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.

FIG. 1

Persistence of viral DNA and hCFTR gene expression in the presence of the wild-type E4 region in the lungs of immunodeficient and immunocompetent mice. The E1/E3 deletion vector expressing hCFTR (AdTG6418 [Table 1]) was administered intratracheally to SCID (A), C3H (B), and C57BL/6 (C) mice at a dose of 1.5 × 10⁹ IU/animal, with six animals per time point for the SCID mice and five animals per time point for the C3H and C57BL/6 mice. The animals were sacrificed on the indicated days, and the persistence of the viral DNA in the lungs was analyzed by Southern blotting. Control lanes contain 10, 5, 1, and 0.1 viral genome copies, each mixed with 10 μg of lung cellular DNA from an untreated mouse (1 viral genome copy is equivalent to 30 pg of viral DNA). Expression of hCFTR in the lung was analyzed by Northern blotting using an hCFTR-specific DNA probe. Control lanes contain 35 and 7 ng of total RNA from AdTG6418-infected 293 cells mixed with 10 μg of lung RNA from an untreated mouse. Lung DNA and RNA were extracted and processed as described in Materials and Methods. Lanes marked n.i. contain DNA or RNA from the lungs of noninfected mice.

FIG. 2

Quantification of the persistence of the viral DNA in the presence or absence of the E4 region in the lungs of immunodeficient and immunocompetent mice. Autoradiograms corresponding to the Southern blots shown in Fig. 1 and in Fig. 4C and D were quantified by densitometry scanning, and the values are reported in panels A and B, respectively. (A) Persistence of the viral DNA in SCID (⧫), C57BL/6 (), and C3H (●) mice injected intratracheally with the E1/E3 deletion vector expressing hCFTR (AdTG6418 [Table 1]). (B) Persistence of the viral DNA in C57BL/6 () and C3H (●) mice injected intratracheally with the E1/E3/E4 deletion vector expressing hCFTR (AdTG5643 [Table 1]).

FIG. 3

Induction of anti-adenovirus and anti-hCFTR cellular responses in C3H and in C57BL/6 mice. Splenocytes were recovered from C3H and C57BL/6 mice injected intravenously 7 days earlier with PBS, with an E1/E3 deletion vector expressing hCFTR (AdTG6418 [Table 1]), or with an E1/E3 deletion vector carrying no transgene (AdE1°). The splenocytes were then analyzed by an ELISPOT assay for the presence of IFN-γ-expressing cells after stimulation with either AdE1° (open bars) or Ad-hCFTR (solid bars) (A) or after stimulation with syngeneic RMA cells (for C57BL/6 splenocytes) or L929 cells (for C3H splenocytes) infected with a vaccinia virus expressing hCFTR (B).

FIG. 4

Shutoff of CFTR expression in the absence of the viral E4 region. The E1/E3/E4 deletion vector carrying the CMV-hCFTR expression cassette (AdTG5643 [Table 1]) was administered intravenously to SCID mice (A) and intratracheally to SCID (B), C3H (C), and C57BL/6 (D) mice, at a dose of 1.5 × 10⁹ IU/animal, with five (A) or four (B through D) animals per time point. The animals were then sacrificed on the indicated days, and the persistence of viral DNA and expression of hCFTR in the lungs and liver were analyzed by Southern and Northern blotting, as described in the legend to Fig. 1.

FIG. 5

Reactivation of CMV-driven transgene expression by E4 gene products. The E1/E3/E4 deletion vector carrying the CMV-hCFTR expression cassette (AdTG5643 [Table 1]) was administered intratracheally to SCID mice at a dose of 1.5 × 10⁹ IU/animal, with five animals per time point. Mice were sacrificed and analyzed at day 3 and day 30 postinjection. At day 45 postinjection, the mice were reinjected intratracheally with an E1/E3/E4 deletion vector with no transgene but retaining E4 ORFs 1 through 4. The presence in the lungs of both vector genomes and of hCFTR mRNA was analyzed as described in the legend to Fig. 1.

FIG. 6

Growth properties of the E4 modification vectors in 293 cells and in 293-E4ORF6,7 cells. Cells were infected at an MOI of 2 IU/cell with E1/E3 deletion vectors either retaining the wild-type (wt) E4 sequences or having specific modifications in the E4 region. At 48 h postinfection, the viral yields were determined by indirect DBP immunofluorescence (see Materials and Methods) on 293-E4ORF6,7 cells.

FIG. 7

Expression of transgene and late viral genes in cells infected with E4 modification vectors. Human A549 cells were infected with the indicated E4 modification vectors at MOIs of 1,000 IU/cell (A) and 100 IU/cell (B and D). In panel C, the single infections were performed at an MOI of 200 IU/cell, while the double and triple infections were performed at an MOI of 100 IU/cell for each vector. Wild-type (wt) Ad5 was used at an MOI of 0.5 IU/cell. Total DNA and RNA were then extracted at 72 h postinfection and processed as described in the legend to Fig. 2 and in Materials and Methods. L3 hexon mRNA was detected with a ³²P-labeled oligonucleotide.

FIG. 8

Transcription of the transgene in the nuclei of A549 cells infected with E4-modification vectors. A549 cells were infected with the indicated vectors at an MOI of 100 IU/cell for 48 h, and nuclei were then isolated and analyzed as described in Materials and Methods. (A) Denatured target DNAs were dotted on a filter and hybridized to radiolabeled RNA isolated from the nuclei of the infected A549 cells. The target DNAs are human β-actin cDNA (panel 1), the ppolyII plasmid backbone (panel 2), and hCFTR cDNA (panel 3). wt, wild type. (B) Quantification by scintillation counting of the labeled nuclear RNA hybridized to the hCFTR probe dotted on the filter shown in panel A.

FIG. 9

E4 ORF3 is required but not sufficient for the persistence of CMV-CFTR expression in the lungs of SCID mice. Vectors with wild-type (wt) E4 sequences or with specific modifications in the E4 region were administered by intratracheal injection to SCID mice at a dose of 1.5 × 10⁹ IU/animal, with four animals per time point. Animals were sacrificed on the indicated days. The persistence of viral DNA (A) and of hCFTR expression (B) in the lung were determined by Southern and Northern blot analysis, respectively. DNA and RNA were extracted and processed as described in the legend to Fig. 1 and in Materials and Methods.

FIG. 10

E4 ORF3 is sufficient for the persistence of CMV-CFTR expression in the livers of SCID mice. Vectors with wild-type (wt) E4 sequences or with specific modifications in the E4 region were administered by intravenous injection to SCID mice at a dose of 1.5 × 10⁹ IU/animal, with five animals per time point. Animals were sacrificed on the indicated days. The persistence of viral DNA (A) and of hCFTR expression (B) in the liver were determined by Southern and Northern blot analysis, respectively. DNA and RNA were extracted and processed as described in the legend to Fig. 1 and in Materials and Methods.