Introduction Type I interferons (IFNs) are rapidly mobilized in response to viral infection and trigger potent antiviral responses. One such response is the induction by IFN of a family of 2′5′ oligoadenylate synthetases (OAS); upon activation by virally encoded dsRNA, these enzymes produce 5′-phosphorylated 2′-5′ linked oligoadenylates (2–5A) from ATP . 2–5A, in turn, is an activator of ribonuclease L (RNase L) , which degrades viral (and cellular) single stranded RNAs . In vivo evidence for the antiviral role of the 2–5A system was provided by studies with RNase L−/− mice, which have enhanced susceptibility to infections by the picornaviruses, encephalomyocarditis virus, and Coxsackievirus B4 [4,5]. Ultimately, sustained activation of RNase L triggers a mitochondrial pathway of apoptosis that eliminates virus-infected cells [4,6–8]. Genetic lesions in RNase L impair this apoptotic response, which has raised interest in the possibility that such mutations might also contribute to malignancy . In this context, several recent studies have linked germline mutations in RNase L to prostate cancer susceptibility [10–13]. Prostate cancer has a complex etiology influenced by androgens, diet, and other environmental and genetic factors . While sporadic prostate cancer displays an age-related increase in prevalence, familial prostate cancer kindreds often display early-onset disease. Such kindreds, defined by having more than three affected members per family, account for 43% of early onset cases ( 98% nt identity overall and >99% amino acid (aa) identity for predicted open reading frames (ORFs), and thus represent the same virus. The full genome of the virus (Figures 2 and S1) is 8,185 nt long and is distinct from all known isolates of MuLV. The genome is most similar to the genomes of exogenous MuLVs, DG-75 cloned from a human B-lymphoblastoid cell line , and MTCR, with which it shares 94% and 93% overall nt sequence identity, respectively. The genome also shares up to 95% nt identity with several full-length Mus musculus endogenous proviruses (Figure 2C). Phylogenetic trees constructed using available mammalian type C retroviral genomes and representative full-length proviral sequences from the mouse genome (Figures 3 and S2) showed that the newly identified virus is more similar to xenotropic and polytropic than to ecotropic genomes. Based on these findings we propose the provisional name Xenotropic MuLV-related virus (XMRV) for this agent. Translation of the XMRV genomic sequence using ORF Finder  identified two overlapping ORFs coding for the full-length Gag-Pro-Pol and Env polyproteins. No exogenous coding sequences, such as viral oncogenes, could be detected in the XMRV genome. The predicted Gag polyprotein is 536 aa long and is most similar to a xenotropic provirus on M. musculus Chromosome 9, with which it shares 97% aa identity (Figure S2A). The Pro-Pol polyprotein is 1,197 aa long and has the highest aa identity with MuLV DG-75 and a xenotropic provirus on M. musculus Chromosome 4, 97% and 96%, respectively (Figure S2B). An amber (UAG) stop codon separates the Gag and Pro-Pol coding sequences, analogous to other MuLVs in which a translational read-through is required to generate the full-length Gag-Pro-Pol polyprotein (reviewed in ). Similar to other MuLVs [23,24,27–31], the Env polyprotein of XMRV is in a different reading frame compared with Gag-Pro-Pol. The Env protein sequence is 645 aa long, and has the highest amino acid identity with the Env protein of an infectious MuLV isolated from a human small cell lung cancer line NCI-417  and MuLV New Zealand Black 9–1 xenotropic retrovirus (NZB-9–1) ), 95% and 94%, respectively. The XMRV Env protein also shares similarly high identity with several murine xenotropic proviruses (Figure S2C). Conserved splice donor (AGGTAAG, position 204) and acceptor (CACTTACAG, position 5,479) sites involved in the generation of env subgenomic RNAs  were found in the same