A3B introduces strand-coordinated C to T clustered mutations in the adenovirus genomes.

HBEC-WT, -GFP, -A3B and -A3B-DD were infected with HAdV-A12, -B3 or -C2 and the intracellular DNA was extracted 48 hours post infection. 3DPCR reactions were conducted on the E1B, L3 and E4 genes. (A) Agarose gels illustrating 3DPCR reactions specific for the E4 gene of the A12 strain are shown. The dotted white line indicates the threshold between mutated and unmutated 3DPCR products. Low denaturation temperature amplicons were recovered in HBEC-A3B cells only. (B) Graphical representation of the 3DPCR results obtained for the 3 viral genes (E1B, L3 and E4), for the 3 viral strains (A12, B3 and C2) and for the 4 cell lines (WT, GFP, A3B and A3B-DD). The lowest denaturation temperatures allowing the production of the expected amplicons were represented on the gradients by colored circles. (C) 3DPCR products generated from HAdV-A12-infected WT and A3B-expressing cells were cloned and sequenced. Hypermutated sequences detected in A3B-expressing cells (A3B) were aligned against the reference viral genome (Ref) and against sequences isolated from WT infected cells (WT). Deamination occurred on the coding (C to T mutations) or the template strand (G to A mutations of the coding strand). The mutations underlined took place within an A3-favored motif with a T in 5’ of the deaminated C. (D) Mutation types recorded on the E1B, L3 and E4 hypermutated sequences. The numbers in brackets indicate the number of bases sequenced. (E) 5’ nucleotide contexts of the deaminated Cs recorded on the E1B, L3 and E4 hypermutated sequences were reported by red bars. 5’ nucleotide context expected values, based on the dinucleotide composition of the DNA sequences were represented by white bars. P-values were calculated by χ2-tests.