No abstract

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...

Hepatitis delta virus (HDV) is a pathogenic human virus whose RNA genome and replication cycle resemble those of plant viroids. However, viroid genomes contain no open reading frames, whereas HDV RNA encodes a single protein, hepatitis delta antigen (HDAg), which is required for viral replication. A cellular gene whose product interacts with HDAg has now been identified, and this interaction was found to affect viral genomic replication in intact cells. DNA sequence analysis revealed that this protein, termed delta-interacting protein A (DIPA), is a cellular homolog of HDAg. These observations demonstrate that a host gene product can modulate HDV replication and suggest that HDV may have evolved from a primitive viroidlike RNA through capture of a cellular transcript.

The phosphoprotein (P protein) of respiratory syncytial virus (RSV) is a key component of the viral RNA-dependent RNA polymerase complex. The protein is constitutively phosphorylated at the two clusters of serine residues (116, 117, and 119 [116/117/119] and 232 and 237 [232/237]). To examine the role of phosphorylation of the RSV P protein in virus replication, these five serine residues were altered to eliminate their phosphorylation potential, and the mutant proteins were analyzed for their functions with a minigenome assay. The reporter gene expression was reduced by 20% when all five phosphorylation sites were eliminated. Mutants with knockout mutations at two phosphorylation sites (S232A/S237A [PP2]) and at five phosphorylation sites (S116L/S117R/S119L/S232A/S237A [PP5]) were introduced into the infectious RSV A2 strain. Immunoprecipitation of 33 P i -labeled infected cells showed that P protein phosphorylation was reduced by 80% for rA2-PP2 and 95% for rA2-PP5. The interaction between the nucleocapsid (N) protein and P protein was reduced in rA2-PP2-and rA2-PP5-infected cells by 30 and 60%, respectively. Although the two recombinant viruses replicated well in Vero cells, rA2-PP2 and, to a greater extent, rA2-PP5, replicated poorly in HEp-2 cells. Virus budding from the infected HEp-2 cells was affected by dephosphorylation of P protein, because the majority of rA2-PP5 remained cell associated. In addition, rA2-PP5 was also more attenuated than rA2-PP2 in replication in the respiratory tracts of mice and cotton rats. Thus, our data suggest that although the major phosphorylation sites of RSV P protein are dispensable for virus replication in vitro, phosphorylation of P protein is required for efficient virus replication in vitro and in vivo.The phosphoprotein (P protein) of human respiratory syncytial virus (RSV), a prototype Pneumovirus of the family Paramyxoviridae, is an essential component of the viral RNA polymerase, along with the large polymerase (L) and nucleocapsid (N) proteins (12,35). Interaction of the RSV P protein with the N and L proteins promotes the formation of a transcriptase complex that is essential for viral RNA transcription and replication (10,19,20). Although L protein is the catalytic RNA polymerase, P protein is essential for transcription and replication of viral RNA (7,14). In addition to the N, P, and L proteins, several viral proteins are required for RSV RNA synthesis. The antitermination function of M2-1 is essential for processive RNA synthesis and suppression of transcription termination in intergenic regions (6, 13). M2-2 has been postulated to have a role in regulating the switch between viral RNA transcription and replication processes (3, 17).The P protein of RSV subgroup A 2 is 241 amino acids in length, which is much shorter than the P proteins of other paramyxoviruses (5, 21), and forms homotetramers (1), similar to the Sendai virus P protein (29, 30). The interaction of the N and P proteins enables proper folding of N protein and enables N protein to encapsidate v...