relative locations as in other MuLV genomes. A multiple sequence alignment of XMRV Env and corresponding protein sequences of other representative MuLVs (Figure 4) showed that within three highly variable regions (VR), VRA, VRB, and VRC, known to be important for cellular tropism [34–36], XMRV has the highest aa identity with xenotropic envelopes from MuLVs NZB-9–1, NFS-Th-1 , and DG-75. Although unique-to-XMRV aa are present in each of the three VRs, based on the overall similarity to the known xenotropic envelopes, we predict that the cellular receptor for XMRV is XPR1 (SYG1), the recently identified receptor for xenotropic and polytropic MuLVs [38–40]. The long terminal repeat (LTR) of XMRV is 535 nt long and has the highest nt identity with the LTRs from xenotropic MuLVs NFS-Th-1 (96%) and NZB-9–1 (94%). The XMRV LTRs contain known structural and regulatory elements typical of other MuLV LTRs [33,41]. In particular, the CCAAT box, TATAAAA box, and AATAAA polyadenylation signal sequences were found in U3 at their expected locations (Figure S3A). U3 also contains a glucocorticoid response element sequence AGA ACA GAT GGT CCT. Essentially identical sequences are present in genomes of other MuLVs. These elements have been shown to activate LTR-directed transcription and viral replication in vitro in response to various steroids including androgens [42–45]. In addition, presence of an intact glucocorticoid response element is thought to be the determinant of higher susceptibility to FIS-2 MuLV infection in male compared with female NMRI mice [46,47]. Despite these similarities, single nt substitutions unique to XMRV and an insertion of an AG dinucleotide immediately downstream from the TATA box are present in U3 (Figure S3A). Consistent with these findings, a phylogenetic analysis based on U3 sequences from XMRV and from representative xenotropic MuLV provirus groups [48,49] showed that XMRV U3 sequences formed a well-separated cluster most similar to the group containing NFS-Th-1 and NZB-9–1 (Figure S3B). The 5′ gag leader of XMRV, defined as the sequence extending from the end of U5 to the ATG start codon of gag, consists of a conserved non-coding region of ~200 nt, containing a proline tRNA primer binding site as well as sequences required for viral packaging [50,51] and the initiation of translation [52,53]. The non-coding region is followed by a ~270-nt region extending from the conserved CTG alternative start codon of gag. This region represents the most divergent segment of the genome compared with other MuLVs (Figures 5 and 2C). Unlike ecotropic MuLVs, where translation from this codon adds an ~90 aa N-terminal leader peptide in frame with the rest of the Gag protein, thus generating a glycosylated form of Gag , XMRV has a stop codon 53 aa residues downstream from the alternative start. Interestingly, both MuLV DG-75 and MTCR gag leader sequences are also interrupted by stop codons, and therefore are not expected to produce full-length glyco-Gag. Furthermore, a characteristic 24-nt deletion was present in this region of the XMRV genome, which is not found in any known exogenous MuLV isolate. However, a shorter deletion of nine nt internal to this region is present in the sequences of several non-ecotropic MuLV proviruses found in the sequenced mouse genome (Figure 5). In cell culture, expression of intact glyco-Gag is not essential for viral replication [55,56]. However, lesions in this region have been associated with interesting variations in pathogenetic properties in vivo [57–61]. For example, an alteration in ten nt affecting five residues in the N-terminal peptide of glyco-Gag was found to be responsible for a 100-fold difference in the frequency of neuroinvasion observed between CasFrKP and CasFrKP41 MuLV strains . In addition, insertion of an octanucleotide resulting in a stop codon downstream of the CUG start codon prevented severe early hemolytic anemia and prolonged latency of erythroleukemia