Specific Inhibition of Type I Interferon Signal ... - ATS Journals

Specific Inhibition of Type I Interferon Signal Transduction by Respiratory Syncytial Virus Murali Ramaswamy, Lei Shi, Martha M. Monick, Gary W. Hunninghake, and Dwight C. Look Department of Internal Medicine, University of Iowa Roy J. and Lucille A. Carver College of Medicine, and Veterans Administration Medical Center, Iowa City, Iowa

Respiratory viruses often express mechanisms to resist host antiviral systems, but the biochemical basis for evasion of interferon effects by respiratory syncytial virus (RSV) is poorly defined. In this study, we identified RSV effects on interferon (IFN)-dependent signal transduction and gene expression in human airway epithelial cells. Initial experiments demonstrated inhibition of antiviral gene expression induced by IFN-␣ and IFN-␤, but not IFN-␥, in epithelial cells infected with RSV. Selective viral effects on type I IFN-dependent signaling were confirmed when we observed impaired type I, but not type II, IFN-induced activation of the transcription factor Stat1 in RSV-infected cells. RSV infection of airway epithelial cells resulted in decreased Stat2 expression and function with preservation of upstream signaling events, providing a molecular mechanism for viral inhibition of the type I IFN JAK-STAT pathway. Furthermore, nonspecific pharmacologic inhibition of proteasome function in RSV-infected cells restored Stat2 levels and IFN-dependent activation of Stat1. The results indicate that RSV acts on epithelial cells in the airway to directly modulate the type I IFN JAK-STAT pathway, and this effect is likely mediated though proteasome-dependent degradation of Stat2. Decreased antiviral gene expression in RSV-infected airway epithelial cells may allow RSV replication and establishment of a productive viral infection through subversion of IFN-dependent immunity.

Human respiratory syncytial virus (RSV) is an enveloped, negative-strand RNA, paramyxovirus that causes the highest incidence of respiratory infection in the winter and early spring (1). RSV has generated intense research interest since its identification over 40 yr ago because it infects virtually 100% of children within the first few years of life, is the most frequent cause of lower respiratory tract infection (pneumonia and bronchiolitis) in infants and young children, and has important roles in exacerbation of several respiratory diseases (2). The success of RSV in establishing productive infections in human airway epithelia depends on viral expression of mechanisms for evasion of innate and acquired immune responses. A central feature of the host response to viral infection in the airway is activation of cellular genes that are important in innate and adaptive immunity by a potent group of mediators termed interferons (IFNs) (3). RSV is a potent inducer of type I IFN from airway epithelial cells and leukocytes, but a relatively poor inducer of type II IFN from (Received in original form November 13, 2003 and in revised form December 23, 2003) Address correspondence to: Dwight C. Look, M.D., University of Iowa Roy J. and Lucille A. Carver College of Medicine, Department of Internal Medicine, 200 Hawkins Drive, C33-GH, Iowa City, IA 52242. E-mail: [email protected] Abbreviations: analysis of variance, ANOVA; human tracheobronchial epithelial, hTBE; IFN-␣ receptor, IFNAR; IFN-␥ receptor, IFNGR; interferon regulatory factor, IRF; interferon-stimulated gene factor 3, ISGF3; intercellular adhesion molecule-1, ICAM-1; Janus family kinase, JAK; Laboratory of Human Carcinogenesis, LHC; lactate dehydrogenase, LDH; major histocompatibility complex, MHC; multiplicity of infection, m.o.i.; not detected, ND; plaque-forming units, PFU; protein kinase R, PKR; respiratory syncytial virus, RSV; signal transducer and activator of transcription, STAT. Am. J. Respir. Cell Mol. Biol. Vol. 30, pp. 893–900, 2004 Originally Published in Press as DOI: 10.1165/rcmb.2003-0410OC on January 12, 2004 Internet address: www.atsjournals.org

