Porcine Adenovirus Type Research Articles

Porcine Adenovirus Type 3 E3 Encodes a Structural Protein Essential for Capsid Stability and Production of Infectious Progeny Virions.

The adenovirus E3 region encodes proteins that are not essential for viral replication in vitro The porcine adenovirus type 3 (PAdV-3) E3 region encodes three proteins, including 13.7K. Here, we report that 13.7K is expressed as an early protein, which localizes to the nucleus of infected cells. The 13.7K protein is a structural protein, as it is incorporated in CsCl-purified virions. The 13.7K protein appears to be essential for PAdV-3 replication, as mutant PAV13.73A expressing a mutated 13.7K could be isolated only in VIDO AS2 cells expressing the 13.7K protein. Analysis of PAV13.73A suggested that even in the presence of reduced levels of some late viral proteins, there appeared to be no effect on virus assembly and production of mature virions. Further analysis of CsCl-purified PAV13.73A by transmission electron microscopy revealed the presence of disrupted/broken capsids, suggesting that inactivation of 13.7K protein expression may produce fragile capsids. Our results suggest that the PAdV-3 E3 region-encoded 13.7K protein is a capsid protein, which appears to be essential for the formation of stable capsids and production of infectious progeny virions.IMPORTANCE Although E3 region-encoded proteins are involved in the modulation of leukocyte functions (N. Arnberg, Proc Natl Acad Sci U S A 110:19976-19977, 2013) and inducing a lytic infection of lymphocytes (V. K. Murali, D. A. Ornelles, L. R. Gooding, H. T. Wilms, W. Huang, A. E. Tollefson, W. S. Wold, and C. Garnett-Benson, J Virol 88:903-912, 2014), none of the E3 proteins appear to be a component of virion capsid or required for replication of adenovirus. Here, we demonstrate that the 13.7K protein encoded by the E3 region of porcine adenovirus type 3 is a component of progeny virion capsids and appears to be essential for maintaining the integrity of virion capsid and production of infectious progeny virions. To our knowledge, this is the first report to suggest that an adenovirus E3-encoded protein is an essential structural protein.

Incorporation of porcine adenovirus 4 fiber protein enhances infectivity of adenovirus vector on dendritic cells: implications for immune-mediated cancer therapy.

One strategy in cancer immunotherapy is to capitalize on the key immunoregulatory and antigen presenting capabilities of dendritic cells (DCs). This approach is dependent on efficient delivery of tumor specific antigens to DCs, which subsequently induce an anti-tumor T-cell mediated immune response. Human adenovirus serotype 5 (HAdV5) has been used in human studies for gene delivery, but has limited infection in DCs, which lack the proper receptors. Addition of the porcine fiber knob (PK) from porcine adenovirus type 4 to HAdV5 allows the virus to deliver genetic material via binding to glycosylated surface proteins and bypasses the coxsackie-and-adenovirus receptor required by wild-type HAdV5. In this study we explored the potential therapeutic applications of an adenovirus with PK-based tropism against cancers expressing mesothelin. Infectivity and gene transfer assays were used to compare Ad5-PK to wild-type HAdV5. Mouse models were used to demonstrate peptide specificity and T-cell responses. We show that the PK modification highly augmented infection of DCs, including the CD141+ DC subset, a key subset for activation of naïve CD8+ T-cells. We also show that Ad5-PK increases DC infectivity and tumor specific antigen expression. Finally, vaccination of mice with the Ad5-PK vector resulted in enhanced T-cell-mediated interferon gamma (IFN-γ) release in response to both mesothelin peptide and a tumor line expressing mesothelin. Ad5-PK is a promising tool for cancer immunotherapy as it improves infectivity, gene transfer, protein expression, and subsequent T-cell activation in DCs compared to wild-type HAdV5 viruses.

An Adenovirus Vector Incorporating Carbohydrate Binding Domains Utilizes Glycans for Gene Transfer

BackgroundVectors based on human adenovirus serotype 5 (HAdV-5) continue to show promise as delivery vehicles for cancer gene therapy. Nevertheless, it has become clear that therapeutic benefit is directly linked to tumor-specific vector localization, highlighting the need for tumor-targeted gene delivery. Aberrant glycosylation of cell surface glycoproteins and glycolipids is a central feature of malignant transformation, and tumor-associated glycoforms are recognized as cancer biomarkers. On this basis, we hypothesized that cancer-specific cell-surface glycans could be the basis of a novel paradigm in HAdV-5-based vector targeting.Methodology/Principal FindingsAs a first step toward this goal, we constructed a novel HAdV-5 vector encoding a unique chimeric fiber protein that contains the tandem carbohydrate binding domains of the fiber protein of the NADC-1 strain of porcine adenovirus type 4 (PAdV-4). This glycan-targeted vector displays augmented CAR-independent gene transfer in cells with low CAR expression. Further, we show that gene transfer is markedly decreased in cells with genetic glycosylation defects and by inhibitors of glycosylation in normal cells.Conclusions/SignificanceThese data provide the initial proof-of-concept for HAdV-5 vector-mediated gene delivery based on the presence of cell-surface carbohydrates. Further development of this new targeting paradigm could provide targeted gene delivery based on vector recognition of disease-specific glycan biomarkers.

