J Vet Sci.  2017 Jun;18(2):217-227. 10.4142/jvs.2017.18.2.217.

Bovine adenovirus type 3 virions cannot be rescued in vivo after full-length viral genome transfection in the absence of detectable polypeptide IX

• Zhang P
• Xue Q
• Ma J
• Ren J
• Xia S
• Zhang L
• Wang W
• Tikoo SK
• Du E

• KMID: 2412575
• DOI: http://doi.org/10.4142/jvs.2017.18.2.217

Abstract

Bovine adenovirus type 3 (BAdV3) is being used in the development of potential vehicles for gene therapy and vectored vaccine. To that end, a more comprehensive description of BAdV3 biology is essential. In this study, we focused on the role of pIX in BAdV3 virion rescue after full-length BAdV3 genome transfection. Initially, pIX deletion or initiation codon mutation abolished the production of progeny virions, which suggested that pIX was essential for the rescue of BAdV3 containing a full-length genome. Moreover, through transfection of a panel of pIX mutant BAdV3 genomes, we observed that the conserved N-terminus and the putative leucine zipper element (PLZP) were essential for virion rescue, whereas the C-terminus following the coiled-coil domain was non-essential. In addition, swap of the PLZP element and its following region of BAdV3 pIX to corresponding domains of human adenovirus type 5 (HAdV5) did not affect virion production, whereas swap of the entire pIX abolished production of progeny virions. We suggest that failure of the full-length BAdV3 pIX swap might be due to species specificity of its N-terminus region before the PLZP element.

Keyword

adenovirus; bovine adenovirus type 3; polypeptide IX; viral rescue

MeSH Terms

Animals
Blotting, Western
Capsid Proteins/genetics/*physiology
Cattle/virology
Genome, Viral/physiology
HEK293 Cells
Humans
Leucine Zippers/genetics
Mastadenovirus/genetics/*isolation & purification/physiology
Transfection/veterinary
Virion/genetics/*isolation & purification/physiology
Capsid Proteins

Figure

• 

  Fig. 1 Strategy used for the generation of recombinant BAdV3 plasmids. (A) The flowchart for construction of plasmid pTK304EP-chloramphenicol acetyltransferase (CAT). Firstly, a 12176 AscI-BstZ17I DNA fragment (nucleotide [nt] 1403 to nt 13578) from pFBAV304a.I-SceI was ligated into AscI-BstZ17I- cleaved 5'- pTKL- 3' to create pTK304. Then, digested plasmid pTK304 with EcoRI and PacI was blunted and self-ligated for eliminating the extra HpaI site to generate pTK304EP. Finally, to construct plasmid pTK304EP-CAT gene, a 1040 bp CAT fragment (containing 50 bp homology arms in 5' and 3' sites and flanked with SbfI enzyme sites) was inserted into HpaI-linearized pTKEP304 in Escherichia coil BJ5185 by homology recombination. (B) The flowchart for construction of shuttle plasmid pTK304-CAT. A 5385 bp fragment isolated from pTK304EP-CAT using AscI and Eco47III was ligated into AscI-Eco47III-cleaved pTK304 to created final shuttle plasmid pTK304-CAT. (C) The flowchart for construction of pIX deleted BAdV3 plasmid pFBAV304a. I-SceI-ΔpIX. AscI-BstZ17I-cleaved pTK304-CAT was cloned into PmeI-linearized pFBAV304a.I-SceI by homology recombination in Escherichia coil BJ5185 to construct pFBAV304a.I-SceI-CAT. Then, the pIX deleted recombination plasmid pFBAV304a.I-SceI-ΔpIX was constructed by digesting the recombinant pFBAV304a.I-SceI-CAT with SbfI and religated to release CAT expression cassette. (D) The flowchart for construction of recombinant BAdV3s expressing genes of variant mutated pIXs. The variant pIX mutations were amplified by polymerase chain reaction using primers and templates (Table 1), then inserted in SbfI site of pFBAV304a.I-SceI-ΔpIX using In-Fusion HD Cloning Kit. CAT with 50 bp homology to pIX ORF upstream and downstream and flanked with SbfI sites. The asterisk represents the site for PacI& EcoRI digestion, blunting, and self-ligation.

