Multiple-Sequence Alignment of Protein Sequences from XMRV and Related MuLVs Spanning SU Glycoprotein VRA, VRB, and VRC, Known to Determine Receptor Specificity

<p>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 [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#ppat-0020025-b077" target="_blank">77</a>] 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.</p

Detection of XMRV Protein in Prostatic Tissues Using Immunostaining

<p>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).</p

XMRV Detection by DNA Microarrays and RT-PCR

<p>(A) Virochip hybridization patterns obtained for tumor samples from 19 patients. The samples (<i>x-</i>axis) and the 502 retroviral oligonucleotides present on the microarray (<i>y-</i>axis) were clustered using hierarchical clustering. The red color saturation indicates the magnitude of hybridization intensity.</p> <p>(B) Magnified view of a selected cluster containing oligonucleotides with the strongest positive signal. Samples from patients with QQ <i>RNASEL</i> genotype are shown in red, and those from RQ and RR individuals as well as controls are in black.</p> <p>(C) Results of nested RT-PCR specific for XMRV <i>gag</i> gene. Amplified <i>gag</i> PCR fragments along with the corresponding human <i>GAPDH</i> amplification controls were separated by gel electrophoresis using the same lane order as in the microarray cluster.</p

Phylogenetic Analysis of XMRV Based on Complete Genome Sequences

<p>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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#s4" target="_blank">Materials and Methods</a>). An unrooted neighbor-joining tree was generated based on this alignment, excluding gaps and using Kimura's correction for multiple base substitutions. Bootstrap values (<i>n</i> = 1000 trials) are indicated as percentages. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E).</p

Detection of XMRV Nucleic Acid in Prostatic Tissues Using FISH

<p>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.</p

Comparison of XMRV Sequences Derived from Tumor Samples of Different Patients

<p>(A) Phylogenetic tree based on the 380 nt XMRV <i>gag</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#s4" target="_blank">Materials and Methods</a>). Bootstrap values (<i>n</i> = 1000 trials) are indicated as percentages. Sequences are labeled as xenotropic (X), polytropic (P), modified polytropic (Pm), or ecotropic (E).</p> <p>(B) Phylogenetic tree based on a 2500-nt <i>pol</i> PCR fragment from the 9 XMRV-positive tumor samples. The tree was constructed as described in (A).</p

Complete Genome of XMRV

<p>(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).</p> <p>(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 (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#ppat-0020025-st002" target="_blank">Table S2</a>) were derived either from the genome of MTCR (black arrows) or from overlapping VP35 clones (blue arrows).</p> <p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#s4" target="_blank">Materials and Methods</a>). The alignments were made using AVID [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#ppat-0020025-b081" target="_blank">81</a>], and plots were generated using mVISTA [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#ppat-0020025-b082" target="_blank">82</a>] with the default window size of 100 nt. <i>Y</i>-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).</p

Characterization of XMRV-Infected Prostatic Cells by FISH and FISH/Immunofluorescence

<p>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.</p> <p>(A) A stromal fibroblast.</p> <p>(B) A dividing stromal cell.</p> <p>(C) A stromal hematopoietic cell. The section was concomitantly stained for XMRV by FISH (green) and cytokeratin AE1/AE3 by immunofluorescence (red).</p

Multiple-Sequence Alignment of 5′ <i>gag</i> Leader Nucleotide Sequences from XMRV and Related MuLVs

<p>Sequences extending from the alternative CUG start codon to the AUG start codon (underlined) of <i>gag</i> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.0020025#s4" target="_blank">Materials and Methods</a>). 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.</p

Viral Discovery and Sequence Recovery Using DNA Microarrays

<div><p>Because of the constant threat posed by emerging infectious diseases and the limitations of existing approaches used to identify new pathogens, there is a great demand for new technological methods for viral discovery. We describe herein a DNA microarray-based platform for novel virus identification and characterization. Central to this approach was a DNA microarray designed to detect a wide range of known viruses as well as novel members of existing viral families; this microarray contained the most highly conserved 70mer sequences from every fully sequenced reference viral genome in GenBank. During an outbreak of severe acute respiratory syndrome (SARS) in March 2003, hybridization to this microarray revealed the presence of a previously uncharacterized coronavirus in a viral isolate cultivated from a SARS patient. To further characterize this new virus, approximately 1 kb of the unknown virus genome was cloned by physically recovering viral sequences hybridized to individual array elements. Sequencing of these fragments confirmed that the virus was indeed a new member of the coronavirus family. This combination of array hybridization followed by direct viral sequence recovery should prove to be a general strategy for the rapid identification and characterization of novel viruses and emerging infectious disease.</p> </div