RESUMEN
Two hypermutated genomes of hepatitis B virus (HBV) were cloned from sera of chronic virus carriers. Twelve percent and 26% of guanosine residues were replaced by adenosine, with the transitions being erratically distributed along the genome. G-->A substitutions showed a strong dinucleotide preference, decreasing in the order GpA > GpG > > GpC > or = GpT. Such traits are typical of retroviral G-->A hypermutation which results from cDNA synthesis coinciding with fluctuations in the intracellular [dTTP]/[dCTP] ratio. The observations offer an explanation for the high prevalence of HBV variants bearing a tryptophan 28-->stop codon in the pre-core region of carriers with chronic active or fulminant hepatitis. The HBV hypermutants indicate that a small proportion of hepatocytes have distorted dNTP pools, which might have implications for the fidelity of hepatocyte DNA replication or repair.
Asunto(s)
Adenina , Portador Sano/virología , Genoma Viral , Guanina , Virus de la Hepatitis B/genética , Hepatitis B/virología , Mutación Puntual , Secuencia de Bases , Cartilla de ADN , ADN Viral/química , Fosfatos de Dinucleósidos/análisis , Fosfatos de Dinucleósidos/química , Mutación del Sistema de Lectura , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Alineación de Secuencia , Eliminación de SecuenciaRESUMEN
The effects of deoxynucleoside triphosphate (dNTP) imbalances on the fidelity of human immunodeficiency virus type 1 (HIV-1) replication were investigated. Using detergent permeabilized virions and biased dNTP concentrations different types of hypermutants were readily produced. However, the mutant spectrum was different from naturally occurring hypermutants demonstrating that the host cell may restrict variation. Using a genetic screen based on the blue/white beta-galactosidase complementation assay, G --> A hypermutants were recovered from HIV-infected thymidine treated U937 cells. Furthermore, hypermutants were recovered from 1 to 2% of resting or activated peripheral blood mononuclear cells indicating that small proportions of primary cells had distorted intracellular [dTTP] and [dCTP]. Such imbalances may underlie a proportion of somatic and germline point mutations and shape to some extent the evolution of mammalian and viral genomes.
Asunto(s)
Variación Genética , VIH-1/genética , Precursores de Ácido Nucleico/genética , Secuencia de Bases , Línea Celular , Desoxirribonucleótidos/genética , Humanos , Líquido Intracelular/metabolismo , Líquido Intracelular/virología , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Datos de Secuencia Molecular , Monocitos/metabolismo , Mutagénesis , Virión/genéticaRESUMEN
The evolution of human immunodeficiency virus (HIV) type 1 nef quasispecies in a patient clonally infected with a contaminated batch of blood clotting factor IX was monitored. nef sequences were derived at 11, 25, and 41 months postinfection from infected peripheral blood mononuclear cells after molecular cloning of PCR-amplified proviral DNA. The phylogenetic relationships among a total of 41 informative sequences were established by split decomposition analysis and used as a basis to establish a substitution matrix and to score synonymous (s) and nonsynonymous (ns) substitutions. The number of observed in-phase stop codons within the nef sequences was comparable to that expected on a random basis. Similarly, the numbers of observed s and ns substitutions did not differ significantly from expected values. No codon position was preferentially mutated. The maximum sequence divergence increased in a linear manner, with approximately 4.4 nucleotide and approximately 3.2 amino acid changes per year. It appears that stochastic processes strongly influence short-term HIV nef quasispecies evolution in vivo.
Asunto(s)
Frecuencia de los Genes , Genes nef , Infecciones por VIH/microbiología , VIH-1/genética , Secuencia de Aminoácidos , Evolución Biológica , Hemofilia A/microbiología , Humanos , Masculino , Datos de Secuencia Molecular , FilogeniaRESUMEN
The vigorous CTL response directed against HIV is considered to be important in reducing HIV viral load, although it is unable to stop ongoing viral replication, which generates new antigenic variants. We analyzed the impact of sequential changes in five epitopes of HIV-1 Nef on CTL recognition in four stable patients. A high rate of variation was found, and in all these patients we could detect CTL specific for 32 out of 36 autologous viral variants occurring in 5 HLA-A2- or HLA-B7-restricted Nef epitopes at two time points. Two distinct patterns for dynamics of CTL responses to viral variation were observed: 1) temporary amplification of viral variants followed by expansion of variant-specific CTL, ultimately leading to the disappearance of 12 out of the 14 initial epitope variants within two years. A second set of viral variants that had replaced the initial ones could also stimulate specific CTL precursors in the context of the same or an alternative HLA molecule; and 2) persistence of 2 viral variants in relatively conserved epitopes despite specific CTL recognition. Therefore, a remarkable flexibility of the immune system allows constant adaptation of CTL to multiple HIV variants and thus elimination of HIV variant-producing cells in slow progressors.
Asunto(s)
Variación Antigénica/genética , Productos del Gen nef/inmunología , Genes nef , VIH-1/genética , Linfocitos T Citotóxicos/inmunología , Secuencia de Aminoácidos , Citotoxicidad Inmunológica , ADN Viral/genética , Progresión de la Enfermedad , Epítopos/genética , Epítopos/inmunología , Estudios de Seguimiento , VIH-1/inmunología , Antígenos HLA-A/inmunología , Humanos , Provirus/genética , Selección Genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) replication is shown to be sensitive to the intracellular concentration of deoxynucleoside triphosphate substrates. Addition of thymidine to established cell lines resulted in a dramatic reduction of virus production. The effect could be substantially alleviated by addition of deoxycytidine, which, alone, enhanced viral titers by a factor of 2 to 3. Hydroxyurea treatment abolished HIV-1 replication in peripheral blood mononuclear cells and could be reversed by deoxyadenosine. These data show that HIV-1 replication occurs under suboptimal DNA precursor conditions.