Recombinant bovine adenovirus-3 co-expressing bovine respiratory syncytial virus glycoprotein G and truncated glycoprotein gD of bovine herpesvirus-1 induce immune responses in cotton rats.

One of the impediments in the development of safe and cost effective vaccines for veterinary use has been the availability of appropriate delivery vehicle. We have chosen to develop and use bovine adenovirus (BAdV)-3 as vaccine delivery vector in cattle. Here, we describe the construction of recombinant E3 deleted BAdV-3 vectors expressing single vaccine antigen (BAV360; bovine respiratory syncytial virus G) or two vaccine antigens (BAV851; bovine herpesvirus-1gDt and bovine respiratory syncytial virus G). Recombinant proteins expressed by BAV360 or BAV851 were recognized by protein-specific monoclonal antibodies. Moreover, intranasal immunization of cotton rats with BAV360 (expressing a single vaccine antigen) or BAV851 (expressing two vaccine antigens) induced strong antigen-specific immune responses. These results suggest that single replication-competent BAdV-3 expressing vaccine antigens of two economically important respiratory pathogens of calves has potential to act as a feasible approach in the development of economically effective veterinary vaccines for cattle.

Bovine adenovirus-3 E1A coding region contain cis-acting DNA packaging motifs.

To elucidate further the regulation of E1 gene transcription and viral DNA packaging, we constructed and analyzed mutant BAdV-3s in which the deletion of sequences between left ITR and E1A ATG codon was combined with the functional blocking of E1A gene expression by introducing deletion mutations into E1A open reading frame (ORF). The results suggest that E1A coding region contains cis-acting packaging motifs for efficient encapsidation of BAdV-3 DNA into preformed empty capsids. In addition, E1A is not required for the transcription of E1B.

Porcine adenovirus type 3 E1B large protein downregulates the induction of IL-8.

Replication-defective (E1-E3 deleted) adenovirus vector based gene delivery results in the induction of cytokines including IL-8, which may contribute to the development of inflammatory immune responses. Like other adenoviruses, E1 + E3 deleted porcine adenovirus (PAdV) 3 induces the production of IL-8 in infected cells. In contrast, no IL-8 production could be detected in cells infected with wild-type or mutant PAdV-3s containing deletion in E1A + E3 (PAV211) or E1Bsmall + E3 (PAV212). Expression of PAdV-3 E1Blarge inhibited the NF-kappaB dependent transcription of luciferase from IL-8 promoter. Imunofluorescence and electrophoretic mobility shift assays suggested that constitutive expression of PAdV-3 E1Blarge inhibited the nuclear translocation of NF-kappaB and its subsequent binding to DNA. These results suggest that E1Blarge interacts with NF-kappaB to prevent transcription and down regulate proinflammatory cytokine IL-8 production.

E1A promoter of bovine adenovirus type 3.

Conserved motifs of eukaryotic gene promoters, such as TATA box and CAAT box sequences, of E1A of human adenoviruses (e.g human adenovirus 5) lie between the left inverted terminal repeat (ITR) and the ATG of E1A. However, analysis of the left end of the bovine adenovirus 3 (BAdV-3) genome revealed that the conserved sequences of the E1A promoter are present only in the ITR. As such, the promoter activity of ITR was tested in the context of a BAdV-3 vector or a plasmid-based system. Different regions of the left end of the BAdV-3 genome initiated transcription of the red fluorescent protein gene in a plasmid-based system. Moreover, BAdV-3 mutants in which the open reading frame of E1A was placed immediately downstream of the ITR produced E1A transcript and could be propagated in non-E1A-complementing Madin-Darby bovine kidney cells. These results suggest that the left ITR contains the sole BAdV-3 E1A promoter.

A newly identified interaction between IVa2 and pVIII proteins during porcine adenovirus type 3 infection.