• 

  Fig. 2 Generation of pIX-deleted Adenoviruses variants. (A) Schematic representation of the pIX mutants. The pIX-deleted mutants were introduced by homology recombination (pFBAV304a.IsceI-ΔpIX, replace pIX by SbfI enzyme site) and enzyme cleavage (pH5LRedΔpIX, deletion pIX by AflII & MunI digestion and blunt ligation). The full name of genome plasmids are listed on the left and the corresponding pIX mutant proteins expressed by the recombinant viruses were listed on the right. “■” represents the CDS of BAdV3's pIX. “” represents the CDS of HAdV5's pIX, and “□” represents the deletion of the pIX's CDS of BAdV3 or HAdV5. “SbfI” indicates the use of SbfI enzyme site to replace the pIX's CDS of BAdV3. (B) Recombinant virions were rescued either in VIDO DT1 cells (BAdV3) or 293A cells (HAdV5). The cytopathic effect and fluorescence were observed at 15 days post-transfection. (C) The cells (VIDO DT1 or 293A) were infected by 2nd passage of wtBAdV3 or BAdV3ΔpIX virions, or by 2nd passage of HAdV5-RED or HAdV5-RED-ΔpIX virions. BF, bright field; FF, fluorescence field; wt, wild-type.

• 

  Fig. 3 Generation of start codon mutant and pIX/10GS/HA fusion gene. (A) Schematic representation of the pIX variants. Plasmid names are listed on the left and the corresponding pIX mutant proteins expressed by recombinant viruses are listed on the right. “■” represents the CDS of BAdV3's pIX, “” represents the 10GS/HA linker. “ATG→CTG” indicates the use of CTG to replace the start codon ATG of the BAdV3 pIX. (B) Generation of BAdV3 mutants. Recombinant adenoviral genomes in panel A were transfected into VIDO DT1 cells. At 15 days posttransfection, the cytopathic effect (CPE) and enhanced yellow fluorescent protein (EYFP) were examined. (C) The VIDO DT1 cells were infected by 2nd passage of wtBAdV3, BAdV3- mATGpIX or BAdV3-pIX/10GS/HA virions and observed CPE and EYFP expression. (D) Analysis of the expression of ATG mutant pIXs. The recombination plasmids pFBAV304a.I-sceIpIX/10GS/HA and pFBAV304a.I-sceI-mATGpIX/10GS/HA were transiently transfected in 293T cells and the cell extracts were detected with anti-HA mAb. Mock was a 293T cell extract. BF, bright field; FF, fluorescence field; wt, wild-type.

• 

  Fig. 4 Conserved amino acid regions in pIX. (A) Amino acid sequence alignment of pIX from Bovine (b) AdV and several human (h) AdV serotypes (Clustal W). Fully conserved residues are shaded black. Residues that occur in more than 50% of the sequences are shaded gray. Conserved sequence elements are boxed. The symbol “♦” denotes identical amino acid residues. (B) Amino acid sequence of BAdV3 pIX. The conserved sequence element and the coiled-coil domain are underlined, and the square bracket delineates the putative leucine zipper (2-ZIP server) [26,27].

