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259 Nonfunctional Hypermutated HIV-1 DNA Sequences Resulting from Infection of Early Activated T Cells Are Abundant in Infected Patients
M. Janini*1, M. Rogers1, D. Birx2, and F. McCutchan1
1Henry M. Jackson Fndn., Rockville, MD, USA and 2Walter Reed Army Inst. of Res., Rockville, MD, USA
Background: HIV-1 G to A hypermutation is a mutational process in which G to A transitions far exceed all other mutations in viral sequences. There is a potential loss of informational content in sequences that are affected by hypermutation. HIV-1-infected patients have not been systematically evaluated for the presence of hypermutants, and the activation status of target cell types in which hypermutation occurs have not been defined.
Methods: An agarose gel electrophoresis system incorporating an AT-binding dye was developed to visualize and separate hypermutated and normal sequences in a 297-bp segment, encoding the protease gene, amplified by nested PCR from PBMC DNA from randomly selected patients. Amplified DNAs were cloned, sequenced, and analyzed for hypermutation and its parameters. Resting CD4+ T cells were infected with cloned NL4-3 virus stocks at various times before and after activation of cells with PHA. Cultures were sampled at intervals and the extracted DNA was evaluated for normal and hypermutated sequences as above.
Results: Among 53 patients, at least 43% of patients harbored abundant hypermutated, along with normal, protease genes. In 70 hypermutated sequences, replacement of G residues ranged from 20 to 94%. Other mutations were not elevated, and G to A replacement was entirely restricted to GA or GG dinucleotides. Sixty-nine of 70 hypermutated and 3 of 149 normal sequences had in-frame stop codons. Virus cultures indicated that hypermutation was pronounced when HIV-1 infection occurred simultaneously with, or a few hours after, PHA activation, but after 12 hours PHA or more, most HIV-1 sequences were normal. Hypermutated sequences generated in culture corresponded exactly in all parameters to those obtained from patient PBMC.
Conclusions: Nonfunctional hypermutated HIV-1 proviruses are abundant in infected patients. In cultures, generation of defective hypermutated genomes is influenced by the stage of the T-cell cycle at the moment of infection. Hypermutation reduces the pool of viable HIV-1 genomes and could potentially be exploited for clinical benefit.
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