The adenovirus IVa2 is an intermediate viral gene product that appears to perform multiple essential roles in viral infection. Using IVa2 as bait in the yeast two-hybrid system, we screened selected open reading frames (ORFs) of porcine adenovirus (PAdV)-3 for potential interaction with IVa2. Interestingly, pVIII showed specific interaction with IVa2. The yeast two-hybrid findings were validated by GST pull-down assays, in vitro binding studies employing cell-free coupled transcription-translation products and in vitro co-immunoprecipitations using protein-specific antibodies. Finally, we demonstrated that IVa2 specifically interacts with pVIII during PAdV-3 infection.

Promoter activity of left inverted terminal repeat and downstream sequences of porcine adenovirus type 3.

Early region 1 (E1) of porcine adenovirus type 3 (PAdV-3) consists of E1A and E1B transcription units. The authentic promoter region of E1A contains a TATA box at nucleotide position (nt) 449 and a bifunctional regulatory element between nt 374 and 431, which enhances the transcription of E1A, but represses that of E1B. Here, we investigated the role of the left inverted terminal repeat (ITR) and its downstream sequences (between nt 151 and 312) in the transcription of early viral genes, and viral replication. Mutant PAdV-3s without the authentic E1A promoter region could be rescued by transfection of mutant genomic DNA into fetal porcine retina cells. Moreover, the mutant PAdV-3s produced E1A-specific mRNA and remained viable in swine testis (ST) cells suggesting that the left-terminal 151 bp including the ITR, can serve as a promoter for E1A expression. However, mutant PAdV-3s containing deletion including authentic E1A promoter region, displayed both reduced steady-state levels of early gene mRNAs (E1A, E1B, E2A, E3, and E4) and decreased rate of viral replication in ST cells. Interestingly, mutant PAdV-3s containing the left-terminal 312 bp displayed increased transcription of early genes including E1A. Our results suggest that the left ITR of PAdV-3 contain the promoter like elements and the sequences (between nt 151 and 312) downstream of left ITR can enhance its promoter activity.

cis-Acting packaging motifs of porcine adenovirus type 3.

The cis-acting packaging domain is required for selective encapsidation of adenovirus DNA into preformed empty capsids late in the viral life cycle. Earlier, it was demonstrated that the cis-acting packaging domain of porcine adenovirus type (PAdV)-3 is located between nucleotide position (nt) 212 and 531 at the left end genome which contains six AT/GC rich motifs. Removal of packaging domain from left end to the right end of the genome produced a viable mutant virus suggesting that the identified cis-acting packaging domain represents the DNA sequences required for selective packaging of PAdV-3 DNA, whose position and orientation appear to be flexible. Here, by constructing and analyzing a panel of virus mutants carrying deletions or linker scanning mutations in AT/GC rich sequences, we examined the significance of the continuous A/T or G/C sequences individually in the viral packaging process. In contrast to consensus bipartite structure (5'-TTTGN8CG-3') described for most of packaging motifs of human adenovirus type 5 (HAdV-5), the packaging motifs I, II, III, and IV of PAdV-3 displayed a tripartite structure in which the continuous A/T nucleotides were flanked by G/C-rich sequences. Mutations in both continuous A/T nucleotides and its flanking GC-rich sequences reduced the packaging efficiency of mutants to varying degrees. In addition, although the continuous A/T sequences were present in all of the packaging motifs, their significance in the packaging process appears to vary within each packaging motif.

Bovine adenovirus type 3 containing heterologous protein in the C-terminus of minor capsid protein IX.

Earlier, we detected pIX of BAdV-3 as a 14-kDa protein in purified virions. Analysis of BAdV-3 pIX using different region antibodies revealed that the N-terminus and central domain of the pIX contain immunogenic sites and are not exposed on the surface of BAdV-3 virion. This suggested that the C-terminus of BAdV-3 pIX (125 amino acid) may be exposed on the virion and may be used as a site for incorporation of heterologous peptides or proteins. We constructed recombinant BAV950 containing a small peptide (21 amino acid), including the RGD motif or recombinant BAV951 containing enhanced yellow-green fluorescent protein (EYFP) fused to the C-terminus of pIX. Western blot analysis demonstrated that the chimeric pIX-RGD was incorporated into virion capsids. Incorporation of the RGD motif into the pIX resulted in significant augmentation of BAdV-3 fiber knob-independent infection of the integrin-positive cells, suggesting that RGD motifs are displayed on the surface of virion capsids and are accessible for binding to integrins. Analysis of BAV951 revealed that the chimeric pIX is incorporated into virion capsids and EYFP containing the C-terminus of pIX is exposed on the surface of the virion. Moreover, insertion of chimeric pIXs was maintained without change through successive rounds of viral replication. These results suggested that in contrast to major capsid proteins (hexon, penton, fiber), the minor capsid protein IX can be use for the incorporation of targeting ligands based on either small peptides or longer polypeptides.