human mononuclear cell preparations and lymphocytes (4–7). The existence of RSV mechanisms for subversion of IFN-dependent immunity is suggested by reports that type I IFN inefficiently inhibits replication of this virus in human epithelial cells and increasing type I IFN levels in the human airway has limited efficacy in adults with RSV respiratory infection (8–11). Despite the importance of type I IFN-dependent immunity in antiviral defense in the airway, the biochemical basis for evasion of IFN effects by RSV is poorly defined. Type I IFN is produced by most nucleated cells through multiple IFN-␣ and one IFN-␤ genes, and type II IFN is produced mainly by T cells and natural killer cells as IFN-␥ (12). IFNs mediate host cell effects by binding to two different type-specific receptor complexes linked to two specific Janus family kinase (JAK)-STAT signaling cascades that have some shared components (13, 14). Activation of the type I IFN-driven pathway is triggered by engagement and multimerization of the IFN-␣ receptor (IFNAR) by IFN-␣ or IFN-␤, phosphorylation of IFNAR1-associated Tyk2 and IFNAR2-associated Jak1 tyrosine kinases, and then phosphorylation of IFNAR1 and IFNAR2 (15, 16). Phosphorylation of the IFNAR1 chain of the IFN-␣ receptor results in recruitment, phosphorylation, and subsequent release of Stat1 and Stat2 from the receptor (17, 18). Activated Stat1 and Stat2 associate with IFN regulatory factor(IRF)-9 to form the transcriptional activator complex IFN-stimulated gene factor 3 (ISGF3), translocate to the nucleus, and bind specific DNA recognition sequences to activate transcription of type I IFN-inducible genes (19). Type II IFN signaling results in activation of Stat1, but not Stat2, by a distinct JAK-STAT pathway, but there are several mechanisms for crosstalk between the two IFN systems (14, 20, 21). Activation of IFN-responsive gene transcription through JAK-STAT signal transduction cascades results in expression of an entire set of immune response genes oriented toward antiviral defense, including genes involved in establishment of a host cell antiviral state and augmentation of immune recognition (3, 20, 22). Based on the specific genes expressed by each type of IFN, type I IFN seems to be particularly important in mediating innate immune responses against viruses, whereas type II IFN has been more extensively studied as a regulator of the adaptive immune system, although there is significant overlap (3, 14). In this report, we demonstrate that RSV specifically inhibits IFN-dependent gene expression in airway epithelial cells through selective effects on the type I IFN JAK-STAT pathway. We show that RSV infection results in specific decrease in epithelial cell Stat2 expression with consequent blockade of type I IFN-dependent downstream signaling and gene expression. Furthermore, pharmacologic inhibition of proteasome function in RSV-infected cells restored Stat2 levels and IFN-dependent activation of Stat1 and Stat2. Our results support the concept that inhibition of type I IFN-dependent signaling results in modulation of immune response genes by RSV, which may allow for evasion of the airway defense response and establishment of a productive infection.

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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 30 2004

Materials and Methods Airway Epithelial Cell Isolation, Culture, and Stimulation Human tracheobronchial epithelial (hTBE) cells were obtained under a protocol approved by the University of Iowa Institutional Review Board. Epithelial cells were isolated from tracheal and bronchial mucosa by enzymatic dissociation and cultured in Laboratory of Carcinogenesis (LHC)-8e medium on plates coated with collagen/albumin for study up to passage 10 as described previously (23–25). Cells were treated with recombinant human IFN-␣2a or IFN-␤ (PBL Biomedical Laboratories, Piscataway, NJ) at a concentration (1,000 U/ml) and for durations that result in maximal effects on type I IFN JAK-STAT pathway component phosphorylation (15 min) or gene expression (6–24 h). Some cells were treated with recombinant human IFN-␥ (a gift from Genentech, San Francisco, CA) at a concentration (100 U/ml) and for durations that result in maximal effects on Stat1 phosphorylation (15 min) or intercellular adhesion molecule-1 (ICAM-1) expression (24 h) as described previously (23–25). In some experiments, cells were treated with the cell-permeable nonspecific proteasome inhibitor carbobenzoxy-L-leucyl-L-leucyl-L-leucinal (MG-132; Calbiochem, San Diego, CA).

Viral Preparation and Infection High concentration and purity RSV strain A2 were obtained from Advanced Biotechnology (Columbia, MD), where it was purified from HEp-2 cell lysates by pelleting through sucrose gradient centrifugation. Control inactivated RSV was prepared by exposure to 18 J of ultraviolet light at 4⬚C at the same concentration in the same diluent as live virus samples. Loss of viral replication capacity in epithelial cells was verified by plaque assay as described previously (26), or by immunoblot analysis or immunofluorescence cytochemical staining for RSV protein production in airway epithelial cells. RSV were incubated with epithelial cells for 0.5–48 h in culture media at 37⬚C in 5% CO2 at a multiplicity of infection (m.o.i.) of 0.005–100.

