 8, 9, and 10 and FIGS. 1A-1 G or similar nucleotide sequences discussed herein.
In one form of the invention, nucleotide sequences are provided that encode a protein that includes an amino acid sequence having at least about 50%, 40%, 60%, 50%, and 40% identity to the amino acid sequence set forth in SEQ ID NOS:1 , 2, 3 , 4 and 5, respectively. Preferably, amino acid sequences have at least about 70%, 60%, 70%, 70%, and 60% identity to the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, respectively, more preferably at least about 80% identity to SEQ ID NOS:1 , 2, 3, 4 and 5 and most preferably at least about 90% identity to SEQ ID NOS:1 , 2, 3, 4, and 5. In one specific form of the invention, the nucleotide sequence encodes a protein having an amino acid sequence set forth in SEQ ID NO:2 from amino acid 1 to amino acid 38, in SEQ ID NO:3 from amino acid 41 to amino acid 163, and in SEQ ID NO:4 from amino acid 106 to amino acid 146 having at least the percentage identity to these sequences described above for SEQ ID NOS:2, 3, and 4. Similarly, the invention also encompasses nucleotide sequences that encode proteins that include amino acid sequences having at least about 50%, 40%, 40%, 30% and 90% identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8, 9, and 10, respectively, that preferably function in viral replication. Preferably, the nucleotide sequences encode a protein having an amino acid sequence that has at least about 60%, 60%, 60%, and 50%, identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively, and further preferably at least about 80%, 80%, 80% and 70% identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8 and 9, respectively. In most preferred embodiments, the nucleotide sequences encode a protein having an amino acid sequence having at least about 90% identity to the amino acid sequences set forth in SEQ ID NOS:6, 7, 8 and 9. In one specific form of the invention, the nucleotide sequence encodes a protein having an amino acid sequence set forth in SEQ ID NO:8 from amino acid 1 to amino acid 367, and in SEQ ID NO:9 from amino acid 1 to amino acid 135, and sequences having at least the percent identity to these sequences described above for SEQ ID NOS:8 and 9.
The nucleotide sequences provided herein are also preferably transcribed to form an RNA nucleotide sequence, such as an mRNA transcript. Therefore, the RNA nucleotide sequences corresponding to the DNA sequences recited herein in SEQ ID NOS: 1 , 2, 3, 4 and 5, as well as in SEQ ID NOS:6, 7, 8, 9, and 10, wherein deoxythymidine ("T") is replaced by uridine ("U"), are also provided herein. RNA nucleotide sequences substantially similar to the transcripts are also encompassed in the invention, as well as RNA transcripts having the percent identity to these sequences as described herein for the DNA sequences.
The term "isolated nucleic acid," as used herein, is intended to refer to nucleic acid which is not in its native environment. For example, the nucleic acid is separated from other contaminants that naturally accompany it, such as proteins, lipids and other nucleic acid sequences. The term includes nucleic acid which has been removed or purified from its naturally- occurring environment or clone library, and further includes recombinant or cloned nucleic acid isolates and chemically synthesized nucleic acid.
The term "nucleotide sequence," as used herein, is intended to refer to a natural or synthetic linear and sequential array of nucleotides and/or nucleosides, including deoxyribonucleic acid and ribonucleic acid, and derivatives thereof. The terms "encoding" and "coding" refer to the process by which a nucleotide sequence, through the mechanisms of transcription and translation, provides the information to a cell from which a series of amino acids can be assembled into a specific amino acid sequence to produce a functional polypeptide, such as, for example, an active enzyme or other protein that has a specific function. The process of encoding a specific amino acid sequence may involve DNA sequences having one or more base changes (i.e., insertions, deletions, substitutions) that do not cause a change in the encoded amino acid, or which involve base changes which may alter one or more amino acids, but do not eliminate the functional properties of the polypeptide encoded by the DNA sequence.
It is therefore understood that the invention encompasses more than the specific exemplary nucleotide sequences provided herein. For example, nucleic acid sequences encoding variant amino acid sequences, as discussed above, are within the scope of the invention. Modifications to a sequence, such as deletions, insertions, or substitutions in the sequence, which produce "silent" changes that do not substantially affect the functional properties of the resulting polypeptide molecule are expressly contemplated by the present invention. For example, it is understood that alterations in a nucleotide sequence which reflect the degeneracy of the genetic code, or which result in the production of a chemically equivalent amino acid at a given site, are contemplated. Thus, a codon for the amino acid alanine, a hydrophobic amino acid, may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine. Similarly, changes which result in substitution of one negatively charged residue for another, such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, may also be expected to produce a biologically equivalent product.
Nucleotide changes which result in alteration of the N-terminal and C-terminal portions of the encoded polypeptide molecule would also not generally be expected to alter the activity of the polypeptide. In some cases, it may in fact be desirable to make mutations in the sequence in order to study the effect of alteration on the biological activity of the polypeptide. Each of the proposed modifications is well within the routine skill in the art.
In a preferred embodiment, the nucleotide sequences have substantial similarity to the sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, and, in other preferred embodiments, to SEQ ID NOS:6, 7, 8, 9, and 10, and variants described herein. The term "substantial similarity" is used herein with respect to a nucleotide sequence to designate that the nucleotide sequence is sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions. This method of determining similarity is well known in the art to which the invention pertains. Briefly, moderately stringent conditions are defined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. Vol. 1 , pp. 101 -104, Cold Spring Harbor Laboratory Press (1989) as including the use of a prewashing solution of 5X SSC (a sodium chloride/sodium citrate solution), 0.5% sodium dodecyl sulfate (SDS), 1 .0 mM ethylene diaminetetraacetic acid (EDTA) (pH 8.0) and hybridization and washing conditions of 55°C, 5x SSC. Thus, encompassed as part of the invention are nucleic acid sequences which are complementary to the sequences specifically shown in the sequence listing. A further requirement of the preferred polynucleotide is that it must encode a polypeptide having similar functionality to the proteins described herein. In a preferred form of the invention, the nucleotide sequences are deoxyribonucleic acid (DNA) sequences. In other forms of the invention, the nucleotide sequences are RNA sequences, preferably an RNA transcript, such as an mRNA transcript, produced from transcribing the nucleotide sequences provided herein. Such RNA sequences, including those transcribed from SEQ ID NOS:1 , 2, 3, 4, and 5, may also be involved in controlling viral latency.