Viral RNAs detected in virions of porcine adenovirus type 3.

It has been demonstrated that cellular and viral RNAs were packaged in the virions of human cytomegalovirus (CMV) and herpes simplex virus 1 (HSV 1), members of the Herpesviridae family, both of which are enveloped double-stranded DNA viruses. Here, we provide evidence suggesting that RNAs are packaged in the virions of porcine adenovirus type 3 (PAdV-3), which is a member of the Adenoviridae family, a non-enveloped double-stranded DNA virus. The RNAs packaged in PAdV-3 virions were enriched in the size range of 300-1000 bases long. By reverse transcription (RT) of RNAs isolated from purified PAdV-3 virions, PCR amplification, and DNA sequence analysis of PCR products, we determined the identities of some viral RNAs contained in PAdV-3 virions. The results indicated that the RNAs representing transcripts from E1A, E1B, DNA binding protein (DBP), DNA polymerase (POL), E4 and some of the late genes including pIIIA, pIII, pV, Hexon, 33 K, and fiber were detected from purified PAdV-3 virions. In contrast, we could not detect the RNAs representing transcripts of precursor terminal protein (pTP), 52 kDa, pX, or 100-kDa protein genes in purified virions. Because the transcripts of pIX, IVa2, E3, protease, pVI, pVII, and pVIII overlap with those of other genes in PAdV-3, we could not definitely conclude that RNAs representing these transcripts were packaged in virions although the expected DNA fragments were produced by RT-PCR in the RNAs isolated from purified virions.

Porcine adenovirus type 3 E1 transcriptional control region contains a bifunctional regulatory element.

We identified a bifunctional regulatory element located between nt 374 and 431 upstream of TATA box of porcine adenovirus (PAV) 3 E1A promoter. Deletion of the element dramatically reduced the steady-state level of E1A mRNA, but increased that of E1B, which lies immediately downstream of E1A. The mutant virus displayed defective replication at early times of infection, but replicated nearly as efficiently as wild-type PAV-3 at late times of infection. This defect was complemented with coinfecting wild-type virus in a mixed infection. The results indicated that the upstream activation sequences (UAS) of E1A overlap the upstream repression sequences (URS) of E1B, although both transcription units are transcribed from different promoters.

Altered tropism of recombinant bovine adenovirus type-3 expressing chimeric fiber.

Recombinant bovine adenovirus-3 (BAV-3) has been used as a gene delivery vector for vaccination of calves. However, its usefulness as a vector for non-bovine species is limited due to poor transduction efficiency. To develop BAV-3 based vector for non-bovine species, we determined the feasibility of making targeted BAV-3 vector by modifying its natural tropism. We constructed a chimeric virus, BAV600, in which the knob region of the BAV-3 fiber protein was replaced with that from human adenovirus type 5 (HAV-5). Unmodified BAV-3 vector (BAV304) was able to transduce and direct the expression of green fluorescent protein (GFP) in non-bovine cells, with low efficiency. In contrast, the transduction efficiency of BAV600 in these cells was increased by 3-67-fold. Although, expression of early and late genes was detected in non-human cells, no progeny virus (BAV600) was detected in these cells. Our results suggest that it is possible to develop BAV-3 vectors with tropism for non-bovine species.

Characterization of cis-acting sequences involved in packaging porcine adenovirus type 3.