Primary Antibodies Chicken polyclonal IgY against human MxA was from BacLab (Muttenz, Switzerland); rabbit polyclonal IgG 3072 against human protein kinase R (PKR), rabbit polyclonal IgG 9172 against total human Stat1, rabbit polyclonal IgG 9171 against phosphorylated human Stat1, and rabbit polyclonal IgG 9321 against phosphorylated human Tyk2 were from Cell Signaling Technology (Beverly, MA); mouse IgG1 mAb clone 84H10 against human ICAM-1 was from Immunotech (Westbrook, ME); goat polyclonal IgG against human RSV proteins was from Biodesign International (Saco, ME); mouse IgG2a mAb clone AC-74 against human ␤-actin was from Sigma-Aldrich (St. Louis, MO); rabbit polyclonal IgG 07-224 against phosphorylated human Stat2, rabbit polyclonal IgG 06-502 against total human Stat2, and rabbit polyclonal IgG 06-638 against total human Tyk2 were from Upstate Biotechnology (Lake Placid, NY); mouse IgG1 mAb clone 6 against human IRF-9 was from BD Transduction Laboratories (Lexington, KY); fluoresceinconjugated goat polyclonal IgG against human RSV was from Virostat (Portland, ME).

Immunoblot Analysis Epithelial cell protein levels were assessed by immunoblot analysis using specific antibodies against human JAK-STAT pathway components as described previously (23, 24). Whole cell protein extracts were prepared by lysis of cell monolayers in 50 mM Tris, pH 7.4, 150 mM NaCl, 1% Nonidet P-40, a protease inhibitor cocktail (Roche Bioscience, Palo Alto, CA), and a phosphatase inhibitor panel (Calbiochem). Protein concentrations were determined using a Coomassie brilliant blue G-250 binding assay (Bio-Rad Laboratories, Hercules, CA) and equal amounts of cell protein were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis in 5–12% polyacrylamide. Resolved proteins were electrophoretically transferred to nitrocellulose membranes (Hybond; Amersham Biosciences, Piscataway, NJ), exposed to 5% nonfat milk or 5% bovine serum albumin in Tris-buffered saline with 0.1% Tween20 to block nonspecific antigens, and then incubated with antibodies against a specific cellular protein. Primary antibody binding was detected using rabbit antichicken IgY (Sigma-Aldrich, St. Louis, MO),

goat antirabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA), goat antimouse IgG (Boehringer Mannheim, Indianapolis, IN), or donkey antigoat IgG (Santa Cruz) conjugated to horseradish peroxidase and an enhanced chemiluminescence detection system (Amersham Biosciences). Reprobing of membranes with a different primary antibody was done after washing in Restore buffer (Pierce, Rockford, IL) for 15 min at 37⬚C.

Enzyme-Linked Immunoassays ICAM-1 protein levels on the surface of hTBE cell monolayers at confluence on 96-well tissue culture plates were determined by an enzyme-linked colorimetric immunoassay as described previously (23–25, 27). IFN-␣ and IFN-␤ concentrations in cell culture media were determined using commercial sandwich enzyme-linked immunosorbent assay kits (PBL Biomedical Laboratories, Piscataway, NJ). The IFN-␣ kit recognizes all but one of the known IFN-␣ forms, has no crossreactivity with other forms of IFN, and has a sensitivity of ⬎ 40 pg/ml according to the manufacturer. The IFN-␤ kit also is highly selective and has a sensitivity of ⬎ 250 pg/ml.

Cytotoxicity Assays Dead and live hTBE cell numbers were quantified by detection of plasma membrane permeability to ethidium homodimers in dead cells and intracellular esterase activity in live cells using a commercial fluorescence-based viability and cytotoxicity kit (Molecular Probes, Eugene, OR). Release of the cytoplasmic enzyme lactate dehydrogenase (LDH) by hTBE cell monolayers was determined by a colorimetric enzyme activity assay of culture media using a commercial kit (Roche, Indianapolis, IN) (24). Cells were treated with the permeabilizing agent 0.1% saponin to cause 100% cytotoxicity.

