Transcription mapping and characterization of proteins produced from early region 4 of porcine adenovirus type 3

Abstract

The early region 4 (E4) of porcine adenovirus 3 (PAdV-3) was characterized by Northern blot, rapid amplification of cDNA ends (RACE), RT-PCR and cDNA sequence analysis. Northern blot analysis revealed three different classes of transcripts, which appeared and peaked at different times post-infection. The RT-PCR, RACE and cDNA sequence analysis identified nine major E4 transcripts, all of which shared a 107-bp 5' leader sequence and a 126-bp 3' terminus. These transcripts have one to three introns removed. Interestingly, of the nine major transcripts, there was one fusion transcript of ORFp1 and ORFp7 (ORFp1/7), which codes for a protein of 119 amino acids. All transcripts initiated at nucleotide 33740 of the PAdV-3 genome. To identify proteins, rabbit antiserum was prepared using a bacterial fusion protein encoding p2, p3, p4 or p7 proteins. Serum against p2, p3 and p4 immunoprecipitated proteins of 13.5, 13.6 and 15.3 kDa, respectively, in in-vitro transcribed and translated mRNA and in PAdV-3-infected cells. Serum against p7 immunoprecipitated a protein of 19.8 kDa in in-vitro transcription and translation analysis but recognized two proteins of 19.8 kDa (encoded by ORFp7) and 14 kDa (encoded by the fusion transcript ORF1/7) in PAdV-3-infected cells. The protein encoded by ORFp2 was localized in the nucleus of PAdV-3-infected cells. The proteins encoded by ORFp3 and ORFp7\ORFp1/7 were detected in the cytoplasm of PAdV-3-infected cells. However, the protein encoded by ORFp4 was observed both in the cytoplasm and nucleus of PAdV-3-infected cells.