A 72-bp internal deletion in the left inverted terminal repeat of the bovine adenovirus type 3 genome does not affect virus replication

Abstract

The genome of bovine adenovirus type 3 (BAV3) is flanked by 195-base pair (bp) inverted terminal repeats (ITR). We isolated a BAV3 mutant (BAV3c29) having an internal deletion within the left ITR. The deletion eliminated 72 bp between nucleotides (nt) 89 and 162, including most of the GC-rich sequences located close to the end of the ITR sequences. This deletion did not seem to have any affect on the virus plaque size or morphology and the kinetics of viral replication compared to wild-type (wt) BAV3. The nt sequence of the right ITR of BAV3c29 remained identical to the right or left ITR of wt BAV3. These results indicate that the cis-acting sequences present within the 72 bp between nt 89 and 162 of the left ITR are not essential for BAV3 DNA replication in cultured cells.

Fig. 1.

Generation of BAV3c29 and restriction enzyme analysis of genomic DNA extracted from wt BAV3 and BAV3c29. A) Diagram representing the generation of the internal deletion in the left ITR during PCR amplification of the EcoRI fragment of BAV3. PCR was performed using an oligonucleotide encoding the PacI recognition site and the first 21 bp of the BAV3 ITR (PacI-1-21) as a forward primer and the nucleotidet sequence from 1138 to 1159 as a reverse primer. The purified PCR product was subsequently cloned in pUC18 to form pMvOBE1E4PacI (van Olphen & Mittal, 1999), and it was subsequently then used with the BAV3 genome to generate plasmid pMvOBAV3c29 by recombination in Escherichia. coli BJ5183. Genome-length DNA obtained from pMvOBAV3c29 was linearized with PacI and transfected into MDBK cells using a lLipofectin-mediated transfection protocol (Life Technologies). Two weeks post-after transfection, an isolated viral plaque was collected and propagated to produce the BAV3c29 stock. The left-end AscI fragment of wt BAV3, pMvOBAV3c29, and BAV3c29 depicting the ntucleotide position of the ITR boundaries, and the locations of EcoRI and AscI recognition sites are shown. ITRs are shown as open-boxes and the position and length of the internal ITR deletion are also shown. B) DNA samples obtained from purified preparations of wt BAV3 and BAV3c29 were digested with restriction enzymes, AscI and EcoRI. The resultant DNA fragments were loaded onto a 1.5% agarose gel andel, separated by electrophoresis and the ethidium bromide-stained bands were visualized with an UV transilluminator. The sizes of the terminal fragments containing the left ITR generated with the digestion of AscI and EcoRI are shown on the left and right side of the panel, respectively. MWM =, 1 kb DNA molecular weight marker.

Fig. 2.

Nucleotide sequence comparison of the left ITR of wt BAV3, BAV3c29 and HAV5. On the basis of sequence similarities with HAV5, the potential binding sites for a number of transcription factors were identified in the left BAV3 ITR. The potential binding motifs for ORP-A, NFI, NFIII, SP1 and ATF are depicted as gray underlined, underlined, double underlined, gray background and thick underlined, respectively. The deleted nt in the left ITR of BAV3c29 are depicted by the symbol ‘∼’. The conserved nt in the binding motif for NFI are shown in bold. The nt substitutions in BAV3c29 are shown in bold face with lines above and below.

Fig. 3.

Development of plaques by BAV3 and BAV3c29 on MDBK cells. Confluent monolayers of MDBK cells in 60 mm dishes were infected with various log dilutions of either BAV3 or BAVc29 and the virus was allowed to adsorb for 1 h at 37°. Subsequently monolayers were covered with an agarose overlay (1 x MEM, 5% fetalCloneIII, 0.5% agarose, 0.05% yeast extract, 50 μg/ml gentamicin and 25mM MgCl₂). Plaques were stained with crystal violet 12 days after infection. Dishes infected with virus dilutions of 10^-8, 10^-9 and 10^-10 are shown.

Fig. 4.

Kinetics of replication of wt BAV3 and mutant BAV3c29. MDBK cell monolayers were grown in 60mm culture dishes and infected with wt BAV3 or BAV3c29 at an MOI of 2 PFU per cell. Infected cells along with the medium were collected 6, 12, 24, 48, 72 and 96 h after infection and the virus titration was done by plaque assay on MDBK cells.