Respiratory syncytial virus (RSV) subverts the antiviral interferon (IFN) response, but the mechanism for this evasion was unclear. Here we show that RSV preferentially inhibits IFN-␣/ signaling by expression of viral NS1 and NS2. Thus, RSV infection or expression of recombinant NS1 and NS2 in epithelial host cells causes a marked decrease in Stat2 levels and the consequent downstream IFN-␣/ response. Similarly, NS1/NS2-deficient RSV no longer decreases Stat2 levels or IFN responsiveness. RSV infection decreased human but not mouse Stat2 levels, so this mechanism of IFN antagonism may contribute to viral host range, as well as immune subversion.Paramyxoviruses appear to effectively antagonize the interferon (IFN) system through either inhibition of IFN generation or blockade of IFN signal transduction components such as Stat1 and Stat2 (8). For Sendai and measles viruses, the viral V and C proteins inhibit Stat1 expression and activation (7,11,14,22), whereas the V protein of human parainfluenza virus 2 affects Stat2 responses (1, 15). However, these proteins are not present in respiratory syncytial virus (RSV), the most common cause of serious respiratory infection in childhood (12). Nonetheless, RSV is also relatively resistant to the IFN-induced antiviral state (2). Recombinant deletion mutants of bovine RSV demonstrated that the nonstructural proteins NS1 and NS2 are cooperatively necessary for this resistance (21). These proteins also appear to inhibit induction of IFN, since deletion mutants of NS1 and NS2 for both human and bovine RSV induce more IFN-␣/ production than does wild-type RSV (23,24). However, these previous approaches have not addressed a possible action of RSV proteins on IFN signaling that might also inhibit the initial induction of an IFN-dependent antiviral state.In the present study, we found that infection of primaryculture human tracheobronchial epithelial cells (hTECs) (13) or a human alveolar epithelial cell line (A549 cells) with a low inoculum (multiplicity of infection [MOI] of 2) of RSV slightly increased baseline Stat1 and Stat2 expression and tyrosine phosphorylation but markedly suppressed any subsequent response to IFN- (Fig. 1A and data not shown). A higher inoculum of RSV (MOI of 10) decreased the level of Stat2 expression and fully inhibited IFN- responsiveness, as described by others (19). Neither level of RSV inoculation caused any significant decrease in Stat1 expression or IFN-␥ responsiveness ( Fig. 1A and data not shown). In concert with these effects, RSV infection also increased baseline ISRE promoter activity without affecting control Renilla luciferase activity and suppressed IFN- responsiveness of the ISRE promoter (Fig. 1B). Thus, at a low inoculum, RSV-driven increases in baseline Stat1 and Stat2 expression and phosphorylation are likely due to endogenous IFN- production acting in a paracrine manner on uninfected cells. A high inoculum of RSV causes selective down-regulation of Stat2 with consequent inhibition of IFN-␣/ signaling that becomes apparen...
In epithelial cells, alternative splicing of fibroblast growth factor receptor 2 (FGFR2) transcripts leads to the expression of the FGFR2(IIIb) isoform, whereas in mesenchymal cells, the same process results in the synthesis of FGFR2(IIIc). Expression of the FGFR2(IIIc) isoform during prostate tumor progression suggests a disruption of the epithelial character of these tumors. To visualize the use of FGFR2 exon IIIc in prostate AT3 tumors in syngeneic rats, we constructed minigene constructs that report on alternative splicing. Imaging these alternative splicing decisions revealed unexpected mesenchymal-epithelial transitions in these primary tumors. These transitions were observed more frequently where tumor cells were in contact with stroma. Indeed, these transitions were frequently observed among lung micrometastases in the organ parenchyma and immediately adjacent to blood vessels. Our data suggest an unforeseen relationship between epithelial mesenchymal plasticity and malignant fitness.alternative splicing ͉ mesenchymal-epithelial transitions ͉ tumor plasticity R egulation of alternative splicing is essential for normal gene expression (1), and alterations of this regulation are linked to disease (2), as illustrated by the association between cancer and splicing defects (3-7). An elegant example of this is provided by the splicing of transcripts encoding fibroblast growth factor receptor 2 (FGFR2). The status of FGFR2 alternative splicing depends on the interplay between several cis-acting elements in the FGFR2 premRNA and transacting factors, some of which are cell-type-specific (8). In mesenchymal cells, exon IIIb is silenced by the action of an exonic splicing silencer (9) and two flanking intronic splicing silencers (10-12). This silencing is mediated by Polypyrimidine tract-binding protein (PTB), hnRNP A1, and heretofore unknown factors. In epithelial cells, exon IIIb silencing is countered by several intronic elements. The best characterized are the intronic activating sequence 2, the intronic splicing activator and repressor (ISAR, also known as IAS3), and several GCAUG repeats (13-18). These elements have a dual function in epithelial cells, because they are also involved in silencing exon IIIc (14-18).FGFR2 splicing has been studied in tumors derived from the R-3327 Dunning rat prostate tumor, which arose spontaneously from the dorsal lobe of the prostate in a Copenhagen rat (19). Some R-3327-derived tumors (DT or DT3) express FGFR2(IIIb), which is consistent with their epithelial phenotype (20), whereas AT tumors (e.g., AT3), which have lost epithelial markers and display many mesenchymal indicators (21), express FGFR2(IIIc) (20). The significance of these alternative decisions for tumor behavior is underscored by the fact that forced expression of FGFR2(IIIb) suppresses tumor progression of AT3 tumors (22). Most importantly, however, the differential splicing of FGFR2 transcripts in these two cell types highlights broad differences in gene expression programs. Arguably, monitoring alternative sp...
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