Figure 1.

Adenovirus construct and enzymatic activity of the transgenes. (a) Schematic representation of the adenoviruses used in this study. Each virus was engineered to express the transgene immediately downstream of a CMV promoter (P) followed by a polyadenylation sequence (pA) using either the AdZ5 recombination system or EnAd. AdZ5 is a replication deficient recombinant adenovirus with E1/E3 deletions. EnAd is a conditionally-replicating chimeric group B adenovirus that contains frequent nonhomologous nucleotide (non-homologous NT) substitutions of Ad3 for Ad11p. In addition, it has a nearly complete E3 deletion and a smaller E4 deletion mapped to E4orf4. Concentrated supernatants of HEK293 cells 48 hours after infection with enzyme-expressing AdZ5 viruses were each tested for their ability to degrade their corresponding substrate. (b) aDNAse I degradation of calf thymus DNA was assessed by agarose gel electrophoresis. (1) Undigested DNA, (2) DNA digested with recombinant DNAse, (3) DNA digested with supernatant (S/N) from uninfected cells, (4) DNA digested with supernatant from AdZ5-infected cells, (5) DNA digested with supernatant from AdPH20 infected cells, (6) DNA digested with supernatant from AdDNAse-infected cells. (c) Agarose gel electrophoresis-stained patterns of hyaluronidase degrading hyaluronic acid (HA). (1) Undigested HA, (2) HA digested with supernatant from uninfected cells, (3) HA digested with supernatant from AdZ5 infected cells, (4) HA digested with supernatant from AdPH20-infected cells. (d) Degradation of chondroitin sulfate by chondroitinase ABC measured by UV absorbance. Mean ± SD is plotted (n = 3). (e) Liberation of Folin-positive amino acids as a result of proteinase K hydrolysis of hemoglobin denatured with urea; tyrosine standard (Std), proteinase K (PK ctrl), uninfected cells (Ctrl). Mean ± SD is plotted (n = 3).