in mice infected with Friend MuLV . While we do not yet know the pathogenetic significance of the lesions in XMRV glyco-Gag, the high degree of sequence divergence suggests that this region may be under positive selective pressure and therefore may be relevant to the establishment of infection within the human host. Association of XMRV Infection and R462Q RNASEL Genotype To further examine the association between presence of the virus and the R462Q (1385G->A) RNASEL genotype, we developed a specific nested RT-PCR assay based on the virus sequence recovered from one of the tumor samples (VP35, see above). The primers in this assay (Figure S1) amplify a 380-nt fragment from the divergent 5′ leader and the N-terminal end of gag. The RT-PCR was positive in eight (40%) of 20 examined tumors from homozygous (QQ) individuals. In addition, one tumor from a homozygous wild-type (RR) patient was positive among 52 RR and 14 RQ tumors examined (Figure 1 and Table 1). Interestingly, this case was associated with the highest tumor grade among all XMRV-positive cases (Table S3). PCR specific for the mouse GAPDH gene was negative in all samples (unpublished data), arguing strongly against the possibility that the tumor samples were contaminated with mouse nucleic acid. Collectively, these data demonstrate a strong association between the homozygous (QQ) R462Q RNASEL genotype and presence of the virus in the tumor tissue (p 98% nt and >98% aa identity to each other. In contrast, the fragments had 97% nt and >97% aa identity to each other. In contrast, the fragments had A) RNASEL variant using a premade TAQMAN genotyping assay (Applied Biosystems, Foster City, California, United States; Assay c_935391_1) on DNA isolated from peripheral blood mononuclear cells. Five nanograms of genomic DNA were assayed according to the manufacturer's instructions, and analyzed on an Applied Biosystems 7900HT Sequence Detection System instrument. Immediately after prostatectomies, tissue cores were taken from both the transitional zone (the site of benign prostatic hyperplasia, BPH) and the peripheral zone (where cancer generally occurs), snap-frozen in liquid nitrogen, and then stored at −80 °C. Remaining prostate tissue was fixed in 10% neutral buffered formalin, processed, and embedded in paraffin for later histological analyses. Frozen tissue cores were transferred from dry ice immediately to TRIZOL reagent (Invitrogen, Carlsbad, California, United States), homogenized with a power homogenizer or manually using a scalpel followed by a syringe, and total RNA was isolated according to the manufacturer's instructions. The prostate tissue RNA was then subjected to RNase-free DNase I (Ambion, Austin, Texas, United States) digestion for 30 min at 37 °C. The sample was then extracted with phenol and the RNA was precipitated with isopropanol overnight at −20 °C followed by centrifugation at 12,000 g for 30 min at 4 °C. Poly-A RNA was isolated from the DNase digested total RNA using the Oligotex mRNA Midi Kit (Qiagen USA, Valencia, California, United States) as instructed by the manufacturer. The poly-A RNA concentration was measured using the RIBOgreen quantitation kit (Molecular Probes, Invitrogen), and the samples were stored at −80 °C. Microarray screening. Virochip microarrays used in this study were identical to those previously described [20–22]. Prostate tumor RNA samples were amplified and labeled using a modified Round A/B random PCR method and hybridized to the Virochip microarrays as reported previously (Protocol S1 in ). Microarrays were scanned with an Axon 4000B scanner (Axon Instruments, Union City, California, United States) and gridded using the bundled GenePix 3.0 software. Microarray data have been submitted to the NCBI GEO database (GSE3607). Hybridization patterns were interpreted using E-Predict as previously described  (Table S1). To make Figure 1, background-subtracted hybridization intensities of all retroviral