In yet another embodiment, nucleotide sequences having selected percent identities to the nucleotide sequence set forth in SEQ ID NO:1 , 2, 3, 4, and 5 are provided. In one preferred form, nucleotide sequences are provided that have at least about 50% identity, preferably at least about 70% identity, more preferably at least about 80% identity, and most preferably at least about 90% identity to the nucleotide sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5. The nucleotide sequences may further have the percent identities mentioned herein to a nucleotide sequence of substantial length within the porcine adenoviral genome. For example, such length may be less than about 100, 200, 500, 600 or 900 nucleotides in SEQ ID NOS:1 , 2, 3, 4, and 5, or may be the length of the entire sequence in these SEQ ID NOS. A further requirement is that the nucleotide sequences described in SEQ ID NOS:1 , 2, 3, 4, and 5 encode proteins or RNA, preferably of adenoviral origin, that function as described herein.
In alternative embodiments, nucleotide sequences are provided that have at least about 50% identity, preferably at least about 70% identity, more preferably at least about 80% identity and most preferably at least about 90% identity to the nucleotide sequences set forth in SEQ ID NOS:6, 7, 8, 9, and 10. The nucleotide sequences may further have the percent identities mentioned herein to a nucleotide sequence of substantial length within the porcine adenoviral genome. For example, such length may be less than about 600, 700 and 1500 nucleotides, for SEQ ID NOS:6, 7, 8, 9 and 10, or may be the length of the entire sequence in these SEQ ID NOS. Furthermore, the nucleotides sequences provided in SEQ ID NOS:6, 7, 8, 9, and 10 must encode proteins or RNA, preferably of adenoviral origin, that function as described herein.
The percent identity may be determined, for example, by comparing sequence information using the Gap program described above. In one specific form of the invention, the nucleotide sequences include those having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:2 from nucleotide 1 to nucleotide 114, those set forth in SEQ ID NO:3 from nucleotide 121 to nucleotide 489 and those set forth in SEQ ID NO:4 from nucleotide 316 to nucleotide 438. Nucleotide sequences having the selected percent identities to these sequences, as discussed for SEQ ID NOS:2, 3 and 4 above, are also provided, as are nucleotide sequences which are substantially similar to these nucleotide sequences.
In another specific form of the invention, nucleotide sequences include those having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:8 from nucleotide 1 to nucleotide 1101 and those set forth in SEQ ID NO:9 from nucleotide 1 to nucleotide 405 .
It is not intended that the present invention be limited to these exemplary nucleotide sequences, but include sequences having substantial similarity thereto and sequences which encode variant forms of the proteins as discussed above and as further discussed below.
A suitable DNA sequence may be obtained by cloning techniques using cDNA libraries. Such cDNA libraries are available commercially or may be constructed using standard methods known in the art. Suitable nucleotide sequences may be isolated from DNA libraries obtained from a wide variety of adenoviral serotypes by means of nucleic acid hybridization or polymerase chain reaction (PCR) procedures, using as probes or primers nucleotide sequences selected in accordance with the invention, such as those set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, nucleotide sequences having substantial similarity thereto, or portions thereof as described herein. Alternately, a suitable sequence may be made by techniques which are well known in the art. For example, nucleic acid sequences encoding the proteins described herein, or acting as templates for the RNA transcripts, may be constructed by recombinant DNA technology, for example, by cutting or splicing nucleic acids using restriction enzymes and DNA ligase. Furthermore, nucleic acid sequences may be constructed using chemical synthesis, such as solid-phase phosphoramidate technology. PCR may be used to increase the quantity of nucleic acid produced. Moreover, if the particular nucleic acid sequence is of a length which makes chemical synthesis of the entire length impractical, the sequence may be broken up into smaller segments which may be synthesized and ligated together to form the entire desired sequence by methods known in the art. RNA sequences may also be produced from the DNA templates by methods known to the skilled artisan. In a further aspect of the invention, recombinant nucleic acid molecules are provided. In certain embodiments, the nucleic acid molecules include nucleotide sequences encoding the proteins described herein. The nucleotide sequences have substantial similarity or identity, both as defined above, to the nucleotide sequences set forth in either SEQ ID NOS:1 , 2, 3, 4, and 5 or SEQ ID NOS:6, 7, 8, 9, and 10, or the selected sequences within these sequences as described above, or combinations thereof. The proteins produced have the amino acid sequences set forth in SEQ ID NOS:1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, or variants thereof as described above. Recombinant nucleic acid molecules may be constructed by incorporating the desired nucleotide sequence within a vector according to methods well known to the skilled artisan and as described for example, in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982) and Current Protocols in Molecular Biology, John Wiley and Sons, edited by Ausubel et al. (1988). A wide variety of vectors are known that may be used in the invention. For example, various plasmid and phage vectors are known that are ideally suited for use in the invention. For example, pET30 and λGT11 may be used in the invention. In one embodiment, the desired recombinant vector may be constructed by ligating DNA linker sequences to the 5' and 3' ends of the desired nucleotide insert, cleaving the insert with a restriction enzyme that specifically recognizes sequences present in the linker sequences and the desired vector, cleaving the vector with the same restriction enzyme, mixing the cleaved vector with the cleaved insert and using DNA ligase to incorporate the insert into the vector as known in the art.
