Upstream AP1- and CREB-binding sites confer high basal activity on the adeno-associated virus type 5 capsid gene promoter

Abstract

In contrast to the prototype adeno-associated virus type 2 (AAV2), the capsid gene P41 promoter of AAV5, within viral constructs that lack inverted terminal repeat sequences, displays a high basal level of expression in 293 cells in the absence of coinfecting adenovirus. Here we demonstrate that this was due to differences in the relative strengths of the core promoter elements and to the presence of active binding sites for the transcription factors CREB and AP1 within the upstream region of P41 that are absent from the AAV2 capsid gene promoter P40. These differences also governed the relative basal activity of the AAV capsid gene promoters within near-full-length viral genomes.

FIG. 1.

Core promoter comparison of AAV2 P40 and AAV5 P41. (A) Sequences of AAV2 P40 core promoter (CP40 nt 1823 to 1887) and AAV5 P41 core promoter (CP41 nt 1880 to 1970). The core promoters start from the TATA box of each promoter, and the luciferase genes from pGL3 are cloned downstream for the reporter assay below. TATA box and initiator sequences are indicated in boldface letters. The vertical lines in the boxes indicate the borders of TATA box and initiator elements in the hybrid promoters (AV2 CP40 V5TATA, AV2 CP40 V5Inr, AV5 CP41 V2TATA, and AV5 CP41 V2Inr) tested below. (B) Luciferase (Luc) activity of the wild-type, mutant, and hybrid promoters in 293 cells. The activity of each promoter is the average of duplicate samples from at least three experiments. All values are standardized to Renilla luciferase (R-Luc) activity generated by a TK promoter-driven Renilla luciferase expression vector used as an internal control.

FIG. 2.

Upstream sequence analysis of the capsid promoters. (A) Upstream sequence alignment of AAV2 P40 promoter (USP40 nt 1655 to 1822) and AAV5 P41 promoter (USP41 nt 1681 to 1879). Identical nucleotides are shaded, and the abbreviations L, S, and min indicate the different lengths of the upstream sequences linked to the core promoters in the luciferase constructs tested below. The consensus AP1 and CRE sites are also indicated in the boxes. (B and C) AAV5 upstream sequence enhances promoter activity greater than AAV2 upstream sequence. Different lengths of AAV2 and AAV5 upstream sequences are linked to core promoter CP40 (B) or CP41 (C), and the activity of each promoter is the average of duplicate samples from at least three experiments. All values are standardized to Renilla luciferase (R-Luc) activity generated by a TK promoter-driven Renilla luciferase expression vector used as an internal control. (D and E) AP1 and CRE are the major elements enhancing AAV5 P41 promoter activity. AAV2 core promoter CP40 is linked with wild-type AAV2, AAV5 upstream sequence, or AAV2 upstream sequence mutated to contain AP1, CRE, or both sites (D). A similar set was tested with AAV5 core promoter CP41 linked with wild-type AAV2, AAV5 upstream sequence, or AAV5 upstream sequence with AP1 and with the CRE site mutated (E).

FIG. 3.

Physical interaction between cellular factors and AP1 and CRE sites. (A and B) EMSA of AP1 and CRE with 293 cell nuclear extract. Free probes (F) and specific shifted (S) bands are indicated on the left. Complexes formed with wild-type and mutant (m) probes are shown in lanes 1 and 2, respectively. Tenfold cold wild-type (lane 3) and labeled mutant (lane 4) probe competition results are also included. The bands marked with an asterisk may represent additional unidentified complexes since they are removed by competition. (C) Supershift assay for CRE probe. In lane 2, after incubation of the probe and nuclear extract, 4 μl anti-CREB (a-CREB) antibody was added to the reaction mixture for an additional 30 min. A supershifted band is indicated by SS. (D) UV cross-link coupled immunoprecipitation to test the binding of c-Fos and c-Jun to the AP1 element. The radioactivity of the immunoprecipitated complexes was measured using scintillation counting. A wild-type AP1 probe with anti-β-actin (α-β-actin) antibody and an AP1 mutant probe (AP1m) with anti-c-Fos (α-c-Fos) or anti-c-Jun (α-c-Jun) antibodies were used as controls. The data shown are the average of duplicate samples from at least three experiments.

FIG. 4.

E1A activates AAV5 P41 promoter, but the activation is not directly mediated by AP1 or CRE sites. Shown is the luciferase activity of P41 reporter constructs with either the wild-type or AP1 and CRE mutant upstream regions, tested in HeLa cells together with cotransfection of different E1A isoforms, E1A mutants, or empty vector SK+. The activity is the average of duplicate samples from at least three experiments, as described in Materials and Methods.

FIG. 5.

AP1 and CRE enhance promoter activity in the viral RepCap background. (A) Illustration of the constructs used in the assay. AAV2 RepStopCapTR contains AAV2 sequence nt 145 to 4626 and a premature stop codon at nt 489. AAV2 RepStopDMCapTR has additional AP1 and CRE sites present in the P40 region. AAV5 RepStopCapTR contains AAV5 sequence nt 185 to 4587 and a premature stop codon at nt 480. AAV5 RepStopDMCapTR has additional AP1 and CRE mutation to knock out those sites in the P41 region. The probes used in the RNase protection assay are also shown. (B) RNase protection assay, performed as previously described (15, 23), using homologous RP probes to protect RNA generated in 293 cells following transfection of the constructs mentioned above. pBluescript SK+ was used as an empty vector control. AAV2 or AAV5 Rep was provided in trans (V2 or V5, respectively), driven by human immunodeficiency virus promoter, as previously described (19, 20). pEGFPC1 (Clontech, Mountain View, CA) was cotransfected as an internal control. Transfection of samples for lanes 2 to 4, 6 to 8, 10 to 12, and 14 to 16 was followed by adenovirus infection to allow activation, as indicated. (C) Quantification, using Fujifilm MultiGauge software, of RNase protection. A representative example is shown. Data from at least three experiments, with standard error bars, are presented as the activation (fold) of P40 (P41) promoters comparing transcription levels. The wild-type P40 (P41) level without Rep cotransfection and adenovirus infection is set as 1.

FIG. 6.

Genome sequence comparison of AAV2 and AAV5-like viruses. The P40/P41 upstream sequence alignments of AAV2, AAV5, bovine AAV (BAAV), and caprine AAV (Go-AAV) are shown. The upstream promoter sequences from each virus were aligned using Vector NTI software (Invitrogen) with AAV5 as the consensus reference. Identical nucleotides in all AAVs are shown in the darkest shade of gray. Nucleotides identical to the AAV5 sequence are shown in the lighter shade of gray. The consensus AP1 and CRE sites are indicated in boxes.