Optimization of bovine coronavirus hemagglutinin-estrase glycoprotein expression in E3 deleted bovine adenovirus-3

Abstract

Adenoviral vectors expressing foreign genes have many desirable properties in applications such as vaccination. Recently, we have generated replication-competent (E3 deleted) bovine adenovirus-3 (BAV-3) recombinants expressing significant amounts of glycoprotein D (gD) of bovine herpesvirus-1 (a DNA virus). However, attempts to express the RNA virus genes using the same strategy were not successful. In an effort to optimize the expression, we have constructed several BAV-3 recombinants carrying the hemagglutinin esterase (HE) gene of bovine coronavirus (BCV) in the E3 region with or without exogenous transcription control elements. The expression studies suggest that the introduction of a 137 bp chimeric intron upstream of the HE cDNA is able to increase the level of HE gene expression. The introduction of a SV40 early promoter or human cytomegalovirus (HCMV) immediate early (IE) promoter into the expression cassette changed the kinetics of the HE expression. However, the recombinant BAV-3 containing HE under the HCMV IE promoter replicated less efficiently than the wild-type BAV-3. These studies should prove useful in expression of other RNA viral genes in the E3 region of BAV-3 expression system.

Fig. 1

Schematic representation of recombinant E3 deleted full length BAV-3 genomic clones. BAV-3 genome (■), HE gene (□), SV40 early promoter (), chimeric intron (), SV40 poly(A) (), HCMV IE promoter (). The locations of early (E) region E1, E3 and E4 are depicted. The arrow represents the direction of transcription. The name given to each recombinant virus is depicted on the right.

Fig. 2

Restriction enzyme analysis of recombinant BAV-3 genomes. (A) The DNAs were extracted from BAV-3 (lane1), BAV3.E3d (lane 2), BAV303 (lane 3), BAV332 (lane 4), BAV333 (lane 5) and BAV334 (lane 6) infected MDBK cells by Hirt method (Hirt, 1967) and digested with BamHI. (B) The fragments shown in panel A were transferred to Nytran membranes and probed with -α³²P-labeled HE probe. Lane M, 1 Kb Plus DNA ladder (Gibco/BRL) used for sizing the viral DNA fragments. Sizes in kb is shown on left.

Fig. 3

Northern blot analysis of HE transcription. Total RNA was isolated from mock (M), BAV303 (lane 1, 2), BAV332 (lane 3, 4), BAV333 (lane 5, 6) and BAV334 (lane 7, 8) infected MDBK cells after 18 (lane 1, 3, 5, 7) or 28 (lane 2, 4, 6, 8) h post infection and analyzed by Northern blot analysis as described under ‘Materials and Methods’ using 1.3 Kb BamHI fragment (containing HE coding sequence) of pCVE3 plasmid (Parker et al., 1989) as a probe. Numbers on the right denote the estimated sizes of RNAs in kilobases.

Fig. 4

Expression of HE protein in MDBK cells infected with recombinant BAV3 viruses. Proteins from lysates of radiolabeled mock infected (lane 1), BAV-3 infected (lane 2), BCV infected (lane 3), BAV303 infected (panel A, lanes 4–6), BAV332 infected (panel B, lanes 4–6), BAV333 infected (panel C, lanes 4–6) or BAV334 infected (panel D, lanes 4–6) MDBK cells were immunoprecipitated with polyclonal anti-BCV serum and analyzed by SDS-PAGE under reducing conditions. The cells were harvested at 12 (lane 4), 24 (lane 5) and 36 h (lane 6) postinfection. The positions of the size markers (in kilodaltons) are shown to the left of each panel.