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328 Hypermutation of HIV-1 Genomes Follows a Defined Pattern Consistent with Dual Initiation Points for First-Strand Viral DNA Synthesis
M. Janini*1, J. Carr1, D. Birx2, and F. McCutchan1
1Henry M. Jackson Fndn., Rockville, MD, USA and 2Walter Reed Army Inst. of Res., Rockville, MD
Background: Hypermutation, which involves excessive G to A substitutions in the dinucleotide context GA or GG, has been sporadically observed in HIV-1 proviral sequences from patient PBMC and virus cultures. Hypermutation occurs when HIV-1 infect T cells in the first few hours after activation when nucleotide pools are being augmented and balanced in preparation for cycle. The majority of reports on HIV-1 hypermutation have focused on short genome fragments, contributing to the notion that hypermutation is erratic; parameters of hypermutation have rarely been studied in whole genomes.
Methods: Full-length HIV-1 genomes were amplified from randomly selected patient PBMC DNA by nested PCR. Amplified genomes were fully sequenced using dye terminators and automated sequencers. The parameters of hypermutation were evaluated with the Hypermut Program Package as implemented at the Los Alamos HIV Sequence Database, Los Alamos, NM.
Results: One full-length normal and one full-length hypermutated HIV-1 sequence was obtained from each of 6 patients infected with subtypes B,C, D (2), CRF01_AE, and a novel CRF01_AE/C recombinant, respectively. G to A replacement in the hypermutated genomes ranged from 19 to 32%. Hypermutation was uneven across the genome and showed a highly consistent pattern. Regardless of the overall level of hypermutation, it was lowest at the 5 end, increasing progressively until the central polypurine tract, where it dropped abruptly to baseline, again rising progressively until the 3 polypurine tract. Although rich in hypermutation-sensitive GA and GG dinucleotides, both polypurine tracts were completely spared.
Conclusions: Hypermutation, far from erratic, is generated by a precise mechanism that modulates intensity across the genome in a consistent pattern. Our results suggest that, contrary to current concepts, there could be dual initiation points for first-strand DNA synthesis, at least under some circumstances. A consistent temporal relationship between nucleotide pool levels, stage of T-cell activation, and progression of first-stand DNA synthesis would explain the observed hypermutation pattern.
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