Construction of nondefective adenovirus type 5 bearing a 2.8-kilobase hepatitis B virus DNA near the right end of its genome

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Abstract
A novel helper-free adenovirus type 5 (Ad5) vector system, which utilizes a cloning site 0.2 kilobase (kb) from the right end of the genome, was developed. To construct a nondefective Ad5 bearing the 2.8-kb DNA fragment of hepatitis B virus (HBV) at this site, the 2.1-kb nonessential E3 fragment from cloned DNA covering the right 1/4 of the Ad5 genome (76-100 map units) was deleted, the HBV DNA was inserted into this site, the recombinant DNA was ligated to the rest of the Ad5 genome, and the ligated DNA was transfected into human embryo kidney cells. Most of the recovered virus clones had only the E3 deletion and no HBV insertion, suggesting that a homologous recombination occurs between transfected DNA species in these cells. The isolated Ad5 virus bearing the HBV DNA (Ad5-HBL) grew without helper virus in HeLa cells as efficiently as wild-type Ad5, although the 1.9-kb major E4 transcript was detected only poorly in the early phase in the Ad5-HBL-infected cells, suggesting that the HBV DNA inserted upstream of the E4 promoter reduces the E4 transcript. HBV mRNA species transcribed from the inserted DNA were at least as abundant as Ad5 early mRNA species in the late phase of Ad5-HBL infected, but the HBV surface antigen was barely detectable in the infected-cell lysate and culture medium. This result suggests that HBV mRNA species can be transcribed from the inserted genes but no protein can be translated from the HBV mRNA species, presumably because of the translation suppression of cellular mRNA species caused by adenovirus in its late phase.