Immunofluorescence Microscopy Cellular localization of specific proteins was detected in hTBE cells grown on a chamber slide system (Lab-Tek; Nalge Nunc International, Napersville, IL) similar to methodology described previously (23, 25). Epithelial cells were fixed and permeabilized in 100% methanol, exposed to 2% fishgel (Sigma-Aldrich) to block nonspecific antibody binding, and incubated with specific antibodies against cellular or viral proteins. If the primary antibody was not conjugated to a fluorochrome, antibody binding was detected by exposure to goat anti-mouse IgG conjugated to fluorescein or goat anti-rabbit IgG conjugated to Cy3. Slides were mounted for fluorescence microscopy (Leitz Diaplan; Wild Leitz USA, Inc., Rockleigh, NJ) using Gel/Mount (Biomeda Corporation, Foster City, CA). Images were acquired using a digital CCD camera system (SPOT; Diagnostic Instruments, Sterling Heights, MI) interfaced with SPOT software version 2.2. Nuclear localization was quantified visually by determining the percent of total cells with nuclear ⬎ cytoplasmic staining.

Statistical Analysis Assays were performed multiple times to assure reproducible results and were analyzed for statistical significance using a one-way ANOVA for a factorial experimental design. The multicomparison significance level for the one-way ANOVA was 0.05. If significance was achieved by one-way analysis, post-ANOVA comparison of means was performed using Scheffe F-tests (28).

Results RSV Infection Inhibits IFN-␣–Dependent Gene Expression

Airway infection with RSV results in subsequent release of IFN-␣ from leukocytes and IFN-␤ from epithelial cells (5, 29). RSV is a relatively poor inducer of IFN-␥ from lymphocytes (4, 6). Human airway epithelial cells under uninfected conditions respond to both type I and II IFNs by expression of antiviral genes. To evaluate RSV effects on IFN responsiveness, we initially examined the expression of sentinel antiviral genes that are induced by type I or type II IFN in hTBE cells after viral infection. MxA is a dynamin superfamily transport GTPase that exhibits antiviral activity against several RNA viruses (9, 22).

Ramaswamy, Shi, Monick, et al.: RSV Inhibition of Type I Interferon Signaling

PKR is a double-stranded RNA-activated serine-threonine kinase that inhibits protein synthesis and mediates other cellular responses to viral infection or inflammatory stimuli (22). Both of these intracellular antiviral proteins are induced by type I, but not type II, IFNs (20). In contrast, ICAM-1 is an important adhesive glycoprotein for leukocytes that is selectively induced by type II IFN (20, 30). Although RSV infection has been observed to induce STAT activation and antiviral gene expression (31, 32), our results indicated that RSV induction of MxA and PKR is delayed and at a markedly lower level than expression induced by IFN-␣ (Figure 1A). Furthermore, we found that infection with RSV inhibited IFN-␣–induced MxA and PKR expression in hTBE cells, whereas RSV inactivated by ultraviolet light treatment did not have this capacity (Figure 1B). RSV infection at inoculums that resulted in inhibition of IFN-␣–induced MxA and PKR had little effect on basal or IFN-␥–induced ICAM-1 expression on hTBE cells (Figure 1C), indicating that another related signaling pathway maintained function and suggesting that viral effects on IFN-␣–induced gene expression were not due to virus-induced epithelial cell cytotoxicity. To support this conclusion, RSV cytotoxic effects on hTBE cells were examined to exclude this mechanism for altered cell signaling. RSV did not have a significant effect on epithelial cell uptake of ethidium homodimers or release of LDH until after 24 h of infection (Figure 1D), indicating that epithelial cell cytotoxicity did not account for loss of IFN-␣–induced gene expression that we observed after shorter durations of RSV infection in hTBE cells. IFN-␣ treatment did not decrease viral protein expression when added after initiation of viral infection in hTBE cells (results not shown), and thus decreased antiviral gene expression was not due to IFN effects on RSV. These results raised the possibility that RSV infection might inhibit antiviral gene expression induced by IFN-␣ (but not IFN-␥) through effects on IFN signal transduction in airway epithelial cells. Therefore, subse-

