Figure 1.
XMRV Detection by DNA Microarrays and RT-PCR
(A) Virochip hybridization patterns obtained for tumor samples from 19 patients. The samples (x-axis) and the 502 retroviral oligonucleotides present on the microarray (y-axis) were clustered using hierarchical clustering. The red color saturation indicates the magnitude of hybridization intensity.
(B) Magnified view of a selected cluster containing oligonucleotides with the strongest positive signal. Samples from patients with QQ RNASEL genotype are shown in red, and those from RQ and RR individuals as well as controls are in black.
(C) Results of nested RT-PCR specific for XMRV gag gene. Amplified gag PCR fragments along with the corresponding human GAPDH amplification controls were separated by gel electrophoresis using the same lane order as in the microarray cluster.
Figure 2.
(A) Schematic map of the 8185 nt XMRV genome. LTR regions (R, U5, U3) are indicated with boxes. Predicted open reading frames encoding Gag, Gag-Pro-Pol, and Env polyproteins are labeled in green. The corresponding start and stop codons (AUG, UAG, UGA, UAA) as well as the alternative Gag start codon (CUG) are shown with their nt positions. Similarly, splice donor (SD) and acceptor (SA) sites are shown and correspond to the spliced 3.2-Kb Env subgenomic RNA (wiggled line).
(B) Cloning and sequencing of XMRV VP35 and VP62 genomes. Clones obtained by probe recovery from hybridizing microarray oligonucleotides (blue bar) or by PCR from tumor cDNA (black bars) were sequenced. Primers used to amplify individual clones (Table S2) were derived either from the genome of MTCR (black arrows) or from overlapping VP35 clones (blue arrows).
(C) Genome sequence similarity plots comparing XMRV VP35 with XMRV VP42, XMRV VP62, MuLV DG-75, MTCR, and a set of representative non-ecotropic proviruses (mERVs) (see Materials and Methods). The alignments were made using AVID [81], and plots were generated using mVISTA [82] with the default window size of 100 nt. Y-axis scale for each plot represents percent nt identities from 50% to 100%. Sequences are labeled as xenotropic (X), polytropic (P), or modified polytropic (Pm).
Figure 3.
Phylogenetic Analysis of XMRV Based on Complete Genome Sequences
Complete genomes of XMRV VP35, VP42, and VP62 (red); MTCR; MuLVs DG-75, 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 (see Materials and Methods). An unrooted neighbor-joining tree was generated based on this alignment, excluding gaps and using Kimura's correction for multiple base substitutions. Bootstrap values (n = 1000 trials) are indicated as percentages. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E).
Figure 4.
Multiple-Sequence Alignment of Protein Sequences from XMRV and Related MuLVs Spanning SU Glycoprotein VRA, VRB, and VRC, Known to Determine Receptor Specificity
Env protein sequence from XMRV (identical in VP35, VP42, and VP62; red); MTCR; MuLVs DG-75, NZB-9–1, NFS-Th-1, MCF247, AKV, Moloney, Friend, and Rauscher; and polytropic proviruses MX27 and MX33 [77] were aligned using ClustalX. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). VRs are boxed. Dots denote residues identical to those from XMRV, and deleted residues appear as spaces.
Figure 5.
Multiple-Sequence Alignment of 5′ gag Leader Nucleotide Sequences from XMRV and Related MuLVs
Sequences extending from the alternative CUG start codon to the AUG start codon (underlined) of gag derived from XMRV VP35, VP42, and VP62 (blue); MTCR, MuLVs DG-75, and Friend; and a set of representative non-ecotropic proviruses (mERVs) were aligned with ClustalX (see Materials and Methods). Predicted amino acid translation corresponding to the VP35 sequence is shown above the alignment (red); asterisk indicates a stop. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E). Dots denote nt identical to those from XMRV, and deleted nt appear as spaces.
Table 1.
XMRV Screening by gag Nested RT-PCR
Figure 6.
Comparison of XMRV Sequences Derived from Tumor Samples of Different Patients
(A) Phylogenetic tree based on the 380 nt XMRV gag RT-PCR fragment from the nine positive tumor samples (red) and the corresponding sequences from MTCR; MuLVs DG-75, MCF1233, Akv, Moloney, Rauscher and Friend; and a set of representative non-ecotropic proviruses (mERVs). The sequences were aligned using ClustalX, and the corresponding tree was generated using the neighbor-joining method (see Materials and Methods). Bootstrap values (n = 1000 trials) are indicated as percentages. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E).
(B) Phylogenetic tree based on a 2500-nt pol PCR fragment from the 9 XMRV-positive tumor samples. The tree was constructed as described in (A).
Figure 7.
Detection of XMRV Nucleic Acid in Prostatic Tissues Using FISH
Prostatic tumor tissue sections from QQ cases VP62 (A–C) and VP88 (D–F) were analyzed by FISH using DNA probes (green) derived from XMRV VP35 (top right enlargements). Nuclei were counterstained with DAPI. The same sections were then visualized by H&E staining (left panels). Scale bars are 10 μm. Arrows indicate FISH positive cells, and their enlarged images are shown in the bottom right panels.
Figure 8.
Characterization of XMRV-Infected Prostatic Cells by FISH and FISH/Immunofluorescence
Using a tissue microarray, prostatic tumor tissue sections from QQ case VP62 were analyzed by FISH (green) using DNA probes derived from XMRV VP35 (left panels). Nuclei were counterstained with DAPI. The same sections were then visualized by H&E staining (middle panels). Arrows indicate FISH-positive cells, and their enlarged FISH and H&E images are shown in the top right and bottom right panels, respectively. Scale bars are 10 μm.
(A) A stromal fibroblast.
(B) A dividing stromal cell.
(C) A stromal hematopoietic cell. The section was concomitantly stained for XMRV by FISH (green) and cytokeratin AE1/AE3 by immunofluorescence (red).
Table 2.
Frequency of XMRV-Infected Prostatic Cells Determined by FISH
Figure 9.
Detection of XMRV Protein in Prostatic Tissues Using Immunostaining
Prostatic tumor tissue sections from QQ cases VP62 (A and B) and VP88 (C and D), as well as an RR case VP51 (E) were stained, then visualized by immunofluorescence (left) or bright field (middle) using a monoclonal antibody to SFFV Gag protein. Nuclei are counterstained with hematoxylin. Enlarged images corresponding to the positive cells are shown on the right. Scale bars are 5 μm in (A), (B), and (E) and 10 μm in (C) and (D).