For VLP secretion studies, 2-3610 6 cells were seeded in 10 cm plates, cultured for 24h, and then induced by culture for 36 h in 1.5 mg/ml tetracycline in DME medium with 10% FCS at 37uC. The culture media were centrifuged at 10,0006g for 30 min to remove cell debris. VLPs were then pelleted through a 0.5 ml sucrose cushion by centrifugation at 54,000 rpm for 2 h at 4uC using a TLS55 rotor. To test the effect of neutralizing the pH of acidic cellular compartments, cells were seeded and induced as above. After 2 h of induction the media were changed to DME medium containing 20 mM HEPES pH 8.0, 2% FCS, and 1.5 mg/ml tetracycline plus 20 mM NH 4 Cl as indicated, and the incubation continued for a total of 36 h. E proteins in the cell lysates were immunoprecipitated using mAb 4G2. VLP and lysate samples were then analyzed by SDS-PAGE and western blot using Sango. Figure S1 Open-book view of pr-E interface. Pr peptide is shown in cyan. DI, DII and DIII of E9 protein are colored red, yellow and blue, and the fusion loop at the DII tip is labeled. The important charged residues in the pr-E interface are numbered and shown as stick drawings in blue (positive) or red (negative). In this structure from DV2 16681, E9 residue 71 is a Glu, while the corresponding residue in NGC E9 protein is an Asp. Figure  prepared from Protein Data Bank accession number 3C5X [34] using PyMOL. Found at: doi:10.1371/journal.ppat.1001157.s001 (0.39 MB PDF) Alternative splicing of CD200 is regulated by an exonic splicing enhancer and SF2/ASF CD200, a type I membrane glycoprotein, plays an important role in prevention of inflammatory disorders, graft rejection, autoimmune diseases and spontaneous fetal loss. It also regulates tumor immunity. A truncated CD200 (CD200(tr)) resulting from alternative splicing has been identified and characterized as a functional antagonist to full-length CD200. Thus, it is important to explore the mechanism(s) controlling alternative splicing of CD200. In this study, we identified an exonic splicing enhancer (ESE) located in exon 2, which is a putative binding site for a splicing regulatory protein SF2/ASF. Deletion or mutation of the ESE site decreased expression of the full-length CD200. Direct binding of SF2/ASF to the ESE site was confirmed by RNA electrophoretic mobility shift assay (EMSA). Knockdown of expression of SF2/ASF resulted in the same splicing pattern as seen after deletion or mutation of the ESE, whereas overexpression of SF2/ASF increased expression of the full-length CD200. In vivo studies showed that viral infection reversed the alternative splicing pattern of CD200 with increased expression of SF2/ASF and the full-length CD200. Taken together, our data suggest for the first time that SF2/ASF regulates the function of CD200 by controlling CD200 alternative splicing, through direct binding to an ESE located in exon 2 of CD200. CD200 is a type 1 membrane glycoprotein, delivering immunoregulatory signals through binding to its receptors (CD200Rs) (1) (2) (3) (4) . It is present on neurons, B cells, activated T cells, thymocytes, dendritic cells and endothelium in mice, rats and human (5, 6) . A large and growing body of studies demonstrates that expression level of CD200 regulates graft survival (7) (8) (9) , susceptibility to autoimmune diseases (10) (11) (12) , fetal loss (13) , inflammation/infection (14) and tumor immunity (15) (16) (17) (18) .