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Figure 1. RSV infection inhibits IFN-␣–dependent gene expression. (A ) MxA and PKR protein levels were determined using immunoblot analysis of extracts from hTBE cell monolayers that were left untreated, or were treated with 1,000 U/ml of IFN-␣ or infected with RSV at m.o.i 15 for the indicated durations. The positions of MxA and PKR are indicated by arrows, and staining of proteins on the membrane with Ponceau S dye was used to verify equivalency of protein isolation and loading. (B ) MxA and PKR protein levels were determined using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV or ultraviolet light–inactivated RSV at the indicated m.o.i. for 6 h, followed by incubation without or with IFN-␣ for 12 h. (C ) ICAM-1 protein levels on the surface of hTBE cells were determined using an enzyme-linked immunoassay with cell monolayers that were left uninfected or were infected with RSV at the indicated m.o.i. for 18 h, followed by incubation without (open bars) or with (filled bars) IFN-␥ for 24 h. Values are expressed as mean ⫾ SD (n ⫽ 3–4), and a significant increase from corresponding levels on cells not incubated with IFN-␥ is indicated by an asterisk. (D ) Dead and live hTBE cell numbers were quantified by detection of plasma membrane permeability to ethidium homodimers in dead cells and intracellular esterase activity in live cells. Values were calculated as dead cells/total cells and each condition represents the mean ⫾ SD for 5 microscopic fields (40–100 cells/field). Extracellular levels of LDH were determined by a spectrophotometric assay of culture media from the same hTBE cell monolayers. LDH values are expressed as mean percent of maximally permeabilized control cells (accomplished by treatment of hTBE cell monolayers with 0.1% saponin) ⫾ SD (n ⫽ 3). Cells were left uninfected or were infected with RSV at m.o.i. 15 for the indicated duration, and a significant difference between levels in uninfected and RSV-infected cells is indicated by an asterisk.

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quent experiments were directed at examining viral mechanisms for specifically modifying the type I IFN JAK-STAT pathway for antiviral gene expression. RSV Infection Specifically Inhibits IFN-␣–Induced Stat1 Phosphorylation

Type I and type II IFN-driven transcription of antiviral genes in airway epithelial cells requires tyrosine phosphorylation of Stat1 that is mediated through specific JAK-STAT pathways (16). Using immunoblot analysis with phosphorylation-specific Stat1 antibodies to detect activation of this transcription factor, we found that RSV infection at an inoculum level that inhibited MxA and PKR expression also inhibited IFN-␣–induced phosphorylation of Stat1 in hTBE cells (Figure 2A). This effect required viral replication, because ultraviolet-inactivated RSV did not affect phosphorylation of Stat1. Based on our previous finding that RSV had little effect on IFN-␥–induced ICAM-1 expression, it seemed likely that RSV would not affect type II IFN signaling in airway epithelial cells. This correlation was confirmed when we found that RSV infection increased rather than inhibited phosphorylation of Stat1 in response to IFN-␥ in hTBE cells (Figure 2B). RSV infection resulted in a moderate increase in total Stat1 levels, but this did not correlate with RSV effects

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Figure 2. RSV infection specifically inhibits IFN-␣-induced Stat1 phosphorylation. (A ) Stat1 phosphorylation was assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV or ultraviolet light–inactivated RSV at m.o.i. 15 for 24 h, followed by incubation without or with IFN-␣ for 15 min. The positions of phosphorylated and total Stat1 are indicated by arrows. (B ) Stat1 phosphorylation was assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV at m.o.i. 15 for 24 h, followed by incubation without or with IFN-␣ or IFN-␥ for 15 min.

on Stat1 activation as was illustrated by the difference in induction of Stat1 phosphorylation in virus-infected cells treated with IFN-␣ versus IFN-␥. These results indicate that RSV infection inhibits IFN-␣ signal transduction resulting in modulation of airway epithelial cell gene expression. RSV Infection Rapidly Inhibits IFN-␣ Signaling

Based on the observation that RSV infection resulted in inhibition of IFN-␣–dependent Stat1 activation in airway epithelial cells, subsequent experiments focused on further defining characteristics of this RSV effect. To allow detection of viral effects on IFN signaling, conditions were established in which the majority of the cultured epithelial cells were infected with RSV. Airway epithelial cell infection with an m.o.i. of at least 15 infectious viral particles per epithelial cell for 24 h resulted in RSV protein expression in virtually 100% of epithelial cells in the sample as assessed by immunofluorescence cytochemical staining (results not shown) and near-maximal levels of RSV G, N, and P protein expression as assessed by immunoblot analysis (Figure 3A). Infection of hTBE cells with different quantities of RSV resulted in inoculum-dependent inhibition of IFN-␣–induced Stat1 phosphorylation, with marked inhibition at ⭓ m.o.i. 15 (Figure 3B). Using hTBE cells infected with this inoculum level for varying durations, we found that IFN-␣–induced Stat1 phosphorylation was significantly decreased after 6–12 h of RSV infection, and this effect persisted for at least 48 h (Figure 3C). Thus, it is likely that RSV inhibition of IFN-␣–dependent activation of Stat1 allows viral subversion of antiviral defense during early stages of viral infection and replication. RSV Infection Selectively Inhibits Stat2 Expression