Crystallographic Structure of Porcine Adenovirus Type 4 Fiber Head and Galectin Domains

Adenovirus isolate NADC-1, a strain of porcine adenovirus type 4, has a fiber containing an N-terminal virus attachment region, shaft and head domains, and a C-terminal galectin domain connected to the head by an RGD-containing sequence. The crystal structure of the head domain is similar to previously solved adenovirus fiber head domains, but specific residues for binding the coxsackievirus and adenovirus receptor (CAR), CD46, or sialic acid are not conserved. The structure of the galectin domain reveals an interaction interface between its two carbohydrate recognition domains, locating both sugar binding sites face to face. Sequence evidence suggests other tandem-repeat galectins have the same arrangement. We show that the galectin domain binds carbohydrates containing lactose and N-acetyl-lactosamine units, and we present structures of the galectin domain with lactose, N-acetyl-lactosamine, 3-aminopropyl-lacto-N-neotetraose, and 2-aminoethyl-tri(N-acetyl-lactosamine), confirming the domain as a bona fide galectin domain.

Crystallization of the head and galectin-like domains of porcine adenovirus isolate NADC-1 fibre.

The porcine adenovirus NADC-1 isolate, a strain of porcine adenovirus type 4, has a fibre with an atypical architecture. In addition to a classical virus attachment region, shaft and head domains, it contains an additional galectin like domain C-terminal to the head domain and connected to the head domain by a long RGD-containing loop. The galectin-like domain contains two putative carbohydrate-recognition domains. The head and galectin-like domains have been independently crystallized. Diffraction data have been obtained to 3.2 angstrom resolution from crystals of the head domain and to 1.9 angstrom resolution from galectin-like domain crystals.

Transcription mapping and characterization of proteins produced from early region 4 of porcine adenovirus type 3

The early region 4 (E4) of porcine adenovirus 3 (PAdV-3) was characterized by Northern blot, rapid amplification of cDNA ends (RACE), RT-PCR and cDNA sequence analysis. Northern blot analysis revealed three different classes of transcripts, which appeared and peaked at different times post-infection. The RT-PCR, RACE and cDNA sequence analysis identified nine major E4 transcripts, all of which shared a 107-bp 5' leader sequence and a 126-bp 3' terminus. These transcripts have one to three introns removed. Interestingly, of the nine major transcripts, there was one fusion transcript of ORFp1 and ORFp7 (ORFp1/7), which codes for a protein of 119 amino acids. All transcripts initiated at nucleotide 33740 of the PAdV-3 genome. To identify proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding p2, p3, p4 or p7 proteins. Serum against p2, p3 and p4 immunoprecipitated proteins of 13.5, 13.6 and 15.3 kDa, respectively, in in-vitro transcribed and translated mRNA and in PAdV-3-infected cells. Serum against p7 immunoprecipitated a protein of 19.8 kDa in in-vitro transcription and translation analysis but recognized two proteins of 19.8 kDa (encoded by ORFp7) and 14 kDa (encoded by the fusion transcript ORF1/7) in PAdV-3-infected cells. The protein encoded by ORFp2 was localized in the nucleus of PAdV-3-infected cells. The proteins encoded by ORFp3 and ORFp7\ORFp1/7 were detected in the cytoplasm of PAdV-3-infected cells. However, the protein encoded by ORFp4 was observed both in the cytoplasm and nucleus of PAdV-3-infected cells.

Bovine adenovirus type 3 internalization is independent of primary receptors of human adenovirus type 5 and porcine adenovirus type 3

Usefulness of adenoviral vectors derived from human adenovirus (HAd) type 5 (HAd5) is mainly limited by wide prevalence of preexisting anti-HAd5 immunity as well as non-specific tissue tropism of these vectors. As an alternative, non-human adenoviral vectors including bovine adenovirus type 3 (BAd3) are currently being investigated. Non-prevalence of BAd3 in humans and its ability to evade preexisting HAd immunity are some of the features that make BAd3 a promising vector for human gene delivery. BAd3 appears to have a tissue tropism distinct from that of HAd5 and also the repertoire of cells efficiently transduced by BAd3 is different. We performed antibody-mediated receptor blocking experiments to show that BAd3 internalization was independent of coxsackievirus–adenovirus receptor, the primary determinant of HAd5 tropism, or integrin α νβ 3, a secondary molecule involved in HAd5 entry. Using homologous and heterologous knob-mediated competition assays with recombinant knobs of HAd5, porcine adenovirus type 3 (PAd3), or BAd3, we observed that BAd3 internalization was independent of the primary receptors of HAd5 and PAd3. These results provide support for further exploration of BAd3 vectors for designing targeted vectors for human gene therapy.