Encapsidation of adenovirus DNA involves specific interactions between cis-acting genomic DNA sequences and trans-acting proteins. The cis-acting packaging domain located near the left inverted terminal repeat is composed of a series of redundant but not functionally equivalent motifs. Such motifs are made up of the consensus sequence 5'-TTTGN(8)CG-3' and 5'-TTTG/A-3' in human adenovirus 5 (HAV-5) and canine adenovirus-2 (CAV-2), respectively. To gain comparative insight into adenovirus encapsidation, we examined the packaging domain of porcine adenovirus-3 (PAV-3). Using deletion mutants, we localized the PAV-3 packaging domain to 319 bp (nt 212 to 531), which contains six cis-acting elements. However, this domain does not contain the consensus motifs identified in HAV-5. In addition, consensus motif found in CAV-2 is present only once in PAV-3. Instead, PAV-3 packaging domain appears to contain AT/GC-rich sequences. The packaging motifs of PAV-3, which are functionally redundant but not equivalent, are located at the left end of the genome.

Identification of cis-acting sequences required for selective packaging of bovine adenovirus type 3 DNA.

The assembly of adenovirus particles is a multistep process, in which viral genomic DNA is selected and subsequently inserted into preformed empty capsids. The selective encapsidation of the adenovirus genome is directed by cis-acting packaging motifs, termed A repeats due to their AT-rich character in DNA sequence. A repeats are usually located at the left end of the viral genome. In this report, the construction and analysis of bovine adenovirus type 3 (BAdV-3) mutants containing deletion mutations introduced into the AT-rich regions are described. The main cis-acting packaging domains of BAdV-3 were localized between nt 224 and 540 relative to the left end of the viral genome. They displayed a functional redundancy and followed a hierarchy of importance. In addition, the results demonstrated that not all of the AT-rich units functioned as cis-acting packaging motifs.

A recombinant E1-deleted porcine adenovirus-3 as an expression vector.

Replication-defective E1-deleted porcine adenoviruses (PAVs) are attractive vectors for vaccination. As a prerequisite for generating PAV-3 vectors containing complete deletion of E1, we transfected VIDO R1 cells (fetal porcine retina cells transformed with E1 region of human adenovirus 5) with a construct containing PAV-3 E1B(large) coding sequences under the control of HCMV promoter. A cell line named VR1BL could be isolated that expressed E1B(large) of PAV-3 and also complemented PAV214 (E1A+E1B(small) deleted). The VR1BL cells could be efficiently transfected with DNA and allowed the rescue and propagation of recombinant PAV507 containing a triple stop codon inserted in the E1B(large) coding sequence. In addition, recombinant PAV227 containing complete deletion of E1 (E1A+E1B(small) + E1B(large)) could be successfully rescued using VR1BL cell line. Recombinant PAV227 replicated as efficiently as wild-type in VR1BL cells but not in VIDO R1 cells, suggesting that E1B(large) was essential for replication of PAV-3. Next, we constructed recombinant PAV219 by inserting green fluorescent (GFP) protein gene flanked by a promoter and a poly(A) in the E1 region of the PAV227 genome. We demonstrated that PAV219 was able to transduce and direct expression of GFP in some human cell lines.

Genetic organization and sequence analysis of pVIII, fiber and early region 4 of bovine adenovirus type 7.

The DNA sequence of 8,810 nucleotides at the right end of bovine adenovirus type 7 (BAV7) genome was determined and compared with similar regions of other adenoviruses. This genomic region of BAV7 consists of sequences encoding partial 33K, pVIII, fiber, putative early region 4 (E4) proteins and other unassigned proteins. However, BAV7 E3 region is not present in the expected location between pVIII and fiber as BAV7 intergenic region between pVIII and fiber genes is only 183 nucleotides. The predicted pVIII and fiber demonstrates highest homology to corresponding proteins of ovine adenovirus 287 (OAV287), bovine adenovirus-4 (BAV4) and egg drop syndrome virus (EDSV). The E4 region encodes three ORFs, which shows significant homology only to corresponding proteins encoded by E4 region of OAV287 and BAV4. Sequence comparisons, phylogenetic analysis and overall genome organization in this region of BAV7 provide further evidence for the inclusion of BAV7 together with OAV287, BAV4, and EDSV in the proposed genus 'Atadenovirus'.