oligonucleotides (205) were used to cluster samples and the oligonucleotides. Average linkage hierarchical clustering with Pearson correlation as the similarity metric was carried out using Cluster (version 2.0) . Cluster images were generated using Java TreeView (version 1.0.8) . Genome cloning and sequencing. Amplified and labeled cDNA from the VP35 tumor sample was hybridized to a hand-spotted microarray containing several retroviral oligonucleotides, which had high hybridization intensity on the Virochip during the initial microarray screening. Nucleic acid hybridizing to two of the oligonucleotides (9628654_317_rc derived from MTCR: TTC GCT TTA TCT GAG TAC CAT CTG TTC TTG GCC CTG AGC CGG GGC CCA GGT GCT CGA CCA CAG ATA TCC T; and 9626955_16_rc derived from SFFV: TCG GAT GCA ATC AGC AAG AGG CTT TAT TGG GAA CAC GGG TAC CCG GGC GAC TCA GTC TGT CGG AGG ACT G) was then individually eluted off the surface of the spots and amplified by PCR with Round B primers. Preparation of the hand-spotted array, hybridization, probe recovery, and PCR amplification of the recovered material were carried out according to Protocol S1. The recovered amplified DNA samples were then cloned into pCR2.1-TOPO TA vector (Invitrogen), and the resulting libraries were screened by colony hybridization with the corresponding above oligonucleotides as probes. Hybridizations were carried out using Rapid-Hyb buffer (Amersham, Piscataway, New Jersey, United States) according to the manufacturer's protocol at 50 °C for 4 h. Eight positive clones were sequenced, of which two (one from each library; clones K1 and K2R1 in Figure 2A) were viral and had 94–95% nt identity to MTCR. To sequence the remainder of the VP35 genome as well as the entire genome from the VP42 tumor, we amplified fragments of the genome by PCR using either amplified (Round B) or unamplified (Round A) cDNA prepared for original Virochip screening. This was accomplished first using a combination of primers derived from the sequence of MTCR and earlier recovered clones of XMRV. The two overlapping fragments from VP62 were amplified by PCR from cDNA generated by priming poly-A RNA with random hexamers. All PCR primers are listed in Table S2. The amplified fragments were cloned into pCR2.1-TOPO TA vector (Invitrogen) and sequenced using M13 sequencing primers. Genome assembly was carried out using CONSED version 13.84 for Linux . PCR. Screening of tumor samples by gag nested RT-PCR was carried out according to Protocol S2. PCR fragments in all positive cases were gel-purified using QIAEX II gel extraction kit (Qiagen), cloned into pCR2.1-TOPO TA vector (Invitrogen), and sequenced using M13 sequencing primers. Pol PCR was carried out using amplified cDNA (Round B material) as the template. Sequence of the primers used for amplification (2670F, 3870R, 3810F, and 5190R) is listed in Table S2. Amplified products were gel-purified using QIAEX II gel extraction kit (Qiagen), and purified products were directly used for sequencing. Phylogenetic analyses. Xenotropic mERV Chromosome 1, xenotropic mERV Chromosome 4, and xenotropic mERV Chromosome 9 were chosen by BLAST querying the NCBI nr database with the complete XMRV genomes and selecting the most similar full-length proviral sequences, all of which happened to have xenotropic envelopes (Figure S2C). Polytropic mERV Chromosome 7 and polytropic mERV Chromosome 11 were chosen by selecting NCBI nr full-length proviral sequences with envelopes most similar to a prototype polytropic clone MX27 . Similarly, modified polytropic mERV Chromosome 7 and modified polytropic mERV Chromosome 12 were selected on the basis of similarity to a prototype-modified polytropic clone MX33 . U3 analysis was performed using previously described reference sequences: Mcv18, Mcv3, Mxv2, Mcv11, Mxv11, and HEMV18 ; CWM-T-15, CWM-T-15–4, CWM-T-25a, and CWM-T-25b . To generate the neighbor-joining tree of complete genomic sequences (Figure 3), the sequences were