Human Papilloma Virus E6 and E7 Proteins Support DNA Replication of Adenoviruses Deleted for the E1A and E1B Genes  Dirk S. Steinwaerder, Cheryl A. Carlson, André Lieber  Molecular Therapy  Volume 4, Issue 3, Pages 211-216 (September 2001) DOI: 10.1006/mthe.2001.0447 Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 1 Detection of E6 and E7 expression by immunoprecipitation. (A) Structure of adenovirus vectors. The ORFs for HPV16 E6 or E7 are under the control of the human CMV promoter. The polyadenylation signal (PA) is derived from the bovine growth hormone gene. The E6 or E7 expression cassettes were inserted into Ad vectors deleted for E1A and E1B genes (ΔE1 removes Ad5 sequences from bp 342 to 3523) and E3 genes (ΔE3 removes Ad5 sequences from bp 28,133 to 30,818). Preparations of Ad.E6 and Ad.E7 were tested for contamination with E1 + adenovirus by PCR for E1A sequences. Only virus preparations containing less than one E1+ (wild-type) viral genome in 109 pfu (2 × 1010 particles) were used in these studies. (B) Expression of E6 and E7 in SK-Hep1 cells after adenoviral gene transfer. SK-Hep1 cells were infected with Ad.E6 or Ad.E7 at an MOI of 100 pfu/cell (4 × 108 particles per ml), which allows for transduction of > 90% of cells [16,38]. Lanes 1, 5, and 6, Ad.E7-infected cells; lanes 2, 3, and 4, Ad.E6-infected cells. Immunoprecipitation was carried out using anti-E6 antibodies (lanes 1, 2, and 6) or anti-E7 antibodies (lanes 3, 4, and 5). The arrows indicate the HPV-16 E6 and E7 proteins migrating at 18 kDa and 21 kDa, respectively. (C) Expression of E6 and E7 in HPV-associated cervical carcinoma cells. HeLa cells were metabolically labeled and cell extracts were analyzed as described above. Note that HeLa cells express HPV-18 E6/E7, which may account for the slightly lighter molecular weight of E7. Molecular Therapy 2001 4, 211-216DOI: (10.1006/mthe.2001.0447) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 2 HPV E6 and HPV E7 enhance AdE1− DNA replication in vitro and in vivo. Confluent SK-Hep1 (A) or primary human fetal astrocytes (B) were infected with methylated virus at a total MOI of 100 pfu/cell (4 × 108 particles per ml), which allows for transduction of 100% of cells [20,38]. Ad.BG is a methylated β-galactosidase expressing vector used as an example of a standard first-generation, E1/E3-deleted Ad vector. “Wt Ad” is a methylated, wild-type (E1+) Ad5 virus. Methylated Ad.E6 or Ad.E7 were applied either separately or in combination (Ad.E6+AdE7, MOI 50 pfu/cell each (2 × 108 particles per ml)). To analyze vector DNA replication, cells were harvested 72 h after infection. Total cellular DNA (10 μg) was extracted from all samples, digested with HindIII and XhoI, and separated in a 0.8% agarose gel. Southern analysis was carried out using as probe a viral 8-kb HindIII fragment containing one methylation–sensitive XhoI site. Differentiation between parental vector DNA and replicated viral DNA is based on the selective demethylation of replicated viral DNA resulting in a 6.5-kb fragment as opposed to an 8-kb fragment derived from parental vector DNA. As a reference, a mixture of purified methylated and unmethylated virus genomes was included (+). (C) We administered 2 × 109 pfu (4 × 1010 particles) of methylated Ad.BG, Ad.E6, Ad.E7, or wild-type Ad5 to C57Bl/6 mice by tail vein infusion. Five days after infusion, hepatocellular DNA was extracted and analyzed by Southern blot as described in (A) Primary human astrocytes were in a quiescent stage during the period of the experiment. Wild-type Ad5 expresses both E1a (12S and 13S) and E1B (55-kDa and 19-kDa) genes. Molecular Therapy 2001 4, 211-216DOI: (10.1006/mthe.2001.0447) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 3 Analysis of hepatocellular DNA synthesis in liver sections by 3H-thymidine incorporation. We administered 2 × 109 pfu (4 × 1010 particles) Ad.BG, Ad.E6, or Ad.E7 to C57/Bl6 mice via tail vein injection. One group of mice was injected with virus dilution buffer (“Mock”). Four days after virus infusion, mice were intraperitoneally injected twice with [methyl-3H]-thymidine (1 μCi/g of body weight) 20 h and 6 h before sacrifice, and liver tissue was analyzed for morphology and 3H-thymidine incorporation as described [21]. (A) Autoradiography on liver sections. To help identify labeled nuclei, one characteristic positive cell per field is marked with an arrow (staining, hematoxilin; magnification, ×200). (B) Quantification of 3H-thymidine-positive hepatocytes (n = 3). Molecular Therapy 2001 4, 211-216DOI: (10.1006/mthe.2001.0447) Copyright © 2001 American Society for Gene Therapy Terms and Conditions

FIG. 4 Replication of AdE1− in cervical carcinoma cells. HeLa cells, SiHa cells, Caski cells, and confluent primary human astrocytes (prim.Ac) were infected with an MOI of 100 pfu/cell (4 × 108 particles per ml; HeLa, SiHa, astrocytes) and 300 pfu/cell (1.2 × 109 particles per ml; Caski) with a methylated, β-gal-expressing AdE1− vector (Ad.BG) for 3 h. Infections were carried out in 6-well dishes using a suspension volume of 1 ml. To demonstrate similar transduction efficiencies, a subset of cells was harvested 3 h after the start of infection (uptake). Another set of cells (replication) was harvested 72 h after infection. Cellular DNA was analyzed for vector DNA synthesis as previously described [19]. Demethylated progeny vector DNA is detected as a 6.5-kb band, whereas parental vector DNA can be seen as an 8.0-kb band. As a reference, a mix of methylated and unmethylated vector DNA was included (+). Molecular Therapy 2001 4, 211-216DOI: (10.1006/mthe.2001.0447) Copyright © 2001 American Society for Gene Therapy Terms and Conditions