Figure 1

Schematic of HAdV-3p E3-9K mutant virus genotypes. Mutant viruses were generated by recombination of bacmid pKSB2Ad3wt with pE3-9K shuttle plasmids carrying the desired mutations of ORF E3-9K, digestion of bacmid with Mlu I to release the mutated HAdV-3 genome, transfection into A549 cells, and propagation of resultant mutant virus as described in the Materials and Methods section. Recombinants contain Sal I and Bam HI restriction enzyme sites and a flippase recognition target (FRT) scar site. The HAdV-3p-WT control virus was generated by linearization of pKSB2Ad3wt by digestion with Mlu I without prior recombination or mutation. HAdV-3p-E-3-9K-rec, the recombination control virus, was generated by recombination of pKSB2Ad3wt with unaltered pE3-9K shuttle plasmid. An E3-9K knock out mutant, HAdV-3p-E3-9K-KO, was generated by recombination of pKSB2Ad3wt with a pE3-9K shuttle plasmid that contained TAG stop codons in place of all the in-frame ATG codons present in ORF E3-9K. HAdV-3p-E3-9K-NULL was generated by recombination of pKSB2Ad3wt with a pE3-9K shuttle plasmid carrying the HAdV-7h-encoded null version of this ORF (GenBank accession # Z48954 and AF321310).