Sequence and phylogenetic analysis of a novel xenotropic XMRV-like MLV B4rv, derived from the highly metastatic human prostate tumor xenograft-derived cell line C2-4 B4 . A. Hypermut plots for PreXMRV-1, Mus musculus chromosome Y BAC clones RP24-163J18 (AC175744.2) and RP24-320A8 (AC182253.3), Pre-B4rv and B4rv, where vertical lines indicate nucleotide mismatches relative to XMRV, demonstrating the likely proviral sequences from which B4rv originated. Red stars indicate a stop codon in env for AC175744.2 and a stop codon in gag for AC182253.3. Six template switches occurred between AC175744.2 and PreXMRV-1 to generate Pre-B4rv. Two template switches between Pre-B4rv and AC182253.3 generated the predicted B4rv recombinant. The predicted B4rv recombinant differs in sequence from B4rv by 21 nucleotide changes (18 nucleotide substitutions, one insertion (i) and two nucleotide deletions (d)). B4rv was identified based on representative difference analysis differential cloning comparing LNCaP human prostate tumor cells versus the C2-4B4 prostate tumor cell line indicated in (B). A schematic depicting the various LNCaP- and Xenograft-derived human prostate tumor cell lines is depicted in (Additional file 1: Figure S1). B. PCR for mouse-specific intracisternal A-type particle (lAP) long-terminal repeat sequences quantifying the relative amount of mouse DNA in the C4-2 B4 cell lines obtained from isolated genomic DNA of the sources indicated. The C4-2 B4 line outlined in red indicates the line from which B4rv was identified. C. Sequence alignment of B4rv and XMRV VP62 followed by a hypermut plot displaying the nucleotide mismatches of XMRV relative to B4rv. Red lines under the hypermut plot indicate regions of varying sequence identity between XMRV and B4rv, followed by a red line spanning the entire genome to indicate total sequence identity. D. Phylogenetic tree analysis of viruses in the MLV family, including xenotrophic MLVs, XMRV VP62 and B4rv, and proviral sequence PreXMRV-1.