Effects of A3G, A3F, and mA3 proteins on XMRV infection in single-replication-cycle assay. (A and D) Schematic outline of the experimental design, along with the proviral plasmids used in the experiment. XMRV was produced upon transfection of XMRV infectious molecular clone VP62 with (A) and without (D) MSCV-IRES-Luc reporter plasmids, in the presence or absence of A3G, A3F, or mA3 in 293T cells. The viruses produced were used to infect LNCaP prostate carcinoma cells, and the relative infectivity of the virus was measured by determining firefly luciferase activity in infected cells (A) or by PCR amplifying and sequencing the proviral DNA (D). CMV, cytomegalovirus promoter; R, repeat; U₅, unique 5′; GAG-PRO-POL, GAG-protease-polymerase polyprotein gene; ENV, envelope gene; U₃, unique 3′; LTF, long terminal repeat; Ψ, packaging signal; IRES, internal ribosomal entry site; Luc, luciferase gene. (B) VP62, the MSCV-IRES-Luc plasmid, and different amounts of A3G, A3F, and mA3 expression plasmids were transfected in 293T cells; 48 h posttransfection, the viruses were used to infect LNCaP cells in triplicate. The infected LNCaP cells were lysed 48 h posttransfection, and the luciferase activity in the cell lysates was determined. The luciferase activity in the vector control was set to 100%. The asterisks indicate that luciferase activity was less than 1% of that of the vector control. The error bars show the standard deviations of the luciferase activity observed in three independent experiments. (C) Western blot analysis of the incorporation of A3G, A3F, or mA3 protein in XMRV virions. Virus particles were produced in 293T cells by transient transfection of VP62 and MSCV-IRES-Luc in the presence of either 0.5, 1, or 2 μg of A3G expression plasmid; 1, 2, or 4 μg of either A3F or mA3 expression plasmid; or 4 μg of an empty-vector plasmid. The viruses from the supernatant were collected 48 h posttransfection, filtered, and concentrated by ultracentrifugation. At the same time point, the transfected cells were lysed. Equal amounts of cell lysate and equal volumes of concentrated virus were analyzed by immunoblotting them with anti-MLV capsid, anti-FLAG, or anti-HA antibodies. To ensure that equivalent aliquots were loaded onto the gels, the same lysates were analyzed using anti-tubulin antibody. (E) The extent of G-to-A hypermutation was analyzed in the presence of 0.5 μg A3G, 1 μg A3F, and 4 μg mA3. A schematic overview of the XMRV genome is shown, and the region sequenced (nt 2465 to 3660) is indicated with a red bar. The Hypermut (http://www.hir.lanl.gov/content/sequence/HYPERMUT/hypermut.html) color code was used to indicate the mutations: GG to AG in red, GA to AA in cyan, GC to AC in green, GT to AT in magenta, gaps in yellow, and all other mutations in black. In total, 2 of 11 clones were hypermutated in the presence of A3G, 5 of 22 with A3F, and 6 of 21 with mA3. (F) Total numbers of mutations and their occurrence in different dinucleotide contexts. The numbers in parentheses indicate percentages of total G-to-A mutations.

A3G and A3F mRNA levels and A3G protein expression in prostate cancer and T-cell lines. (A) The A3G and A3F mRNA copy numbers were determined by real-time RT-PCR assays and normalized to PBGD. The reactions were prepared in duplicate, and the average and standard error of 3 independent extractions are shown. Copy numbers below the detection limit of 100 copies are marked with asterisks. (B) Equal amounts of protein (5 μg) from the prostate cancer cell lines 22Rv1, LNCaP, and DU145 and the human T-cell lines CEM, CEM-SS, and H9 were analyzed by Western blotting to detect A3G and α-tubulin, which served as a loading control.

Single-replication-cycle drug susceptibility assays. Phenotypic drug susceptibility testing for AZT (A), 3TC (B), ddI (C), d4T (D), ABC (E), TDF (F), raltegravir (G), and foscarnet (H) was performed with HIV-1 (pNLuc + VSV-G) (open circles) and XMRV (VP62+MSCV-IRES-Luc) (filled circles) reporter viruses in LNCaP cells. The intersections of the vertical lines with the drug concentration axis show the IC₅₀ for each curve. Representative graphs are shown. (I) The mean IC₅₀s and standard deviations were calculated using SIGMAPLOT 8.0 as described in Materials and Methods.

Rare hypermutation in the XMRV 22Rv1 provirus. (A) Mutations in proportion to the XMRV genome that were found while sequencing XMRV-22Rv1. In total, 20 clones each of fragments 1 and 2 were sequenced completely. One clone of fragment 1 (clone 1.1) and 2 clones of fragment 2 (clones 2.1 and 2.2) were found to be hypermutated. The Hypermut color code is described in the legend to Fig. 1. (B) The total number and the sequence context of mutations are shown for each clone. Most of the mutations occurred in the GA dinucleotide context.

XMRV hypermutation in prostate cancer and T-cell lines. Mutations found in 22Rv1 (A), LNCaP (B), DU145 (C), CEM-SS (D), CEM (E), and H9 (F) cells in comparison to the XMRV-22Rv1 consensus sequence are marked with vertical bars at their positions. The Hypermut color code is described in the legend to Fig. 1. Below each set of sequences, a detailed table with the total numbers of single-nucleotide mutations is shown.