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′-TTTGN 8CG-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.

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.

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 8CG-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.

Analysis of early region 1 of porcine adenovirus type 3.

To identify the proteins encoded by the porcine adenovirus 3 (PAV-3) E1 region, rabbit antisera were prepared using a bacterial fusion protein encoding E1A, E1B(small), or E1B(large) protein. Sera against E1A, E1B(small), and E1B(large) immunoprecipitated a protein of 35, 23, and 53 kDa, respectively, in in vitro translated and transcribed mRNA and PAV-3 infected cells. To determine the role of E1 proteins in PAV-3 replication, we constructed vectors with a deletion(s) in the E1 region. Mutant PAV211, containing deletions in E1A and E3, grew to titers similar to wild-type in VIDO R1 cells (E1A complementing) but not in swine testicular (ST) cells. No early protein (E1B(small), DNA binding protein) expression could be detected in PAV211 infected ST cells by Western blots. Mutant PAV212, containing deletions in E1B(small) and E3, grew to wild-type titers in VIDO R1 or ST cells. These deletions were successfully rescued, resulting in recombinant PAV214, containing deletions in E1A, E1B(small), and E3. However, mutant PAV-3, containing a triple stop codon inserted in the E1B(large) coding sequence, could not be isolated. Next, we constructed a recombinant PAV216 by inserting the green fluorescent protein gene flanked by a promoter and a poly(A) in the E1A region of the PAV214 genome. Both PAV214 and PAV216 replicate as efficiently as wild-type in VIDO R1 cells. These results suggested that (a) E1A is essential for virus replication and is required for the activation of other PAV-3 early genes, (b) E1B(small) is not essential for replication of PAV-3, and (c) E1B(large) is essential for virus replication. Moreover, the PAV216 vector can be used for the expression of a transgene.

Characterization of DNA Binding Protein of Porcine Adenovirus Type 3

To identify and characterize the protein encoded by the E2A region of porcine adenovirus (PAV)-3, DNA sequence coding for a portion (amino acids 102–457) of the DNA binding protein (DBP) open reaching frame was cloned and expressed in Escherichia coli as a fusion protein with glutathione S-transferase protein of Schistosoma japonica. The affinity-purified fusion protein was used to immunize rabbits. Immunoprecipitation/Western blot analysis demonstrated that the antisera specifically recognized a protein of 50 kD in PAV-3-infected cells. Immunoperoxidase staining detected the DBP protein predominantly in the nucleus of the cells. Western blot analysis demonstrated that DBP was detected as early as 6 h after infection and remained detectable throughout the infection. Based on these results, a novel assay for quantitation of PAV-3 was established. The assay is less time consuming and can be performed in different porcine cells. In addition, virus titers determined by this assay are comparable to the standard plaque assay.

Sequence Analysis of Porcine Adenovirus Type 3 E1 Region, pIX and pIVa2 Genes, and Two Novel Open Reading Frames

The porcine adenovirus type 3 (PAd3) genome between map units 0 and 13.7 was sequenced and compared with similar regions of other adenoviruses. This region consists of the left inverted terminal repeat sequences involved in DNA packaging, the entire early region 1 (E1) and the protein IX (pIX) transcription unit. The lower strand contains the C-terminal end of IVa2 of the E2A transcriptional unit and two novel open reading frames (ORFs). The E1 transcription unit consists of ORFs for proteins homologous to the E1A, E1B-17k and E1B-55k of both human adenovirus type 5 (HAd5) and bovine adenovirus type 3 (BAd3). The predicted PAd3 pIX demonstrated homology with the N-terminal portion of the pIXs of HAd5 and BAd3. On the lower strand, immediately after the putative IVa2 ORF, there are two unique ORFs of 208 and 203 amino acid residues that showed homology with Epstein-Barr virus nuclear antigens and other cellular transcription factors.

