Mutational Analysis of Early Region 4 of Bovine Adenovirus Type 3

Abstract

The primary objective of characterizing bovine adenovirus type 3 (BAV3) in greater detail is to develop it as a vector for gene therapy and vaccination of humans and animals. A series of BAV3 early region 4 (E4) deletion-mutant viruses, containing deletions in individual E4 open reading frames (Orf) or combinations of Orfs, were generated by transfecting primary fetal bovine retinal cells with E4-modified genomic DNA. Each of these mutants was further analyzed for growth kinetics, viral DNA accumulation, and early-late protein synthesis. Mutant viruses carrying deletions in Orf1, Orf2, Orf3, or Orf4 showed growth characteristics similar to those of the E3-deleted BAV3 (BAV302). DNA accumulation and early/late protein synthesis were also indistinguishable from those of BAV302. However, mutant viruses carrying a deletion in Orf5, Orfs 1-3 (BAV429), or Orfs 3-5 (BAV430) were modestly compromised in their ability to grow in bovine cells and express early/late proteins. E4 mutants containing larger deletions, Orfs 1-3 (BAV429) and Orfs 3-5 (BAV430), were further tested in a cotton rat model. Both mutants replicated as efficiently as BAV3 or BAV302 in the lungs of cotton rats. BAV3-specific IgA and IgG responses were detected in serum and at the mucosal surfaces in cotton rats inoculated with mutant viruses. In vitro and in vivo characterization of these E4 mutants suggests that none of the individual E4 Orfs are essential for viral replication. Moreover, successful deletion of a 1.5-kb fragment in the BAV3 E4 region increased the available insertion capacity of replication-competent BAV3 vector (E3-E4 deleted) to approximately 4.5 kb and that of replication-defective BAV3 vector (E1a-E3-E4 deleted) to approximately 5.0 kb. This is extremely useful for the construction of BAV3 vectors that express multiple genes and/or regulatory elements for gene therapy and vaccination.

Production and Formulation of Adenovirus Vectors

“…Changes in the E2 region have been explored which further down-regulate viral DNA replication and gene expression [66][67][68][69][70]. Also, changes in the E4 region have been explored [71][72][73][74] as well as both E2 and E4 deletions [75]. However, expression of the E2 and E4 proteins at levels high enough to adequately complement viral replication can be toxic, especially in E4 complementing lines.…”

Isolation, identification, and complete genome sequence of a bovine adenovirus type 3 from cattle in China

“…The proteins encoded by the E4 region are involved at several levels of regulation of cellular and viral gene expression, viral DNA replication, late viral assembly, E2 expression, and adeno-associated virus helper function [15-18]. It has been suggested that individual ORFs of the BAV-3 E4 can be deleted and are nonessential for viral replication [19]. But the deletion of HLJ0955 strain in E4 region did not result in any deletion of five ORF products of HLJ0955 strain.…”

Bovine adenovirus-3 as a vaccine delivery vehicle

“…The other sites for potential insertion of foreign ORF have been identified in transcriptionally active E4 region of BAdV-3 [58]. Isolation of replication competent BAdV-3 containing deletion of N-terminus 1.501 kb or C-terminus 1.342 kb ( Fig.…”

“…Isolation of replication competent BAdV-3 containing deletion of N-terminus 1.501 kb or C-terminus 1.342 kb ( Fig. 2) in E4 region suggested that these regions are not essential for replication of BAdV-3 [58], thus increasing the potential insertion capacity of E3-E4 deleted BAdV-3 to 4.5 kb [58].…”