To examine type I IFN JAK-STAT pathway components that are required for activation of Stat1 by IFN-␣, we began by assessing the expression and phosphorylation of Stat2. Activation of Stat2 at the type I IFN receptor complex forms a binding site for Stat1 that is required for Stat1 phosphorylation (17, 18). Using epithelial cells infected for varying durations with an RSV inoculum that caused inhibition of Stat1 activation, we found that RSV also inhibited IFN-␣–induced Stat2 phosphorylation after 6–12 h of infection (Figure 4A). However, total Stat2 expression was also significantly decreased in hTBE cells infected with RSV, and this effect persisted for at least 48 h. This effect did not appear to be mediated by epithelial cell release of a soluble extracellular inhibitor because medium transferred from RSV-infected epithelial cells to uninfected hTBE cells did not block Stat1 activation by IFN-␣ (Figure 4B). Moreover, RSV

Figure 3. RSV infection rapidly inhibits IFN-␣ signaling. (A ) RSV protein levels were determined using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV at the indicated m.o.i. for 24 h. The positions of RSV G, N, and P proteins and ␤-actin (to verify equivalency of protein isolation and loading) are indicated by arrows. (B ) Stat1 phosphorylation was assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV at the indicated m.o.i. for 24 h, followed by incubation without or with IFN-␣ for 15 min. (C ) Stat1 phosphorylation was assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV at m.o.i. 15 for the indicated duration, followed by incubation without or with IFN-␣ for 15 min.

infection did not inhibit IFN-␣–dependent phosphorylation of Tyk2 (Figure 4C), revealing that receptor activation that occurs before STAT activation in the type I IFN signaling cascade was not affected by the virus. We also examined the effects of loss of Stat2 expression on interaction with another type I IFN signaling protein. Although IRF-9 has a nuclear localization signal that mediates constitutive nuclear translocation if expressed in the absence of Stat2, the presence of unactivated Stat2 serves to retain a significant portion of IRF-9 in the cytoplasm (33). Constitutive Stat2 interaction with IRF-9 was confirmed when we observed loss of cytoplasmic retention of IRF-9 in hTBE cells infected with RSV (Figure 5A). In these experiments, the lack of nuclear translocation of Stat1 verifies that IRF-9 localization in the nucleus after RSV infection was not due to endogenous release of type I IFN resulting in JAK-STAT pathway activation and formation of ISGF3 with consequent nuclear localization of both IRF-9 and Stat1 (Figure 5B). Based on these findings, modulation of Stat2 levels appears to be an important mechanism for inhibition of IFN-␣ signaling in airway epithelial cell infected with RSV, resulting in intact upstream signaling events, but loss of Stat2-dependent functions.

Ramaswamy, Shi, Monick, et al.: RSV Inhibition of Type I Interferon Signaling Figure 4. RSV infection selectively inhibits Stat2 expression. (A ) Stat2 protein expression and phosphorylation were assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV at m.o.i. 15 for the indicated duration, followed by incubation without or with IFN-␣ for 15 min. The positions of phosphorylated and total Stat2 are indicated by arrows. (B ) Stat1 phosphorylation was assessed using immunoblot analysis of extracts from two sets of hTBE cell monolayers. In the first set, extracts were isolated from monolayers that were left uninfected or were infected with RSV at m.o.i. 15 for 24 h, followed by incubation without or with IFN-␣ for 15 min. Aliquots of media from monolayers infected with RSV but not incubated with IFN-␣ (the monolayers used for lane 4) were harvested after completion of the incubation period and residual viral particles were inactivated by ultraviolet light treatment. Media aliquots were then placed on a second set of uninfected hTBE cell monolayers, which were incubated with IFN-␣ for 15 min, followed by isolation of cellular proteins for immunoblot analysis. (C ) Tyk2 protein expression and phosphorylation were assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV at m.o.i. 15 for 24 h, followed by incubation without or with IFN-␣ for 15 min. The positions of phosphorylated and total Tyk2 are indicated by arrows.

