Nucleosomal Arrangement of HIV-1 DNA: Maps Generated from an Integrated Genome and an EBV-based Episomal Model

• Published in: Journal of molecular biology 1996-03, Vol.256 (3), p.503-516
• Main Authors: Stanfield-Oakley, Sherry A., Griffith, Jack D.
• Format: Article
• Language: English
• Subjects: AIDS/HIV; B-Lymphocytes; Base Sequence; Cell Line; chromatin; DNA Primers; DNA, Viral - metabolism; Edetic Acid - analogs & derivatives; Epstein-Barr virus; Genes, gag - genetics; Genes, pol - genetics; Genome, Viral; Herpesvirus 4, Human - genetics; HIV; HIV-1 - genetics; human immunodeficiency virus 1; Humans; Molecular Sequence Data; MPE-iron (II); nucleosome; Nucleosomes; Plasmids - genetics; Proviruses - genetics; T-Lymphocytes
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1006/jmbi.1996.0104

ACH-2 cells, an immortalized human T-cell line, contain a single integrated copy of the HIV-1 provirus. Here, the structure of HIV-1 chromatin was probed using a DNA cleavage reagent. Nuclei were isolated from ACH-2 cells and treated with methidiumpropyl-EDTA (MPE)- iron(II) to produce limited DNA cleavage. Primers were selected at ∼300 bp intervals along the HIV-1 DNA, and sites of preferential cleavage were mapped by carrying out 50 cycles of primer extension using a thermo-stable DNA polymerase in the presence of [ 32P]dATP. By comparing the resulting cleavage pattern with patterns derived from human cell lines not containing HIV-1 sequences, it was possible to map the arrangement of nucleosomes across the integrated HIV-1 genome. Particularly regular spacing was seen in the 3′ end of the pol and env coding regions, and several extended blocks spared of nucleosomes were found in gag and pol, the largest being a |similar|450 bp region in gag. For comparison, and to examine nucleosome placement on HIV-1 DNA when it is not integrated, overlapping segments of HIV-1 DNA were cloned into an EBV-oriP plasmid, grown as stable episomes in a human B lymphoblastoid cell line, and the same analysis using MPE-iron(II) cleavage and primer extension carried out. The major features of nucleosome placement on these EBV/HIV minichromosomes was very similar to that observed in the integrated HIV-1 genome arguing for a strong sequence dependence for nucleosome placement along HIV-1 DNA.