first manually edited to make all genomes the same length, i.e., R to R. The edited sequences were then aligned with ClustalX version 1.82 for Linux [78,79] using default settings. The tree was generated based on positions without gaps only; Kimura correction for multiple base substitutions  and bootstrapping with n = 1000 were also used. All other trees were generated as above, except sequences were first trimmed to the same length, gaps were included, and Kimura correction was not used, as using these parameters did not have any significant effect on the trees. Antibodies. Monoclonal antibody to SFFV Gag protein was produced from R187 cells (; ATCC: CRL-1912) grown in DMEM (Media Core, Cleveland Clinic Foundation, Cleveland, Ohio, United States) with 10% ultra-low IgG FBS (Invitrogen) until confluent. Conditioned media was collected every three days from confluent cultures. Five ml of conditioned media per preparation was centrifuged at 168 × g for 5 min at 4 °C. Supernatant was filtered through a 0.22-μm syringe filter unit (Millipore, Billerica, Massachusetts, United States) and concentrated 16-fold in an Amicon ultrafiltration unit with a 100-kDa molecular weight cutoff membrane (Millipore). Sodium azide was added to a final concentration of 0.02%. Concomitant XMRV FISH/cytokeratin immunofluorescence was performed using a mouse anti-cytokeratin AE1/AE3 (20:1 mixture) monoclonal antibody (Chemicon International, Temecula, California, United States) capable of recognizing normal and neoplastic cells of epithelial origin. FISH. The XMRV-35 FISH probe cocktail was generated using both 2.15-kb and 1.84-kb segments of the viral genome obtained by PCR with forward primer-2345, 5′ ACC CCT AAG TGA CAA GTC TG 3′ with reverse primer-4495, 5′ CTG GAC AGT GAA TTA TAC TA 3′ and forward primer-4915, 5′ AAA TTG GGG CAG GGG TGC GA 3′ with reverse primer-6755, 5′ TTG GAG TAA GTA CCT AGG AC 3′, both cloned into pGEM-T (Promega, Madison, Wisconsin, United States). The recombinant vectors were digested with EcoRI to release the viral cDNA fragments, which were purified after gel electrophoresis (Qiagen). The purified viral cDNA inserts were used in nick translation reactions to produce SpectrumGreen dUTP fluorescently labeled probe according to manufacturer's instructions (Vysis Inc., Des Plaines, Illinois, United States). Freshly baked slides of prostatic tissues or tissue microarray arrays with ~4-μm thick tissue sections were deparaffinized, rehydrated, and subjected to Target Retrieval (Dako, Glostrup, Denmark) for 40 min at 95 °C. Slides were cooled to room temperature and rinsed in H2O. Proteinase K (Dako) at 1:5000 in Tris-HCl (pH 7.4) was applied directly to slides for 10 min at room temperature. Adjacent tissue sections were also probed with SpectrumGreen dUTP fluorescently labeled KSHV-8 DNA (nts 85820–92789) as a negative control or, as a positive control with SpectrumGreen and SpectrumOrange labeled TelVysion DNA Probe cocktail (Vysis), specific for subtelomeric regions of the P and Q arms of human Chromosome 1 as a positive control to ensure the tissue was completely accessible to FISH. FISH slides were examined using a Leica DMR microscope (Leica Micro-Systems, Heidelberg, Germany), equipped with a Retiga EX CCD camera (Q-Imaging,Vancouver, British Columbia, Canada). FISH images were captured using a Leica TCS SP2 laser scanning confocal with a 63× oil objective numerical aperature 1.4 (Leica Micro-Systems) microscope. XMRV nucleic acids were visualized using maximum intensity projections of optical slices acquired using a 488-nm argon-laser (emission at 500–550 nm). TelVysion DNA Probes were visualized using maximum intensity projections of optical slices acquired using a 488-nm argonlaser (emission at 500–550 nm) and 568-nm krypton-argon-laser (emission at 575–680 nm). DAPI was visualized using maximum intensity projections of optical slices acquired using a 364–nm UV-laser (emission at 400–500 nm). Slides