Preferred vectors are plasmid vectors. In a further preferred form of the invention, the plasmid vectors include at least one of the nucleotide sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, and preferably an adenoviral origin of replication. In a more preferred embodiment, a plasmid vector includes at least one nucleotide sequence selected from the nucleotide sequences set forth in SEQ ID NOS:3 and 4. Such vectors may advantageously be used to introduce the nucleotide sequences into a cell for production of adenoviral vectors as described below.
The vectors may include other nucleotide sequences, such as those encoding selectable markers, including those for antibiotic resistance or color selection. The vectors also preferably include a transcriptional regulatory nucleotide sequence, such as a promoter. The desired nucleic acid insert is preferably operably linked to the promoter. A nucleic acid is "operably linked" to a another nucleic acid sequence, such as a promoter sequence, when it is placed in a specific functional relationship with the other nucleic acid sequence. The functional relationship between a promoter and a desired nucleic acid insert typically involves the nucleic acid and the promoter sequences being contiguous such that transcription of the nucleic acid sequence will be facilitated. Two nucleic acid sequences are further said to be operably linked if the nature of the linkage between the two sequences does not (1 ) result in the introduction of a frame-shift- mutation; (2) interfere with the ability of the promoter region sequence to direct the transcription of the desired nucleotide sequence, or (3) interfere with the ability of the desired nucleotide sequence to be transcribed by the promoter sequence region. Typically, the promoter element is generally upstream (i.e., at the 5' end) of the nucleic acid insert coding sequence. A wide variety of promoters are known in the art, including cell- specific promoters, inducible promoters, and constitutive promoters. The promoters may further be selected such that they require activation by activating elements known in the art, so that production of the gene products, including the protein encoded by the nucleic acid sequence insert as well as the RNA transcript, may be regulated as desired. In further preferred embodiments, the promoter is a foreign promoter. A "foreign promoter" is defined herein as a promoter other than the natural, or native, promoter involved in transcribing a particular region of DNA. The vectors may further include other regulatory elements, such as enhancer sequences, which cooperate with the promoter to achieve transcription of the nucleic acid insert coding sequence. By "enhancer" is meant nucleotide sequence elements which can stimulate promoter activity in a cell, such as a bacterial or eukaryotic host cell. Moreover, the vectors may include another nucleotide sequence insert that encodes a protein that may aid in purification of the desired protein encoded by the desired nucleotide sequence. The additional nucleotide sequence is positioned in the vector such that a fusion, or chimeric, protein is obtained. For example, a protein described herein may be produced having at its C-terminal end several histidine molecules joined to the protein. Therefore, the additional nucleotide sequence may include, for example, the nucleotide sequence encoding multiple histidines. The protein may be isolated on a nickel chromatography column, which will bind the histidine in the protein being purified. After purification procedures known to the skilled artisan, the additional amino acid sequence may be cleaved with an appropriate enzyme, such as a protease. The desired protein may then be isolated from the other proteins, or fragments thereof, by methods known in the art.
The preferred recombinant vectors may be used to transform, or introduce nucleic acid into, a host cell, and thus may include a nucleic acid having a nucleotide sequence, for example, that encodes a protein of interest. Such methods include, for example, those described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, New York (1982), as well as Current Protocols in Molecular Biology, John Wiley and Sons, edited by Ausubel et al. (1988). Once the desired nucleic acid has been introduced into the host cell, the host cell may produce the desired protein, RNA, or variants thereof as described above. Accordingly, in yet another aspect of the invention, a host cell is provided that includes the preferred recombinant vectors described above. In a preferred embodiment, the recombinant vectors are introduced into a host cell to advantageously form an adenoviral vector as described below. The adenoviral vector may then be used to introduce a desired nucleotide sequence into a cell.
Although a wide variety of host cells may be used in the invention, including prokaryotic and eukaryotic host cells, eukaryotic host cells are preferred. Further preferred are animal host cells, including swine, bovine, murine and avian host cells. Human host cells are most preferred. The host cells may include cell lines that transiently or stably produce the adenoviral vectors herein described.
A further aspect of the invention provides recombinant adenoviral vectors. Such adenoviral vectors are preferred vectors for introducing into a host cell a nucleotide sequence encoding a protein of interest. In one form of the invention, an adenoviral vector may include the nucleotide sequences described herein, especially the nucleotide sequences set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, that is preferably replication-defective so that it will not reproduce when introduced into a host cell. For example, the adenoviral vector may include at least one of the nucleotide sequences described herein, such as those set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, preferably a nucleotide sequence encoding a desired protein product and typically a promoter, preferably at least one, as described above.
In another form of the invention, a recombinant adenoviral vector, preferably a porcine adenovirus type 3 vector, is provided that includes an adenoviral genome having a functional deletion in its E1 region. By "functional deletion" is meant a deletion that results in a replication- defective adenovirus. Thus, various portions of the E1 region may be deleted, including, for example, portions of the E1 A and E1 B genes. In certain forms of the invention, the entire E1 region may be deleted. In preferred embodiments of the invention, the deleted portions of the E1 region, or other applicable region of the genome, are advantageously replaced with a nucleotide sequence of interest. The nucleotide sequence preferably includes a gene that encodes a protein of interest. A promoter sequence may also be operably linked to a terminal 5' end of the nucleotide sequence encoding a protein of interest, as discussed above. The vector having the functional E1 deletion in its genome may further include at least one of the introduced nucleotide sequences described herein, especially those set forth in SEQ ID NOS:1 , 2, 3, 4, and 5, and preferably includes at least one promoter sequence operably linked to a terminal 5' end of these nucleotide sequences.
