Regulation of Adenovirus-Mediated Transgene Expression by the Viral E4 Gene Products: Requirement for E4 ORF3

Abstract

In a previous study we showed that multiple deletions of the adenoviral regulatory E1/E3/E4 or E1/E3/E2A genes did not influence the in vivo persistence 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 expression was evaluated by using as a model system the human cystic fibrosis transmembrane conductance regulator (CFTR) cDNA transcribed from the cytomegalovirus (CMV) promoter. We show that the viral E4 region is indispensable for persistent expression from the CMV promoter in vitro and in vivo, with, however, a tissue-specific modulation of E4 function(s). In the liver, E4 open reading frame 3 (ORF3) was necessary and sufficient to establish and maintain CFTR expression. In addition, the E4 ORF3-dependent activation of transgene expression was enhanced in the presence of either E4 ORF4 or E4 ORF6 and ORF6/7. In the lung, establishment of transgene expression was independent of the E4 gene products but maintenance of stable transgene expression required 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 promoter was severely reduced in the absence of E4 functions but could be partially 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 proteins in the transcriptional regulation of heterologous transgenes. We also report that C57BL/6 mice are immunologically weakly responsive to the human CFTR protein. This observation implies that such mice may constitute attractive hosts for the in vivo evaluation of vectors for cystic fibrosis gene therapy.

Efficiency, efficacy, and adverse effects of adenovirus- vs. liposome-mediated gene therapy in cardiac allografts

“…The observation of the relatively high intensity of the antiviral cellular immune response manifested by the earlier and severe infiltration in allografts than isografts suggests the interaction between the antiviral and allogenic immune responses (3). Early-occurring functional alterations followed by structural alteration in the integrity of the cardiac myocytes may be also attributable to the transient transgene expression pattern of the viral vector (14).…”

Functions of the adenovirus E4 proteins and their impact on viral vectors

“…The effect can be rescued by subsequent infection with a vector that contains E4 genes, suggesting that the E4 product(s) activates transcription from the silenced vector (119). Expression from the CMV promoter was shut-down in vectors that lacked E4, but retention of E4orf3 resulted in longevity of expression in murine lung and liver (120,121). Silencing appears dependent upon the promoter and tissue type of the targeted cells, and in some organs there may also be a contribution from other E4 gene products (121,122).…”

“…Expression from the CMV promoter was shut-down in vectors that lacked E4, but retention of E4orf3 resulted in longevity of expression in murine lung and liver (120,121). Silencing appears dependent upon the promoter and tissue type of the targeted cells, and in some organs there may also be a contribution from other E4 gene products (121,122). The exact mechanism by which E4orf3 regulates activity from heterologous promoters remains unclear, although a contribution may come from increased rates of transcription (121).…”

“…Silencing appears dependent upon the promoter and tissue type of the targeted cells, and in some organs there may also be a contribution from other E4 gene products (121,122). The exact mechanism by which E4orf3 regulates activity from heterologous promoters remains unclear, although a contribution may come from increased rates of transcription (121). Vectors that retain a functional E4orf3 may thus provide long-term gene expression combined with the desire for low toxicity and inflammatory response (123).…”

Gene therapy and tissue engineering in repair of the musculoskeletal system

“…The second‐generation adenoviral vectors were products of attempts to reduce the immunogenicity by removing some or all of the open reading frames encoding transacting regulatory proteins [Wang and Finer, 1996; Christ et al, 1997]. This resulted in decreased immunogenicity and subsequently increased transgene expression duration; however, as duration did increase, there was also a decrease in overall expression for the second generation adenoviral vectors [Armentano et al, 1997; Christ et al, 1997; Lusky et al, 1999]. In another attempt to create a less immunogenic adenoviral vector, all the viral genes were removed with only the inverted terminal repeats and the packaging signal remaining [Kochanek et al, 1996; Hammerschmidt, 1999; Kochanek, 1999].…”