were subsequently washed in 2× SSC (0.3 M sodium chloride and 0.03 M sodium citrate, [pH 7.0]) to remove coverslips, and H&E stained for morphological evaluation. IHC. IHC on human tissues was performed on a Benchmark Ventana Autostainer (Ventana Medical Systems, Tucson, Arizona, United States). Unstained, formalin-fixed, paraffin-embedded prostate sections were placed on electrostatically charged slides and deparaffinized followed by a mild cell conditioning achieved through the use of Cell Conditioner #2 (Ventana Medical Systems). The concentrated R187 monoclonal antibody against SFFV p30 Gag was dispensed manually onto the sections at 10 μg per ml and allowed to incubate for 32 min at 37 °C. Endogenous biotin was blocked in sections using the Endogenous Biotin Blocking Kit (Ventana Medical Systems). Sections were washed, and biotinylated ImmunoPure Goat Anti-Rat IgG (Pierce Biotechnology, Rockford, Illinois, United States) was applied at a concentration of 4.8 μg per ml for 8 min. To detect Gag protein localization, the Ventana Enhanced Alkaline Phosphatase Red Detection Kit (Ventana Medical Systems) was used. Sections were briefly washed in distilled water and counterstained with Hematoxylin II (Ventana Medical Systems) for approximately 6 min. Sections were washed, dehydrated in graded alcohols, incubated in xylene for 5 min, and coverslips were added with Cytoseal (Microm International, Walldorf, Germany). Negative controls were performed as above except without the addition of the R187 monoclonal antibody. Concomitant XMRV FISH/cytokeratin IHC was performed on slides of prostate tissue from patient VP62. First, sections were immunostained for cytokeratin AE1/AE3 using the Alexa Fluor 594 Tyramide Signal Amplification Kit (Molecular Probes, Invitrogen). Briefly, unstained, formalin-fixed, paraffin-embedded sections cut at ~4 μm were placed on electrostatically charged slides, baked at 65 °C for at least 4 h, deparaffinized in xylene, and rehydrated through decreasing alcohol concentrations. Slides were incubated in Protease II (Ventana Medical Systems) for 3 min at room temperature and washed in phosphate-buffered saline (PBS) in peroxidase quenching buffer (PBS + 3% H2O2) for 60 min at room temperature, then incubated with 1% blocking reagent (10 mg/ml BSA in PBS) for 60 min at room temperature. The slides were incubated with cytokeratin AE1/AE3 antibody diluted in 1% blocking reagent for 60 min at room temperature and rinsed 3× times in PBS. Goat anti-mouse IgG-horseradish peroxidase (Molecular Probes, Invitrogen) was added and incubated for 60 min at room temperature. The slides were rinsed 3× in PBS. The tyramide solution was added to the slides for 10 min at room temperature and the slides were rinsed 3× in PBS. Slides were then placed in Target Retrieval solution (Dako) for 40 min at 95 °C. FISH for XMRV was performed as described above except in the absence of proteinase K treatment. After FISH, the slides were mounted with Vectashield Mounting Medium plus DAPI (Vector Labs, Burlingame, California, United States) and examined using fluorescence microscopy. Immunofluorescence images were captured using a Texas red filter with a Leica DMR microscope (Leica Micro-Systems), equipped with a Retiga EX CCD camera (QImaging). Supporting Information Figure S1 Complete Nucleotide Sequence of XMRV VP35 Numbers to the left indicate nt coordinates relative to the first nt. Predicted open reading frames for Gag, Gag-Pro-Pol, and Env polyproteins are shown below the corresponding nt. Characteristic 24-nt deletion in the 5′ gag leader is indicated with a triangle. Other genome features as well as primers used in the nested gag RT-PCR are shown as arrows. (558 KB PDF) Click here for additional data file. Figure S2 Phylogenetic Analysis of XMRV Based on Predicted Gag, Pro-Pol, and Env Polyproteins Predicted Gag (A), Pro-Pol (B), and Env (C) sequences of XMRV VP35, VP42, and VP62 (red) as well as the corresponding sequences from MTCR; MuLVs DG-75, MCF1233, Akv, Moloney, Friend, and Rauscher; feline leukemia virus (FLV); koala retrovirus (KoRV); gibbon ape leukemia virus (GALV), and a set of representative non-ecotropic proviruses (mERVs) were aligned using ClustalX. The resulting alignments were used to generate unrooted neighbor-joining trees (see Materials and Methods). Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). (186 KB EPS) Click here for additional data file. Figure S3 Comparison of XMRV U3 Region to Representative Non-Ecotropic Sequences (A) Multiple sequence alignment of U3 sequences from XMRV VP35, VP42, and VP62; MuLVs NZB-9–1 and NFS-Th-1; and from representative non-ecotropic proviruses [37,48,49]. The sequences were aligned using ClustalX (see Materials and Methods). Only sequences most similar to XMRV are shown. Glucocorticoid response element (GRE), and TATA and CAT boxes are indicated by lines. Direct repeat regions (boxed) are numbered according to the existing convention [37,49]. Triangle indicates a 190 nt insertion in polytropic proviruses . XMRV-specific AG dinucleotide insertion is shown in red. Dots denote nt identical to those from XMRV, and deleted nt appear as spaces. (B) Phylogenetic tree based on U3 nt sequences. Multiple sequence alignment from (A) was used to generate an unrooted neighbor-joining tree (see Materials and Methods). Bootstrap values (n = 1000 trials) are shown as percentages. U3 sequences from XMRV are shown in red. (188 KB EPS) Click here for additional data file. Protocol S1 Probe Recovery from Hand-Spotted Microarrays by “Scratching” (83 KB PDF) Click here for additional data file. Protocol S2 XMRV gag Nested RT-PCR (172 KB PDF) Click here for additional data file. Table S1 Computational Viral Species Predictions Using E-Predict for the Virochip Microarrays Shown in Figure 1 (48 KB DOC) Click here for additional data file. Table S2 PCR Primers Used for Sequencing of XMRV Genomes (45 KB DOC) Click here for additional data file. Table S3 Age, Clinical Parameters, and Geographical Locations of XMRV-Positive Prostate Cancer Cases (39 KB DOC) Click here for additional data file. Video S1 Confocal Optical Image Planes of a Representative XMRV FISH Positive Cell Optical image planes (0.5 μm step-size) of the XMRV FISH positive cell from Figure 1A acquired using a Leica TCS SP2 laser scanning spectral confocal microscope (Leica, Heidelberg, Germany) were reconstructed into a 3D volume set using Volocity 3.5 (Improvision, Lexington, Massachusetts, United States). Using Volocity's movie sequence editor, each volume was rotated along horizontal and vertical axes, adjusting nuclei stained DAPI (blue) channel brightness to visualize underlying XMRV FISH (green) nucleic acid signal. The resulting image frames were exported as a movie sequence. Underlying grid represents glass slide to which tissue was placed for FISH analysis. Each square unit within grid represents 4 μm in height and width. (237 KB WMV) Click here for additional data file. Accession Numbers Accession numbers from Gen Bank (http://www.ncbi.nlm.nih.gov/Genbank) are: AKV MuLV (J01998), feline leukemia virus (NC_001940), Friend MuLV (NC_001372), gibbon ape leukemia virus (NC_001885), koala retrovirus (AF151794), modified polytropic mERV Chromosome 7 (AC127565; nt 64,355–72,720), modified polytropic mERV Chromosome 12 (AC153658; nt 85,452–93,817), Moloney MuLV (NC_001501), MTCR (NC_001702MuLV DG-75 (AF221065); MuLV MCF 1233 (U13766), MuLV NCI-417 (AAC97875), MuLV NZB-9–1 (K02730), polytropic mERV Chromosome 7 (AC167978; nt 57,453–65,805), polytropic mERV Chromosome 11 (168–229,176,580), prototype polytropic clone MX27 (M17327), Rauscher MuLV (NC_001819), xenotropic mERV Chromosome 1 (AC083892, nt 158,240–166,448), xenotropic mERV Chromosome 4 (AL627077; nt 146,400–154,635), xenotropic mERV Chromosome 9 (AC121813; nt 37,520–45,770), XMRV VP35 (DQ241301), XMRV VP42 (DQ241302), and XMRV VP 62 (DQ399707).