Further provided herein are methods for making such adenoviral vectors. General methods for making adenoviral vectors that include selected deletions of, and additions to, its genome are well known in the art, including direct cloning and homologous recombination methods. As non-limiting examples, methods for producing such vectors may be found in the following references: Yeh, P. and Perricaudet, M. (1997) FASEB J. 11 :615-623; Wang, Q. et al. (1997) Gene Ther. 4:393-400; Gilgenkrantz, H. et al. (1995) Hum. Gene Ther. 6:1265-1274; and U.S. Patent Nos. 5,518,913; 5,585,362 and 5,698,202. In one form of the invention, a method typically includes introducing into a cell a transcriptional regulatory nucleotide sequence and an isolated nucleic acid molecule as described herein, such as SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10. In other forms of the invention, a method typically involves making a functional deletion in the E1 region of the adenoviral genome and may also include replacing the deleted region of the genome with a nucleotide sequence of interest as described above.
In yet another aspect of the invention, methods of introducing a desired nucleic acid molecule into a cell is provided. In one embodiment, a method includes introducing into a host cell a nucleic acid molecule encoding a desired protein and an isolated nucleic acid molecule having a nucleotide sequence having the percent identity or similarity as described herein to the nucleotide sequence selected from SEQ ID NOS:1 , 2, 3, 4, and 5 and SEQ ID NOS:6, 7, 8, 9, and 10, and combinations thereof. Each of the nucleic acid molecules independently preferably include a foreign promoter, further preferably at least one foreign promoter, operably linked to their terminal 5' end. The nucleotide sequences are preferably carried by an adenoviral vector as described above. In preferred embodiments, the desired nucleic acid molecule is introduced by infecting the cell with an adenoviral vector as described herein. The adenoviral vector may be constructed as described herein. For example, the adenoviral vector may include an adenoviral genome with a functional deletion in its E1 region which is replaced by the nucleotide sequence of interest, and may also include one or more of the viral nucleotide sequences described herein. Alternatively, the vector may include one or more of the nucleotide sequences described herein, especially those selected from SEQ ID NOS:1 , 2, 3, 4, and 5, an inverted terminal repeat (ITR) sequence, an adenoviral origin of replication and the nucleotide sequence, such as a transgene, of interest. After introducing the nucleic acid into the host cell, the cells may be cultured under conditions effective to achieve production of the RNA transcript, and/or expression of the desired protein, depending on whether RNA or protein is desired. In other aspects of the invention, methods of expressing a desired protein are provided that include introducing into a host cell an isolated nucleic acid molecule that includes a first nucleotide sequence described herein, especially those set forth in SEQ ID NOS:1 , 2, 3, 4, or 5, or combinations thereof, and further preferably includes at least one promoter sequence, such as a foreign promoter sequence, operably linked to a terminal 5' end of the isolated nucleotide sequence. A second nucleotide sequence encoding a desired protein is also introduced into the host cell. The protein may typically be one that provides some benefit to the host cell, including replacing the function of a non-functional protein, or one of decreased function, in the host cell. The nucleotide sequence encoding the desired protein preferably includes a promoter sequence operably linked to a terminal 5' end of the nucleotide sequence. The nucleotide sequences are preferably introduced into the host cell by first incorporating the sequences into an adenoviral vector. The desired cells may be infected with the adenoviral vector according to known procedures in the art so that the desired nucleotide sequence may be introduced into the cell. The host cells are then cultured under conditions, well known to the skilled artisan, effective to achieve expression of the desired polypeptide. The desired polypeptide may then be purified using conventional techniques. In a further preferred form of the invention, the transgene may be expressed by utilizing a promoter as described herein while the virus is in a latent state, and, when it is desired that the virus be cleared from the system, the virus is then returned to a non-latent state by regulating production of the RNA and/or proteins described herein. This provides a method of sequential expression of the transgene according to the need.
In a further aspect of the invention, methods of expressing viral proteins are provided. In one form, a method includes introducing into a host cell an isolated nucleotide sequence as described herein, preferably having at least one foreign promoter operably linked to its terminal 5' end. The cells may then be cultured under conditions to achieve expression of the protein.
Reference will now be made to specific examples illustrating the invention described above. It is to be understood that the examples are provided to illustrate preferred embodiments and that no limitation to the scope of the invention is intended thereby.
EXAMPLE 1 SEQUENCE ANALYSIS OF PORCINE ADENOVIRUS Cell Culture and Virus
The swine testicle (ST) cell line obtained from American Type Culture Collection (ATCC), was grown as monolayer cultures using Eagles minimum essential medium (MEM) [Life Technologies Inc.] supplemented with 10% fetalClone III (HyClone Laboratories, Inc.) and 50 μg/ml gentamicin. PAd3, kindly supplied by Dr. W. Mengeling, USDA, Agricultural Research Service, National Animal Disease Center, Ames, IA, was grown in ST cells and purified by cesium chloride density-gradient centrifugation [Graham F. L. and Prevec L. (1991 ) in Murray E. J. (ed): Methods in Molecular Biology: Gene Transfer and Expression Protocols, Clifton, Humana Press, Vol. 7, pp 109-128].