RSV Infection Also Inhibits IFN-␤ Signaling

Epithelial cells tend to produce IFN-␤ in response to RSV infection (Figure 6A), whereas leukocytes tend to produce IFN-␣ (5, 29), and thus both forms of type I IFN may be generated in the airway after RSV infection. IFN-␤ release from hTBE cells after RSV infection was not detectable until 24 h after initiation of infection, and thus viral inhibition of Stat2 expression is established before potential activation of antiviral genes by IFN-␤. Because both IFN-␣ and IFN-␤ require Stat2 for antiviral gene expression that is mediated by signaling through their shared type I IFN JAK-STAT pathway (16), it seemed likely that IFN␤–dependent gene expression and Stat1 activation would also be inhibited by RSV. This possibility was confirmed when we found that RSV infection inhibited IFN-␤–induced MxA expression (Figure 6B), as well as IFN-␤–dependent Stat1 activation that correlated with loss of Stat2 expression in hTBE cells (Figure 6C). RSV Infection Likely Induces Proteasome-Dependent Inhibition of Stat2 Expression

One mechanism for modulation of IFN signaling by paramyxoviruses may be through activation of proteasome-dependent degradation of JAK-STAT pathway components, as has been

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Figure 5. RSV infection inhibits Stat2 effects on IRF-9. (A ) IRF-9 and Stat1 cellular locations were assessed by immunofluorescence microscopy of hTBE cell monolayers that were left uninfected, were infected with RSV at m.o.i. 15 for 24 h, or were incubated with IFN-␣ for 15 min. Primary antibody binding to IRF-9 and Stat1 were detected using fluorescein- or Cy3-conjugated secondary antibody. Scale bar, 20 ␮m. (B ) IRF-9 (open bars) and Stat1 (filled bars) cellular location was quantified by determining the percent of total cells with nuclear ⬎ cytoplasmic staining. Each condition represents the mean ⫾ SD for five microscopic fields from experiment shown in A (40–80 cells/field), and a significant difference from uninfected and untreated cells is indicated by an asterisk.

demonstrated for simian virus 5–mediated downregulation of Stat1 levels and human parainfluenza virus type 2–mediated downregulation of Stat2 levels (10, 34, 35). To assess the possibility of RSV induction of proteasome-mediated degradation of Stat2 in airway epithelial cell, we pretreated hTBE cells with the cell-permeable nonspecific proteasome inhibitor MG-132 and then compared Stat2 levels in cells left uninfected or infected with RSV. We found that RSV lost the capacity to modulate Stat2 expression in hTBE cells pretreated with MG-132 (Figure 7A). Maintenance of Stat2 expression in infected cells was not due to loss of viral gene expression as demonstrated by equal levels of RSV G protein in cells treated with vehicle control or MG-132. Similarly, epithelial cells pretreated with MG-132 were resistant to RSV effects on IRF-9 nuclear localization (Figure 7B), although MG-132 did not itself affect IRF-9 cellular location (Figure 7C). Lastly, pretreatment of hTBE cells with MG-132 at levels that restored epithelial cells Stat2 levels but did not inhibit type I IFN signaling restored IFN-␣–dependent phosphorylation of Stat1 and Stat2 in cells infected with RSV (Figure 7D). Taken together, the results indicate that RSV acts on epithelial cells in the airway to directly modulate the type I IFN JAK-STAT pathway, and this effect is likely mediated through proteasomedependent degradation of Stat2.

Discussion Epithelial cells are often targeted for respiratory virus infection in the airway, but actively participate in the antiviral response by releasing and responding to IFNs. Because IFN-dependent effects are critical for limiting and clearing viral infections, it has been proposed that a prerequisite for successful viral invasion and replication in host cells is a mechanism for avoiding effects of IFNs (22, 36). This report extends our understanding of paramyxovirus effects on host cells and indicates that RSV selectively inhibits the type I IFN JAK-STAT signaling pathway in airway epithelial cells, resulting in reduced and delayed antiviral gene expression. In contrast, we found that RSV has little effect on type II IFN-induced signaling or gene expression. RSV effects on type I IFN signaling appears to be mediated through degradation of Stat2 that is likely proteasome-dependent. Decreased

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Figure 6. RSV infection also inhibits IFN-␤ signaling. (A ) IFN-␣ and IFN-␤ protein levels released from hTBE cells were determined using enzyme-linked immunoassays with media from cell monolayers that were left uninfected or were infected with RSV at m.o.i 15 for the indicated durations. Values are expressed as mean ⫾ SD (n ⫽ 3), and ND ⫽ none detected. (B ) MxA, total Stat2, and ␤-actin protein levels were determined using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV or ultraviolet light–inactivated RSV at m.o.i 15 for 6 h, followed by incubation without and with 1,000 U/ml of IFN-␤ for 12 h. (C ) Phosphorylated and total Stat1 and Stat2 and ␤-actin protein levels were assessed using immunoblot analysis of extracts from hTBE cell monolayers that were left uninfected or were infected with RSV or ultraviolet light–inactivated RSV at m.o.i. 15 for 24 h, followed by incubation without or with IFN-␤ for 15 min.

