Bovine adenovirus type 3 E1B(small) protein is essential for growth in bovine fibroblast cells

In order to study the function of bovine adenovirus type 3 (BAV-3) E1A and E1B(small) proteins, we constructed two mutants: (a) BAV102A carries an in- frame deletion in the coding region for the E1A protein (nt 831-1080); (b) BAV102B carries an insertion of triple stop codons in the E1B region (nt 16 54, 178 by downstream of the E1B(small) start codon), which stops the trans lation of the E1B(small) gene. BAV102A virus could grow to the wild-type BA V-3 titer in transformed cell line VIDO R2 (HAV-5 E1 transformed) cells, bu t no progeny virus could be found in fetal bovine retina cells (FBRC). RT-P CR and western blot analysis showed that neither mRNA transcripts nor prote in expression of early genes [E1B(small) and DNA binding protein (DBP)] cou ld be detected in BAV102A infected FBRC. The BAV102B grew 1.5 log less than wild-type BAV-3 in FBRC; however, no BAV102B progeny virus could be observ ed in bovine fibroblast (BFB) cells. No appreciable difference was observed in DBP transcript synthesis between wild-type BAV-3- or BAV102B-infected F BRC. However, compared to wild-type BAV-3, BAV102B viral DNA synthesis and fiber gene expression were found to be slightly reduced in FBRC. In contras t, compared to wild-type BAV-3, DBP transcripts and viral DNA synthesis wer e drastically reduced in BAV102B-infected BFB cells. In addition, no fiber gene expression could be detected in BAV102B-infected BFB cells. These resu lts suggest that BAV-3 E1A is essential for virus replication and is requir ed for activating the transcription of other BAV-3 early genes. However, th e requirement for E1B(small) protein for BAV-3 replication appears to be ce ll type-dependent. (C) 2001 Academic Press.