Trex1 degrades retroviral cDNA. (A) Schematic of circularization of retroelement. As a nonfunctional by-product, retroelement cDNA is circularized by the cell's DNA repair system. The circles are covalently closed. Red and green arrows represent PCR primers used to amplify the part of circular DNA that contains the site where the two LTR ends of the linear sequence are joined to form a circle. (B) Sequences of 2-LTR amplified from NIH/3T3 cells and mouse embryonic fibroblasts (MEF) that are Trex1 sufficient or deficient (Trex1 k.o.) 24 h after infection with MoMLV. Sequences are from the 0.5-kb band that contains the 2-LTR junction (X): (red) inverted repeat sequences, (green) Cru sequences from NIH/3T3 cells. Cru6 and Cru8 have the prototype sequence with perfect joint; dots indicate nucleotides missing, as compared to prototype sequence. “MEF Trex1-sufficient” sequences are from MEF Trex1 KO cells transduced with Trex1 using a vector based on the mouse stem-cell virus. Numbers in green are from cells not treated with raltegravir; in blue, with raltegravir. From Trex1-sufficient cells, the 14 sequences with insertions/mutations/deletions (i.e., nonprototype) plus one prototype are displayed, of 46 total; from Trex1 KO cells, the 13 nonprototype sequences plus one prototype, of 45 total. Nucleotides in light blue are insertions/point mutations. (C) Proposed schematic of 3′ end-processing by integrase, followed by Trex1 degradation. The processed ends identify the DNA as generated by reverse transcriptase.

Accumulation of preintegration circular DNA of endogenous retrovirus in B/W leukemia cells cultured with raltegravir. (A) One-letter amino acid sequence of part of the proline-rich region of the gp70 envelope protein of various MLVs found in B/W leukemia. NZBX, NZB xenotropic virus NZB-9–1; PMV, polytropic murine virus; MPMV, modified polytropic murine virus; MCF, mink cell focus-forming virus. Protein sequences were deduced from the sequence of RT-PCR-amplified mRNAs isolated from peripheral blood lymphocytes. Dashes indicate identity to xenotropic NZB-9–1. (B) Amplification, with NZB primers (NZB primers A), of circular DNA isolated from NYC cells without (NYC−) or with (NYC+) raltegravir, all done in duplicates for each sample of circular DNA isolation triplicates. SacI, SacI digest of circular DNA from NYC− and NYC+ cells, to show similar input; SacI linearizes the 16.3-kb mitochondrial DNA and digests circular DNA of various sizes.

Raltegravir activity on MLV. (A) Inhibition of MLV integration into host DNA by raltegravir. Flow cytometry graphs displaying GFP intensity: y axis, cell number; x axis, fluorescence intensity on a logarithmic scale. A MoMLV-based vector encoding GFP was added to NIH/3T3 cells with 0-, 10-, 30-, 100-, or 300-nM raltegravir. (B) Amplification of circular DNA isolated from MEF cells infected with MoMLV, done in duplicates; primers used covered sequences of MoMLV (primers M). MoV was transduced with MoMLV in the presence (+) or absence (−) of raltegravir in Trex1-sufficient (Trex +) or -deficient (Trex −) cells.

Effect of raltegravir on B/W and NZB mice. (A) Kaplan–Meier survival curve of B/W mice fed raltegravir. Cohorts of 14 mice fed (red curve; exact 95% confidence limits in magenta) and 11 mice not fed (black curve) with raltegravir. y axis, percent survival; x axis, age of mice, in days. (B) Mean fluorescence intensities in flow cytometry of RBC from individual mice (B/W mice from the cohorts, as shown in A) reacted with anti-IgG antibodies as a function of age of mice, in weeks (23 and 33 weeks): (red circles) individual mice fed with raltegravir (B/W+, 14 mice; B/c+, 10 mice; and NZW+, 4 mice); (black circles) without raltegravir (B/W−, 11 mice; RAG−, 3 mice; BL/6−, 3 mice; B/c−, 5 mice; and NZW−, 4 mice); (bar) mean fluorescence intensities of RBC, averaged over all mice in the cohort. B/W, female B/W mice; RAG, RAG-2-deficient mice on C57BL/6 background; BL/6, C57BL/6 mice; B/c, BALB/c mice; NZW, NZW mice. y axis, relative fluorescence intensity. (C) Development of anti-RBC reactivity over time in individual NZB mothers fed raltegravir (red curves) or not (black curves). y axis, mean fluorescence intensities in flow cytometry of RBC reacted with anti-IgG; x axis, age of mice, in weeks. Data taken from Table S1.