Plasmid Construction
DNA was extracted from purified virions following a technique described previously [Graham F. L. and Prevec L. (1991) in Murray E. J. (ed): Methods in Molecular Biology: Gene Transfer and Expression Protocols, Clifton, Humana Press, Vol. 7, pp 109-128]. The terminal protein attached to PAd3 DNA was removed by sodium hydroxide treatment. PAd3 DNA was digested with BamHI, separated on an agarose gel by electrophoresis and a 4.8 kb fragment containing the left-end of PAd3 genome between m.u. 0 and 13.7 was purified and inserted at the Hincll-BamHI site of pUC18 to obtain pPAd-E1. A large scale purification of pPAd-E1 was performed by alkaline lysis and cesium chloride purification [Sambrook J. et al. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press].
DNA Sequencing and Sequence Analysis
Initially, pPAd-E1 was sequenced using universal and reverse primers and then the entire sequence on both strands was obtained by primer walking following the dideoxy nucleotide chain termination technique [Sanger F. et al (1977) Proc. Natl. Acad. Sci. USA 74:5463-5467] using an Applied Biosystems 373A automated sequencer. All sequence analyses were done using the GCG sequence analysis program.
Results A sequence of 4769 nucleotides (nt) of PAd3 genome between m.u. 0 and 13.7 was obtained. This sequence was compared with the similar sequences reported for HAd5 [Chroboczek J. et al. (1992) Virology 186:280-285], bovine adenovirus type 3 (BAd3) [Zheng B. et al (1994) Virus Res. 31 :163-186; Elgadi M. et al. (1993) Intervirology 36:1 13-120; Reddy P. S. et al (1998) J. Virol. 72:1394-1402] and PAd4 [Kleiboeker S. B. (1995) Virus Res. 31 :17-25]. On the basis of protein homology, ORFs homologous to HAd5 E1A, E1 B-19k, E1 B-55k, plX, plVa2 and five novel ORFs (184R, 97R, 162R, 163R* and 288R) were identified in the PAd3 genome (Fig. 1A, 1 B and 2). Our PAd3 sequence yielded approximately 41 % and 99% homology at the nucleotide level with the E1 regions of PAd4 [Kleiboeker S. B. (1995) Virus Res. 31 :17-25] and recently published sequence of PAd3 [Reddy P. S. et al. (1998) Virus Res. 58:97-106], respectively.
Left-end Untranslated Region The inverted terminal repeat (ITR) sequence from nt 1-144 was identical to the published sequence of PAd3 ITR [Reddy P. S. (1995) Virology 212:237-239]. The PAd3 ITR also contains a 10-nt (8-17 nt) sequence that may be important for virus DNA replication [Shinagawa M. et al (1987) Gene 55:85-93]. It has been demonstrated that there are seven A-repeat packaging domains located within nt 194-380 of the Had5 genome which are involved in viral DNA packaging [Grable M. and Hearing P. (1990) J. Virol. 64:2047-2056; Schmid S. I. and Hearing P. (1997) J. Virol. 71 :3375-3384]. PAd3 at least has two TTTTG domains that are usually a part of A-repeat packaging domains and are located at nt 37-41 and 265-269. In the PAd3 ITR, the consensus nuclear factor III (NFIII)- binding site [Hatfield L. and Hearing P. (1993) J. Virol. 67:3931-3939] is located at nt 130-140. Four GC-rich regions similar to the SP-I binding site were present at nt 68-75, 77-82, 198-204, and 213-219.
E1A Region Within the 288R E1A protein of HAd, binding sites for a number of cellular proteins including p300, retinoblastoma protein (Rb), cyclin A, an associated protein kinase pSS""2 and other unassigned proteins have been defined [Whyte P. et al. (1988) Nature 334:124-129; Whyte P. et al. (1989) Cell 56:67-75; Lundblad J. R. et al. (1995) Nature 374:85-88; Arany Z. et al. (1995) Nature 374:81-84]. The predicted PAd3 E1A ORF contains 170 amino acid residues (R) with an initiation codon at nt 533 followed by a stop codon at nt 1046 (Fig. 1A and 2). This ORF showed approximately 27%, 26%, and 41% amino acid identity and approximately 34%, 35%, and 45% amino acid similarity with E1A of BAd3, HAd5 and PAd4, respectively. A probable intron of 100 bp with its splice donor and splice acceptor sites at nt 1044, and 1145, respectively may result in continuation of E1A ORF until a stop codon appears at 1221. The resultant putative PAd3 E1 A is expected to be of 195R. The putative E1A 'TATA' box and the polyadenylation signal are located at nt 449 and 1287, respectively. The conserved region 3 (CR3) of the 288R E1A of HAd5 includes the activation region, the metal binding region, and the promoter binding region [Lillie J. W. and Green M. R. (1989) Nature 338:39-44]. The predicted activation, metal binding, and the promoter binding regions of PAd3 E1A showed homology with similar E1A sequences of HAd5, BAd3, and PAd4 (Fig. 3A-C). There was a complete conservation of the metal binding (C-X2-C-X13-C-X2-C) motif of PAd3 E1A. The putative PAd3 E1A region between amino acids 112 and 126 had sufficient homology with the Rb binding region [Dyson N. et al. (1990) J. Virol. 64:1353-1356] of E1A of HAd5 and BAd3 (Fig. 3D). However, the putative PAd4 E1A [Kleiboeker S. B. (1995) Virus Res. 31 :17-25] did not seem to contain either the promoter binding domain or the Rb binding domain.
