The N and C termini of Xpr1 are located inside the cell membrane. BHK and E36 cells expressing Xpr1 with FLAG tag at the N terminus (FLAGXpr1) or HA tag at the C terminus (Xpr1HA) were established as described in Materials and Methods. (A) The expression of FLAG on the cell surface was detected by flow cytometry analysis after staining with FITC-conjugated anti-FLAG antibody for 1 h (areas under bold line). Shaded areas represent isotype controls. (B) Same as in panel A, but with anti-HA-Alexa Fluor 488 to detect Xpr1HA expression on the surface of BHKXpr1HA or E36Xpr1HA cells (areas under the bold line). (C) Western blots for FLAGXpr1 expression in E36, E36FLAGXpr1, BHK, and BHKFLAGXpr1 cells (lanes 1 to 4, respectively) were performed with anti-FLAG-HRP antibody. Molecular mass markers in kilodaltons are to the left. The same cell number equivalents of whole-cell lysates from each cell line were loaded. (D) As described for panel C, except that anti-HA monoclonal antibody was used to detect Xpr1HA expression in BHK, BHKXpr1HA, E36, and E36-Xpr1HA cells (lanes 1 to 4, respectively). (E) Predicted topology of Xpr1. Shown is a topological model for human Xpr1 obtained by the HMMTOP method with constrained prediction: the N and C termini of the protein are designated as intracellular, as experimentally validated by labeling with FLAG and HA tags, and the predicted receptor binding domains (35) are extracellular. The transmembrane regions are represented by the gray bars and numbered 1 through 8. The lines on top of the designated transmembrane regions correspond to extracellular regions, with dotted lines representing the proposed receptor binding domains.

PiT1 protein expressed on the surface of BHK cells shares a similar configuration to PiT1 expressed on susceptible MDTFPiT1 cells. The top lane shows a representative Western blot of BHK cell lysates expressing PiT1-13HA mutants containing the individual cysteine substitutions V19C, S97C, Q147C, G222C, V554C, S621C, and L677C reacted with B-mal. BHK cells expressing the functional but cysteineless PiT1-13HA mutant were used as a negative control for B-mal reactivity. MDTF cells expressing PiT1-13HA mutant S97C were used as a positive control. The same cell number equivalents of whole-cell lysates from each cell line were loaded. The bottom lane shows the topological model for SLC20A1 (PiT1) expressed in MDTF cells validated by SCAM experimentally (10). The gray bars represent transmembrane regions. The extracellular regions are numbered 1 through 7. Individual residues presented in Western blots are localized in each ECR outside the cell membrane of MDTF cells.

Hybrids formed between nonpermissive CHOK1 and BHKXpr1 cells are susceptible to XMRV infection. Retroviral vector pBABE-puro was used to make puromycin-resistant CHOK1 (CHOK1-puro) cells. BHK or CHOK1 cells exposed to pLNSX-Xpr1 were used to generate the G418-resistant BHKXpr1 and CHOK1Xpr1 cells. After fusion of CHOK1-puro with BHKXpr1, puromycin and G418 were used for double selection of the CHOK1/BHKXpr1 hybrid. CHOK1-puro, BHKXpr1, CHOK1/BHKXpr1, and CHOK1Xpr1 cells were infected with XMRV-enveloped retroviral vector expressing nuclear localized β-galactosidase. Micrographs were obtained with a 20× objective.

(A) PiT1 protein is expressed on the BHK cell surface. The expression of HA on the cell surface of BHK cells expressing PiT1HA was detected by flow cytometry after staining with Alexa Fluor 488-conjugated anti-HA antibody for 1 h (areas under bold line). Isotype controls are in shaded areas. (B) BHKPiT1HA can bind GALV envelope protein. V5-tagged GALV SU was used to bind BHK or BHKPiT1HA cells as described in Materials and Methods. FITC-conjugated anti-V5 antibody was used to detect GALV SU binding on the cell surface, as represented by the areas under the bold line; shaded areas represent isotype controls.

Expression of Xpr1 in BHK cells confers XMRV envelope protein binding. The binding of XMRV envelope to BHK or BHKXpr1 cells was determined with HA-tagged XMRV SU. HA antibody conjugated with Alexa Fluor 488 was used to detect XMRV binding on cell surface (areas under bold line). Shaded areas represent isotype controls.

Hybrids formed between nonpermissive MDTF and BHKPiT1 cells are susceptible to GALV infection. Hygromycin-resistant MDTF (MDTF-hygro) cells were generated by transducing MDTF cells with retroviral vector pLHCX, and BHKPiT1 and MDTFPiT1 cells were constructed with pLNSX-PiT1, which confers G418 resistance. The MDTF/BHKPiT1 hybrid was produced by coselection with hygromycin and G418 after fusion of MDTF-hygro with BHKPiT1 cells. MDTF-hygro, BHKPiT1, MDTF/BHKPiT1 hybrid, and MDTFPiT1 cells were exposed to GALV-enveloped retroviral vectors expressing nuclear localized β-galactosidase. Micrographs were taken with a 40× objective on a phase-contrast microscope.

Binding of XMRV is heparin independent. (A) After CHOK1Xpr1 cells were treated with (areas under dotted line) or without (areas under bold line) heparinase II, the expression of HSPGs on the cell surface was detected by flow cytometry analysis after staining with anti-human heparan sulfate monoclonal antibody-FITC conjugate for 1 h. (B) Binding of XMRV envelope to CHOK1Xpr1 cells was determined with HA-tagged XMRV SU, similar to in Fig. 3. Areas under the dotted line show XMRV binding to CHOK1Xpr1 after treatment with heparinase II. Areas under the bold line represent XMRV binding to CHOK1Xpr1 without heparinase II treatment. Shaded areas represent isotype controls.