Analysis of Early Region 1 of Porcine Adenovirus Type 3

Abstract

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.

Porcine adenovirus type 3 E1Blarge protein downregulates the induction of IL-8

“…To determine the effect of PAdV-3 E1 proteins on the induction of chemokines, HeLa cells were infected with PAV211 (E1A nt [530–1230] + E3 [nt 28112–28709] deleted), PAV212 (E1B small [nt 1460–1820] + E3 [nt 28112–28709] deleted), PAV227 (E1A + E1B small + E1B large [nt 524–3274] + E3 [nt 28112–28709] deleted) or PAV300 (E3 [nt 28112–28709] deleted) at an MOI of 100 infectious units [24]. The construction and characterization of the mutant PAdV-3s has been described [19,20]. At 6 h post infection, the cells were harvested and processed for the isolation of total RNA using TRIZOL (Invitrogen) as per manufacturer's protocol.…”

“…Recombinant PAdV-3 bearing deletions in the E1 region were generated as described previously [20]. PAV211 contains deletions in the E1A + E3 regions, PAV212 contains deletions in the E1B small + E3 regions, PAV227 contains deletions in E1 + E3 regions and PAV300 contains deletion in E3 region.…”

“…PAV211 contains deletions in the E1A + E3 regions, PAV212 contains deletions in the E1B small + E3 regions, PAV227 contains deletions in E1 + E3 regions and PAV300 contains deletion in E3 region. Viruses were propagated and titrated as described [19,20,24]. HeLa cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal calf serum (FCS).…”

“…Availability of the complete nucleotide sequence and transcription map of PAdV-3 [18] genome has facilitated the construction of recombinant PAdV-3s [16,17,19,20] and their use as vaccine delivery vehicles [21]. Earlier, analysis of early region 1 (E1) of PAdV-3 suggested that while E1A [20] and E1B large [19] are essential for virus replication, E1B small is not essential for virus replication [20]. Here, we report that E1B large can impair the induction of inflammatory cytokine IL-8 by inhibiting the NF-κB dependent gene transcription.…”

Bovine adenovirus‐3 protein VIII associates with eukaryotic initiation factor‐6 during infection

“…The recombinant GST‐fusion proteins were expressed in Escherichia coli BL21 cells as described (Zhou & Tikoo, ). The purification of GST and GST‐pVIII fusion proteins has already been described (Ayalew, ).…”