• 

  Fig. 5 Generation of pIX truncated BAdV3s (BAdV3-pIXΔ1, BAdV3-pIXΔ2, and BAdV3-pIXΔ3). (A) Sequence of the BAdV3 pIX gene. (B) The schematics of truncated pIX mutants. The amino acid positions above the polyline depict the truncated region of pIX. Deletions of the three conserved domains were introduced by using the In-Fusion HD Cloning Kit. The names of genome plasmids are listed on the left and the corresponding names of the pIX mutant proteins expressed by rescued viruses are listed on the right. (C) The genome plasmids in panel B were transfected into VIDO DT1 cells for viral genome rescue, respectively. At 15 days post-transfection, cytopathic effect (CPE) and enhanced yellow fluorescent protein (EYFP) expression were examined. (D) The VIDO DT1 cells were infected by second passage of wtBAdV3, BAdV3-pIXΔ1, BAdV3-pIXΔ2, or BAdV3-pIXΔ3 virions and the CPE and EYFP expression examined. (E) Viral DNAs were extracted from VIDO DT1cells infected with BAdV3-pIXΔ3 or wtBAdV3. After SalI digestion, a 8216 bp fragment in wtBAdV3 viral DNA (lane 2) was cleaved into two fragments (4690 bp and 3550 bp) in BAdV3-pIXΔ3 viral DNA (lane 1), and post-KpnI digestion, a 6730 bp fragment in wtBAdV3 viral DNA (lane 4) was cleaved into two fragments (3578 bp and 3176 bp) in BAdV3-pIXΔ3 viral DNA (lane 3). Marker (M) sizes are shown in bases. (F) Dynamics of wtBAdV3 and BAdV3-pIXΔ3 growth in VIDO DT1 cells. The virus titers were determined at indicated times by plaque-forming unit (PFU) analysis. BF, bright field; FF, fluorescence field; wt, wild-type.

• 

  Fig. 6 Generation of chimeric BAdV3s (BAdV3-HpIX, BAdV3-pIXBH1 and BAdV3-pIXBH2). (A) The schematics of chimeric pIXs. “” represents the pIX sequence of BAdV3, “□” represents the pIX sequence of BAdV3 replaced by corresponding region of HAdV5 and “■” represented the 10GS/HA linker. The start amino acid position for chimeric pIX was marked. The names of genome plasmids are listed on the left and the corresponding names of chimeric pIX proteins expressed by rescued viruses are listed on the right. (B) The genome plasmids in panel A were transfected into VIDO DT1 cells for viral genome rescue, respectively. At 15 days post-transfection, the cytopathic effect (CPE) and enhanced yellow fluorescent protein (EYFP) were examined. (C) The VIDO DT1 cells were infected by second passage of wtBAdV3, BAdV3-HpIX, BAdV3-pIXBH1 or BAdV3-pIXBH2 virions and the CPE and EYFP observed. (D) Restriction enzyme analysis of rBAdV3s genome. Viral DNAs were extracted from infected VIDO DT1 cells. After viral DNAs were digested with SalI, a 8216 bp fragment in wtBAdV3 viral DNA (lane 3) was cleaved into two fragments (4700 bp and 3764 bp) in BAdV3-pIXBH1 (lane 1) or two fragments (4694 bp and 3715 bp) in BAdV3-pIXBH2 (lane 2) viral DNA. Post-KpnI digestion, a 6730 bp fragment in wtBAdV3 (lane 6) viral DNA was cleaved into two fragments (3792 bp and 3186 bp) in BAdV3-pIXBH1 (lane 4) or two fragments (3780 bp and 3186 bp ) in BAdV3-pIXBH2 (lane 5) viral DNA. Marker (M) sizes are shown in bases. (E) Western blot analysis of chimeric pIXs. Proteins from uninfected VIDO DT1, BAdV3-pIXBH1-infected VIDO DT1, BAdV3-pIXBH2-infected VIDO DT1, or wtBAdV3-infected VIDO DT1 were separated by SDS-PAGE (15% gel) and transferred to a polyvinylidene fluoride membranes. The separated proteins were probed in western blot by using anti-HA antibody. (F) Dynamics of rBAdV3s growth in VIDO DT1 cells. The virus titers were determined at indicated times by plaque-forming unit (PFU) analysis. BF, bright field; FF, fluorescence field; wt, wild-type.

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