E1 B Region
The E1B transcription unit of HAd5 consists of two major messages to produce 19k and 55k proteins. Both of these proteins independently inhibit p53-mediated apoptosis by different mechanisms [Wold W. S. M. et al. (1994) Trends Microbiol. 2:437-443]. Coexpression of both E1A and E1B (19k and or 55k) is required for high frequency transformation of cultured rodent cells [Wold W. S. M. et al. (1994) Trends Microbiol. 2:437-443]. The putative first PAd3 E1B ORF begins at nt 1462 to the termination codon at nt 2068. This ORF defines a polypeptide of 202R that had approximately 23%, and 26% amino acid identity and approximately 31%, and 39% amino acid similarity with BAd3 E1 B-19k (Fig. 4A) and HAd5 E1B-19k, respectively. The two other regions of HAd5 E1 B-19k that are conserved in PAd3 E1B-202R are also shown (Fig. 4B & C). It was surprising to note that the published PAd4 E1 region sequence [Kleiboeker S. B. (1995) Virus Res. 31 :17-25] did not contain an ORF similar to PAd3 E1 B-202R.
The putative second PAd3 E1 B ORF starts at nt 1830 and terminates at nt 3255. This ORF defines a polypeptide of 475R that had approximately 32%, 28% and 30% amino acid identity and approximately 43%, 37%, and 36% amino acid similarity with E1 B-55k homologs of BAd3, HAd5 and PAd4, respectively (Fig. 4D). The putative E1 B TATA motif and the polyadenylation signal are located at nt 1330 and 3340, respectively. plX ORF
The HAd5 plX protein is a minor structural protein that is essential for the packaging of full-length genomes [Ghosh-Choudhury G. et al. (1987) EMBO J 6:1733-1739]. An ORF starting at nt 3395 and terminating at nt 3992 represents a protein of 198R. This putative PAd3 plX showed 22%, and 23% amino acid identity and 30%, and 30% amino acid similarity with plX of BAd3 and HAd5, respectively. The N-terminal portion of plX of both HAd5 and BAd3 showed extensive homology with the predicted PAd3 plX (Fig. 5). The putative PAd3 plX TATA motif and the polyadenylation signal are present at nt 3339 and 4085, respectively. The sequence information for the putative PAd4 plX ORF is not available.
IVa2 and Novel ORFs The HAd IVa2 message transcribed from the lower-strand, is involved in adenovirus replication [Swaminathan S. and Thimmapaya B. (1995) in Doerfler W. and Bohm P. (ed) The Molecular Repertoire of Adenoviruses III: Biology and Pathogenesis. Curr Topics Microbiol. Immunol., Berlin, Spring- Verlag, Vol 1999/111, pp 177-194]. The lower-strand of PAd3 genome between m.u. 13.7 and 1 1.7 contains an incomplete ORF representing an approximately 50% C-terminal portion of the predicted PAd3 IVa2 protein (SEQ ID NO:10 and FIG. 2). This ORF demonstrated approximately 77%, and 72% identity with IVa2 proteins of HAd5 and BAd3, respectively (FIG. 6). The putative PAd3 IVa2 polyadenylation signal is located at nt 4075. The sequence information for the putative PAd4 IVa2 ORF is not available. The E1A, E1 B-202R, E1 B-475R showed 100% protein identity whereas plX and IVa2 showed approximately 92 and 99% protein identity respectively with the recently published sequence of PAd3 [Reddy P. S. et al. (1998) Virus Res. 58:97-106]. Following the IVa2 ORF, there are four unique ORFs of 97R, 162R ,
163R* and 288R. Following the plX ORF, there is a novel ORF of a 184R putative protein that exhibits 31% identity with bovine Na+/Ca+,K+- exchanging protein (FIG. 7A,1) [Reilaender H. et al. (1992) EMBO J 11 :1689- 1695] and 34% identity with bovine herpesvirus type 1 immediate early transcriptional control protein (BIC4) (Fig. 7A, 2) [Schwyzer M. et al. (1993) Virology 197:349-357]. The 97R predicted protein has 33% identity with equine herpesvirus (EHV) membrane glycoprotein (Fig. 7A, 3.) [Kirisawa R. et al. (1996) J. Equine Sci. 7:79-87] and 43% identity with EHV transcriptional control protein 57 (FIG. 7A, 4.) [Telford E. A. R. et al. (1995) J. Mol. Biol. 249:520-528]. The 162R putative protein has shows 28% identity with (EHV) membrane glycoprotein (Fig. 7A, 5.) [Kirisawa R. et al. (1996) J. Equine Sci. 7:79-87] and 32% identity trans-acting transcriptional protein ICP0 of herpes simplex virus (Fig. 7A, 6.) [Mcgeoch D. J. et al. (1991) J. Gen. Virol. 72:3057- 3075]. The C-terminal of putative 288R protein demonstrated 44% identity with herpesvirus papio Epstein- Barr virus nuclear antigen 1 (EBNA1 ) (Fig. 7B, 7.) [Yates J. L. et al. (1996) Virology 222:1-13] and 36% identity with the GATA-4 transcription factor of mice (Fig. 7B, 8.) [Arceci R. J. et al. (1993) Mol. Cell Biol. 13:2235-46]. Herpesvirus papio infects baboons and is similar to Epstein-Barr virus (EBV) of human [Heller M. and Kieff E. (1981) J. Virol. 37:821-826]. EBNA1 is involved in maintaining the EBV genome during a latent phase by providing a stable and regulated replication [Yates J. L. et al. (1984) Proc. Natl. Acad .Sci. USA 81 :3806- 3810; Yates J. L. et al. (1985) Nature 313:812-815]. GATA-4 is a retinoic acid-inducible GATA-binding transcription factor expressed in endodermally-derived tissues and heart and is involved in gene expression in these tissues [Arceci R. J. et al. (1993) Mol. Cell. Biol. 13:2235-46]. On the lower strand, there is a unique ORF of 163R* without a start codon. The 163R* predicted protein contains a proline- and alanine-rich region (83-179 residues) that showed 54% identity with the EBV nuclear antigen (FIG. 7B- 9) (EBNA2) [Sample J. et al. (1986) Proc. Natl. Acad. Sci. USA 83:5096- 6000] and 40% identity with a human transcription initiation factor II (TFII) (Fig. 7B, 10.) [Tanese N. Et al. (1996) Proc. Natl. Acad. Sci. USA 93:13611-13616]. EBNA2 is responsible for transforming B lymphocytes [Jones M. D. et al. (1984) EMBO J 3:813-821] and also associated with the survival of the EBV genome during latency [Sample J. et al. (1992) J. Virol. 66:4654-4661]. ORFs similar to the potential PAd3 184R, 97R, 162R, 163R* and 288R proteins in the HAd5 or BAd3 genome between m.u. 0 and 15 were not identified. These proteins may thus be involved in maintaining the adenovirus genome in the latent state and/or may act as transcription factors in controlling adenovirus gene expression.