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 high-affinity binding of SW15 to this site. We show that the two proteins bind DNA cooperatively, and we take advantage of this cooperativeness to purify the stimulatory factor. In another report (5a), we demonstrate that this stimulatory factor is encoded by the GRFJO gene, and therefore, we will refer to this factor that stimulates SWI5 binding as GRF10. The GRF10 gene is also known as PH02 and BAS2. PH02 was identified as a transcriptional activator of PH05 (33), and BAS2 is required for the basal transcriptional activation of the HIS4 gene (3). The name GRF1O (for general regulatory factor) was chosen as a new gene designation since GRFIO plays a role in the transcriptional regulation of diverse genes (49). GRF1O is also required for the full expression of the HO gene (5a).MATERIALS AND METHODS Yeast strains. Yeast strain DY411 (A Ta swiS::hisG ade2-1 canl-100 his3-11,15 leu2-3,112 trpl-1 ura3-52) is a derivative of DY150 (9) which contains a hisG (2) disruption of SWI5. DY472 is strain DY411 which contains plasmid YCp5O:pGAL:SfW5, which overexpresses SWI5 (43)

The SWI5 gene encodes a zinc finger DNA-binding protein required for the transcriptional activation of the yeast HO gene. There are two Swi5p binding sites in the HO promoter, site A at ؊1800 and site B at ؊1300. Swi5p binding at site B has been investigated in some detail, and we have shown that Swi5p binds site B in a mutually cooperative fashion with Pho2p, a homeodomain protein. In this report, we demonstrate that Swi5p and Pho2p bind cooperatively to both sites A and B but that there are differences in binding to these two promoter sites. It has been shown previously that point mutations in either Swi5p binding site only modestly reduce HO expression in a PHO2 strain. We show that these mutant promoters are completely inactive in a pho2 mutant. We have created stronger point mutations at the two Swi5p binding sites within the HO promoter, and we show that the two binding sites, separated by 500 bp, are both absolutely required for HO expression, independent of PHO2. These results create an apparent dilemma, as the strong mutations at the Swi5p binding sites show that both binding sites are required for HO expression, but the earlier binding site mutations allow Swi5p to activate HO, but only in the presence of Pho2p. To explain these results, a model is proposed in which physical interaction between Swi5p proteins bound to these two sites separated by 500 bp is required for activation of the HO promoter. Experimental evidence is presented that supports the model. In addition, through deletion analysis we have identified a region near the amino terminus of Swi5p that is required for PHO2-independent activation of HO, suggesting that this region mediates the long-range interactions between Swi5p molecules bound at the distant sites.The Saccharomyces cerevisiae HO gene encodes an endonuclease which is responsible for initiating mating type switching in yeast (for reviews, see references 8 and 20). When yeast cells divide, they do so asymmetrically, producing a large mother cell and a small daughter cell. Only the mother cell is able to switch its mating type, and the ability to switch mating type requires transcription of the HO gene. Daughter cells do not express HO and are therefore not capable of switching their mating type. The SWI5 gene was identified originally as a positive transcriptional regulator of HO transcription, and SWI5 has been implicated in the asymmetric expression of HO (19,21,27). Recent work has shown that the ASH1 gene product is required to repress HO transcription in daughter cells (2,25).SWI5 encodes a 709-amino-acid protein which contains three zinc finger DNA-binding domains near its carboxy terminus (26). Swi5p binds DNA in vitro via these zinc finger motifs to a site in the HO promoter located approximately 1,300 nucleotides (nt) upstream of the HO ATG start codon (16,26). Recently, a second Swi5p binding site within the HO promoter has been identified that is also required for proper transcriptional regulation of HO in vivo (27).The new Swi5p binding site is located approximately...

SWIS encodes a zinc-finger protein required for expression of the yeast HO gene. Using SwiS protein that was purified from a bacterial expression system, we previously isolated a yeast factor that stimulates binding of Swi5 to the HO promoter. N-terminal amino acid sequence analysis identified the SwiS stimulatory factor as the product of the GRF1O gene, which encodes a yeast homeodomain protein. GRFIO, also known as PH02 and BAS2, is a transcriptional activator of the PH05 acid phosphatase gene and the HIS4 histidine biosynthesis gene. GrflO protein purified from a bacterial expression system binds DNA cooperatively with Swi5 in vitro. Analysis of disasciation rates indicates that the GrflO-Swi5-DNA complex has a longer half-life than protein-DNA complexes that contain only Swi5 or GrflO. Finally, we show that HO expression is reduced in yeast strains containing grflO null mutations and that full expression of a heterologous promoter containing a SWIS-dependent HO upstream activation sequence element requires GRFIO.