Nucleotide Sequence and Transcription Map of Porcine Adenovirus Type 3

The complete nucleotide sequence of porcine adenovirus type 3 was determined and a transcriptional map for the genome was constructed. The size of the genome is 34094 bp in length with an unusually high G + C content (63.7%), the highest thus far reported for any adenovirus. Overall organization of the genome is similar to that for previously sequenced adenoviral DNAs, but there also were distinct differences. The late regions genes are organized into six families, instead of five as they are in human adenovirus type 2. In contrast to bovine adenovirus type 3 and ovine adenovirus, which lack virion-associated RNA genes, the nucleotide sequence analysis of the viral genome indicates that it encodes one short VA RNA species. With the exception of the fiber and a 33-kDa nonstructural protein, the predicted amino acid sequences of the open reading frames in the late regions and the E2 region and IVa2 exhibited a high level of homology, whereas the deduced amino acid sequences of ORFs in E1, E3, and E4 regions, and the pIX showed a lesser homology with the corresponding proteins of other adenoviruses. The proteins V, VII, and IX are unusually long, and the protein VII lacks the consensus protease cleavage site. Genomic and cDNA sequence analysis has identified promoters, cap sites, intron–exon boundaries, polyadenylation signals, and polyadenylation sites in the viral genome.

Characterization of the early region 4 of porcine adenovirus type 3.

The nucleotide sequence of a 3028 bp DNA segment, located between map co-ordinates 100 and 92 in the genome of porcine adenovirus type 3 (PAV-3), was determined. The segment includes the entire early region 4 (E-4) and the right inverted terminal repeat sequences. There were two TATA boxes and one canonical polyadenylation signal on the 1 strand. Homology searches of the GenBank data base for the predicted amino acid sequences revealed that, of the eight open reading frames (ORFs) on the 1 strand, and four ORFs on the r strand, only ORF 8 on the 1 strand showed homology with the 34 kDa E-4 protein of human adenovirus types 2, 12 and 34. Northern blot analysis showed that transcription from the E-4 region of PAV-3 began 4 h after infection, peaked at 8 h and declined after 10 h, before DNA replication began 16 h after infection. The E-4 region of PAV-3 was further characterized by 5' and 3' end mapping of the transcription unit.

Restriction endonuclease analysis and physical mapping of the genome of porcine adenovirus type 5

The HNF61 and HNF70 isolates of porcine adenovirus type 5 (PAV-5) were cultivated in PK-15 cells, and viral DNA was extracted from the infected cells by a modified Hirt procedure. The DNAs were digested by each of 9 restriction endonucleases, and fragments representing the entire genomes were cloned. Based on the sizes of the restriction enzyme fragments, the genome of each isolate was estimated to be 33.2 kb. Physical maps for the 9 restriction endonucleases were constructed. The physical maps of the two isolates were identical for 5 of the restriction endonucleases, but 4 enzymes revealed differences in restriction sites occurring mainly between map unit 78 and 83, which may include the E3 region of the genome. There were no similarities between the physical maps of PAV-5 and those described for the other 4 serotypes of PAV.

Sequence analysis of putative pVIII, E3 and fibre regions of porcine adenovirus type 3

Sequence analysis of a region of the genome of porcine adenovirus type 3 from map unit 79.5 to map unit 92 was performed. Homology studies revealed genes coding for the hexon-associated protein pVIII on the left and for the fibre protein on the right of the sequenced region. By analogy with the genomic organization of other adenoviruses, the 1179 bp sequence between the pVIII and fibre open reading frames, extending from map unit 81.3 to map unit 84.7, was identified as the equivalent of the E3 region of human adenoviruses. The deduced amino acid sequence of one of the three open reading frames of the putative E3 region showed homology with the 13.3K E3 protein of canine adenovirus type 2. The primary structure of the putative fibre protein was similar to that described for human adenovirus types 2 and 5, with a 14 pseudorepeat motif in the shaft region of the fibre. A 742 bp tandem repeat starting in the middle of the fibre gene and extending beyond the termination codon of this gene was observed.

Molecular cloning and physical mapping of porcine adenovirus types 1 and 2.

The 25R strain of porcine adenovirus type 1 (PAV-1) and the A47 strain of PAV-2 were propagated in ST cells, and DNA was extracted from the infected cells by a modified Hirt method. The DNA of each virus was digested by each of nine restriction endonucleases, and restriction enzyme fragments representing the entire genome were cloned. The genomic size of each virus was approximately 33 kb. Physical maps for the nine restriction endonucleases were constructed from the results of double digestion and Southern blot hybridization experiments, and oriented with respect to the PAV-3 genome. PAV-1 and PAV-2 were found to be related genetically to PAV-3, and there was a closer relationship between PAV-1 and PAV-3 than between PAV-1 and PAV-2 or between PAV-2 and PAV-3.