Detalle de: Characterization of minority subpopulations in the mutant spectrum of HIV-1 quasispecies by successive specific amplifications

RNA viruses do not replicate as defined genomic nucleotide sequences but rather as complex distributions of mutant genomes termed viral quasispecies. Quasispecies dynamics has a number of relevant biological consequences in ribo- and retroviruses, among these the possible presence of memory genomes as minority components of their mutant spectra. Minority memory genomes reflect those viral subpopulations that were dominant at an earlier phase of viral evolution, and can quickly re-emerge to react to certain selective pressures, as it was documented with HIV-1 in vivo. Therefore, an adequate clinical management of HIV-1 requires the development of experimental methods for the detection and quantification of minority viral subpopulations, even at levels of less than 1% of the total quasispecies. We describe a new approach based on successive, highly specific PCR amplifications, which allows the genetic characterization of minority genomes present in increasingly smaller proportion in viral populations. We have coined the term 'quasispecies diving' to reflect the progressive draw on minority or 'deeper' genomes in the mutant spectrum of the quasispecies. In the case of the multidrug-resistant HIV-1 strain analyzed here, quasispecies diving allowed the detection of mutant minority genomes at an unprecedented level of 0.0054% of the amplified viral population. This approach represents a general strategy for the genetic characterization of smaller minority genomes in complex molecular populations.