The expression and function of the adenovirus type 5 E4 region
Human adenovirus type S is a dsDNA virus which replicates in the nucleus of the infected cell, exploiting a number of host cell mechanisms. This close association with the eukaryotic cell has made adenovirus the target of numerous studies attempting to understand how cellular systems function. This study focuses on the E4 transcription unit, which has the potential to encode at least 7 distinct polypeptides from reading frames accessed by differential splicing of a single primary transcript. In this study, the pattern of expression of these mRNAs during lytic infection was examined, and two distinct temporal classes were defined; early and late. It had been previously shown that adenovirus mRNAs produced late in the infection depended on a virus-coded RNA transport regulator, EIb-S5K, for optimal cytoplasmic accumulation. However, only one of the E4 late class mRNAs was dependent on this EI b protein for cytoplasmic accumulation leading to the hypothesis that for an E4 late mRNA to be dependent on EIb, it had to retain intact splice sites or intronic sequences. To examine this hypothesis, a virus was built lacking an important splice site of the E4 region to see if, by removing this splice site, the mRNA could leave the nucleus in the absence of the El b complex. The results of initial experiments reported here supported this hypothesis. Two of the E4 open reading frames (ORFI and ORF2) identified in Ad2 were disrupted in the published AdS E4 sequence, but these differences were subsequently found to be sequencing artefacts. The presence of these two proteins in the infected cell had never been previously demonstrated so polyclonal antisera were generated against bacterially expressed ORFI and ORF2. The ORF2 antiserum allowed the identification of the ORF2 protein in the cytoplasm of infected cells, from early stages of the infection. No associations of ORF2 with other infected cell components were detected. In contrast, the ORFI antiserum only reached a low titre and no ORFI protein was detected in infected cells. Now that ORF2 has been found in the infected cell more work needs to be undertaken to elucidate its function.