Since these novel ORFs showed homology either with EBNA antigens or other cellular and viral transcription factors, these putative proteins appear to be stimulating or inhibiting viral replication. For example, PAd3 162R and 97R putative proteins may be trans-activating adenovirus replication similar to trans-activating immediate early proteins of herpesviruses. These predicted proteins may play a role in initial viral replication, virus latent phase or virus replication during reactivation. Some of these PAd3 putative proteins may function as a complex with other viral or cellular protein/s.
EXAMPLE 2
Expression of PAd3 162R in Bacteria Experimental
A 709 bp Hindlll fragment containing the ORF of 162R was isolated from pPAd-E1 and cloned into the Hindlll site of pUC18. The resultant plasmid was digested with BsmBI and cohesive ends were filled in using
T4 DNA polymerase. The resulting fragment was digested with Hindlll, a 0.7 Kb fragment was isolated and was inserted into the EcoRV - Hindlll site of pET-30a(+) expression vector. The resultant plasmid pET162R was used for protein expression in DE3 bacteria. The protein expression was induced with IPTG. The His-tag fusion protein was purified on Nickel affinity chromatography. Results The ORF of putative protein 162R was expressed in a bacterial expression vector. The expression of His-tag fusion protein was induced by IPTG and purified by Nickel affinity chromatography. The protein samples were analyzed by SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and visualized by staining with Coomassie blue (FIG. 8.). The fusion protein was identified by western blotting using S-protein coupled to horseradish peroxidase (FIG.9.) suggesting that the 162R putative proteins may be expressed in virus infected cells. While the invention has been illustrated and described in detail in the drawings and foregoing description, the same is to be considered as illustrative and not restrictive in character, it being understood that only the preferred embodiment has been shown and described and that all changes and modifications that come within the spirit of the invention are desired to be protected. In addition, all references cited herein are indicative of the level of skill in the art and are hereby incorporated by reference in their entirety.

Claims

CLAIMSWhat is claimed is:
1. An isolated nucleic acid molecule, comprising:
(a) a first nucleotide sequence selected from the group consisting of a nucleotide sequence having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID
NO:3, SEQ ID NO:4, and SEQ ID NO:5; and (b) at least one foreign promoter, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
2. The nucleic acid molecule of claim 1 , wherein said first nucleotide sequence consists essentially of a nucleotide sequence selected from the group consisting of a nucleotide sequence having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
3. The molecule of claim 1 , wherein said nucleotide sequence encodes a protein comprised of an amino acid sequence selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, and an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5.
4. The molecule of claim 3, wherein said protein has an amino acid sequence selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
5. The molecule of claim 1 , wherein said nucleotide sequence is selected from the group consisting of the nucleotide sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO: 4, and SEQ ID NO:5.
6. The molecule of claim 1 , wherein said nucleotide sequence consists essentially of a nucleotide sequence having substantial similarity to a nucleotide sequence selected from the group consisting of nucleotide 1 to nucleotide 114 of SEQ ID NO:2, nucleotide 121 to nucleotide 489 of SEQ ID NO:3 and nucleotide 316 to nucleotide 438 of SEQ ID NO:4.
7. The molecule of claim 1 , wherein said first nucleotide sequence is a DNA sequence.
8. A nucleic acid molecule, comprising an RNA nucleotide sequence corresponding to the DNA nucleotide sequence of claim 7.
9. An isolated nucleic acid molecule, comprising:
(a) a first nucleotide sequence encoding a protein, said protein having an amino acid sequence selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, and an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5; and
(b) at least one foreign promoter, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
10. An isolated nucleic acid molecule, comprising:
(a) a first nucleotide sequence selected from the group consisting of a nucleotide sequence having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO:10; and
(b) at least one foreign promoter, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
11. A recombinant nucleic acid molecule, comprising: (a) an isolated first nucleotide sequence having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof; and
(b) at least one foreign promoter, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
12. The molecule of claim 11 , wherein said nucleotide sequence encodes a protein comprised of an amino acid sequence selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, and an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5.
13. The molecule of claim 11 , wherein said promoter is selected from the group consisting of a constitutive promoter, an inducible promoter, and a cell-specific promoter.
14. The molecule of claim 1 1 , wherein said molecule further comprises a nucleotide sequence encoding a desired protein.
15. The molecule of claim 11 , wherein said recombinant nucleic acid molecule comprises a plasmid.
16. The molecule of claim 15, wherein said plasmid comprises an adenoviral origin of replication.
17. The molecule of claim 11 , wherein said nucleotide sequence is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof
18. A host cell, comprising:
(a) an isolated and introduced first nucleic acid molecule having a nucleotide sequence having substantial similarity to the nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and combinations thereof; and
(b) at least one foreign promoter, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
19. The host cell of claim 18, wherein each of said nucleotide sequences encode a protein, said protein having an amino acid sequence selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5, and combinations thereof.
