Regulation of adenovirus-mediated transgene expression by the viral E4 gene products: Requirement for E4 ORF3

In a previous study we showed that multiple deletions of the adenoviral reg ulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persisten ce of the viral genome or affect the antiviral host immune response (Lusky et al., J. Virol, 72:2022-2032, 1998). In this study, the influence of the adenoviral E4 region on the strength and persistence of transgene expressio n was evaluated by using as a model system the human cystic fibrosis transm embrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovi rus (CMV) promoter. We show that the viral E4 region is indispensable for p ersistent expression from the CMV promoter in vitro and in vivo, with, howe ver, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and mainta in CPTR expression. In addition, the E4 ORF3-dependent activation of transg ene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 an d ORF6/7. In the lung, establishment of transgene expression was independen t of the E4 gene products but maintenance of stable transgene expression re quired E4 ORF3 together with either E4 ORF4 or E4 ORF6 and ORF6/7. Nuclear run-on experiments showed that initiation of transcription from the CMV pro moter was severely reduced in the absence of E4 functions but could be part ially restored in the presence of either ORF3 and ORF4 or ORFs 1 through 4. These results imply a direct involvement of some of the E4-encoded protein s in the transcriptional regulation of heterologous transgenes. We also rep ort that C57BL/6 mice are immunologically weakly responsive to the human CF TR protein. This observation implies that such mice may constitute attracti ve hosts for the in vivo evaluation of vectors for cystic fibrosis gene the rapy.