Retroviral integration.

<p><i>(A)</i> Viral cDNA is depicted by a thin line and host target DNA is indicated by a thick line. Base pairs in the host target DNA are numbered. The HIV LTR ends are covalently joined to the target DNA 5 base pairs apart. The intervening host DNA denatures yielding an integration intermediate with two 5 base pair gaps. <i>(B)</i> The sequence preference observed at HIV integration sites. The numbering is identical to (A) and the points of joining are indicated by “IN”. Base pairs in green are favored and base pairs in red are disfavored at HIV integration sites.</p

Oxidative damage during HIV infection.

<p><i>(A)</i> OGG1 and MYH both recognize 8-oxo-dG damage (shown as <i>G_o</i>). OGG1 repairs 8-oxo-dG to G. Replication of 8-oxo-dG results in an 8-oxo-dG:A mispair, which is recognized by MYH. The MYH glycosylase initiates repair of the mispaired A to C yielding an 8-oxo-dG:C base pair. The product of the MYH repair reaction must still be repaired by OGG1. <i>(B)</i> Wild type cells were infected with an HIV based vector expressing GFP. Cells were treated with increasing concentrations of H₂O₂ immediately prior to infection. Viability in the absence (open diamonds) or presence of HIV (closed diamonds) was measured by trypan blue exclusion. Error bars indicate the standard deviations from three independent experiments.</p

G:C content surrounding HIV integration sites.

<p>The percentage of HIV integration sites compared to the percentage of G:C base pairs within a 5 kb window is shown for wild type cells compared to (<b>A</b>) <i>OGG1</i> null cells, (<b>B</b>) <i>MYH</i> null cells, (<b>C</b>) wild type cells treated with 10 µM H₂O₂, and (<b>D</b>) wild type cells treated with 30 µM H₂O₂. The frequency of G:C base pairs in the mouse genome is 0.41. There is no significant difference between HIV integration sites in untreated wild type cells and <i>OGG1</i> null (p = 0.96), <i>MYH</i> null (p = 0.99), 10 µM H₂O₂ treated (p = 0.67), or 30 µM H₂O₂ treated cells (p = 0.88).</p

HIV integration near genomic elements.

<p>HIV integration sites were mapped to genomic elements in <i>OGG1</i> null cells, <i>MYH</i> null cells, untreated wild type cells, and wild type cells treated with 10 µM or 30 µM H₂O₂. (<b>A</b>) HIV has a preference for integration to transcription units. (<b>B</b>) HIV shows no preference for integration to promoters. (<b>C</b>) There is no preference for HIV integration within 5 kb of CpG islands.</p

Effects of 8-oxo-dG specific glycosylases or hydrogen peroxide on HIV integration site sequence preference.

<p>Wild type, <i>OGG1</i> null, <i>MYH</i> null, and wild type cells treated with 10 µM or 30 µM H₂O₂, were infected with an HIV based retroviral vector at 0.8 MOI. 10 µM H₂O₂ is less than the 50% lethal dose for the wild type cells and 30 µM H₂O₂ is greater. After 7 days, genomic DNA was purified. HIV integration sites were subcloned, sequenced, and mapped to the mouse genome. The random frequency of G or C in the mouse genome is 0.205 and A or T is 0.295. The differences in observed base frequencies relative to the random frequencies are shown. Base numbering relative to the HIV points of joining is as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103164#pone-0103164-g001" target="_blank">Figure 1</a>. Boxes indicate the known HIV integration base preferences for wild type cells. Green, red, and gray highlights indicate a statistically significant difference of >0.10 from random frequency with a p value of <0.005. Green highlights are positive differences and red highlights are negative previously published palindromic prefered bases. Deletion of the <i>OGG1</i> gene leads to a loss of HIV sequence preference at positions −2 and −1. Deletion of the <i>MYH</i> gene also leads to loss of the HIV sequence preference at positions −2 and 7. While treatment of wild type cells with 10 µM H₂O₂ did not dramatically alter the sequence preference at integration sites, treatment with 30 µM H₂O₂ led to the disfavor of C at position −3 and disfavor of G at position 8, highlighted in red.</p