20. A purified protein, comprising an amino acid sequence selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, and an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5.
21. The protein of claim 20, wherein said amino acid sequence is selected from the group consisting of the amino acid sequence set forth in SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, and SEQ ID NO:5.
22. An adenoviral vector, comprising:
(a) an introduced first nucleic acid molecule comprising a first nucleotide sequence having substantial similarity to the nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and combinations thereof; and
(b) at least one foreign promoter, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
23. The vector of claim 22, wherein said vector further comprises a second nucleotide sequence encoding a protein of interest.
24. The vector of claim 22, wherein said vector further comprises an adenoviral origin of replication and an inverted terminal repeat sequence.
25. A replication-defective porcine adenoviral type 3 vector, comprising a porcine adenovirus type 3 genome having a functional deletion in the E1 region.
26. The vector of claim 25, further comprising:
(a) a nucleotide sequence encoding a protein of interest, said nucleotide sequence inserted into said E1 region; and (b) a promoter operably linked to a terminal 5' end of said nucleotide sequence encoding said protein of interest.
27. A method of introducing a first desired nucleic acid molecule into a host cell, comprising infecting said host cells with an adenoviral vector, said vector comprising:
(a) a first isolated nucleic acid molecule, said isolated nucleic acid molecule comprising a first nucleotide sequence having substantial similarity to the nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof; (b) at least one first foreign promoter operably linked to a terminal 5' end of said first isolated nucleic acid molecule;
(c) a second desired nucleic acid molecule encoding a desired protein; and (d) a second foreign promoter operably linked to a terminal 5' end of said second desired nucleic acid molecule.
28. The method of claim 27, wherein said first nucleotide sequence encodes a protein having an amino acid sequences selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, and an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5.
29. The method of claim 27, wherein said amino acid sequence is selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.
30. A method of making an adenovirus vector, comprising introducing into a cell a foreign promoter and an isolated nucleic acid molecule, said isolated nucleic acid molecule comprising a first nucleotide sequence having substantial similarity to a nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof, said foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence.
31. The method of claim 30, wherein said isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein having an amino acid sequence selected from the group consisting of an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:1 , an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:2, an amino acid sequence having at least about 60% identity to the amino acid sequence set forth in SEQ ID NO:3, an amino acid sequence having at least about 50% identity to the amino acid sequence set forth in SEQ ID NO:4, an amino acid sequence having at least about 40% identity to the amino acid sequence set forth in SEQ ID NO:5.
32. A method of expressing a desired protein, comprising: (a) infecting a host cell with an adenoviral vector having an isolated nucleic acid molecule comprising a first nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof, a second nucleotide sequence encoding a desired protein, at least one foreign promoter operably linked to a terminal 5' end of said first nucleotide sequence and a second promoter operably linked to a terminal 5' end of said second nucleotide sequence; and
(b) culturing under conditions to achieve expression of said protein.
33 The method of claim 32, wherein said promoters are inducible promoters.
34. The method of claim 32, further comprising inducing said second promoter to achieve expression of said desired protein.
35. The method of claim 32, wherein said desired protein is an antigen.
36. A method of expressing a viral protein, comprising: (a) introducing into a host cell an isolated first nucleotide sequence comprising a nucleotide sequence selected from the group consisting of SEQ ID NO:1 , SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and combinations thereof, and a foreign promoter, said foreign promoter operably linked to a terminal 5' end of said isolated nucleotide sequence; and
(b) culturing under conditions to achieve expression of said protein
37. The method of claim 36, wherein said nucleotide sequence comprises a nucleotide sequence having substantial similarity to a nucleotide sequence selected from the group consisting of nucleotide 1 to nucleotide 114 of SEQ ID NO:2, nucleotide 121 to nucleotide 489 of SEQ ID NO:3 and nucleotide 316 to nucleotide 438 of SEQ ID NO:4.
PCT/US2000/004711 1999-02-24 2000-02-24 Adenoviral vectors, proteins and methods for their use WO2000050076A1 (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040305A2 (en) * 2001-09-24 2003-05-15 University Of Saskatchewan Porcine adenovirus e1 and e4 regions
US7335353B2 (en) 1998-04-15 2008-02-26 University Of Saskatchewan Porcine cells comprising an adenovirus E3 gene region

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
KLEIBOEKER S.B.: "identification and sequence analysis of the E1 genomic region of a porcine adenovirus", VIRUS RESEARCH,, vol. 36, 1995, pages 259 - 268, XP002926764 *
KLEIBOEKER S.B.: "Sequence analysis of the fiber genomic region of a porcine adenovirus predicts a novel fiber protein", VIRUS RESEARCH,, vol. 39, 1995, pages 299 - 309, XP002926766 *
REDDY ET AL.: "Sequence and transcription map analysis of early region-1 of porcine adenovirus type-3", VIRUS RESEARCH,, vol. 58, 1998, pages 97 - 106, XP002926765 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7335353B2 (en) 1998-04-15 2008-02-26 University Of Saskatchewan Porcine cells comprising an adenovirus E3 gene region
WO2003040305A2 (en) * 2001-09-24 2003-05-15 University Of Saskatchewan Porcine adenovirus e1 and e4 regions
WO2003040305A3 (en) * 2001-09-24 2004-03-18 Univ Saskatchewan Porcine adenovirus e1 and e4 regions
AU2002363489B2 (en) * 2001-09-24 2009-01-15 University Of Saskatchewan Porcine adenovirus E1 and E4 regions
US7569217B2 (en) 2001-09-24 2009-08-04 University Of Saskatchewan Porcine adenovirus E1 and E4 regions

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