Figure 7. RSV infection likely induces proteasomedependent inhibition of Stat2 expression. (A ) Stat2 expression was assessed using immunoblot analysis of extracts from hTBE cell monolayers that were pretreated with control DMSO or 2.1 ␮M MG-132, and then were left uninfected or were infected with RSV at m.o.i. 15 for 24 h. (B ) IRF-9 cellular location was assessed by immunofluorescence microscopy of hTBE cell monolayers that were pretreated with control DMSO or 2.1 ␮M MG-132, and then were left uninfected or were infected with RSV at m.o.i. 15 for 24 h. IRF-9 was detected using anti-IRF-9 primary antibody and fluorescein-conjugated secondary antibody. Scale bar, 20 ␮m. (C ) IRF-9 cellular location was quantified by determining the percent of total cells with nuclear ⬎ cytoplasmic immunostaining. Each condition represents the mean ⫾ SD for five microscopic fields from experiment shown in B (40–80 cells/field), and a significant difference from uninfected and untreated cells is indicated by an asterisk. (D ) Stat1 and Stat2 expression and phosphorylation were assessed using immunoblot analysis of extracts from hTBE cell monolayers that were pretreated with control DMSO or 0.7 ␮M MG-132, and then were left uninfected or were infected with RSV at m.o.i. 15 for 24 h, followed by incubation without or with IFN-␣ for 15 min.

Stat2 levels in epithelial cells infected with RSV are seen as early as 6 h after initiation of infection (before IFN-␤ generation by airway epithelial cells), and persist for at least 48 h (until initial viral replication is complete). Therefore, RSV effects on IFN-dependent gene expression appear to correlate with initial airway infection, with establishment before IFN-induced antiviral systems have been activated (Figure 8). The capacity to inhibit IFN effects in host cells has coevolved in several pathogens, and is currently an area of intense research (12, 37). Viruses use several targeted strategies to modify antiviral effects of IFNs including suppression of IFN production, release of soluble inhibitory factors, blockade of IFN signaling cascade component expression or function, or direct modulation of specific IFN-inducible antiviral proteins (12, 22). Examples include inhibition of IFN-␤ production by hepatitis B virus, generation of a soluble receptor that blocks binding of IFN-␣ to cell surface receptors by vaccinia virus, expression of proteins that bind and inactivate intracellular Tyk2 and IRF-9 by human papilloma virus, and production of a protein that binds and inactivates PKR by hepatitis C virus (36, 38–41). Furthermore, adenovirus type 5 inhibits type II IFN-dependent gene expression or function by downregulation of the level of the IFNGR2 chain of the IFN-␥ receptor, expression of the E1A oncoprotein that interacts with and functionally inactivates Stat1 and its transcriptional coactivator p300/CBP, and expression of the E3 protein

Figure 8. Model for RSV effects on type I IFN signal transduction. During initial infection, RSV inhibits type I IFN-dependent gene expression by decreasing Stat2 levels. This viral effect is established prior to RSV induction of type I IFN release by cells in the airway, allowing subversion of antiviral effects of IFN-␣ and IFN-␤.

Ramaswamy, Shi, Monick, et al.: RSV Inhibition of Type I Interferon Signaling

that inhibits MHC class I transport to the cell surface (23, 24, 42, 43). Thus, the same virus may generate multiple mechanisms for modulation of IFN-dependent immunity. The family Paramyxoviridae includes parainfluenza viruses, mumps virus, measles virus, simian virus 5, Sendai virus, and Nipah virus, as well as RSV (11). Although genotypically related, these viruses have evolved a variety of different strategies to counteract the antiviral effects of IFNs, and these viral mechanisms have recently been defined in some members of the family (10, 11). For example, human parainfluenza virus type 2 inhibits type I IFN signaling by decreasing host cell levels of Stat2 (as we have shown for RSV), whereas mumps virus and simian virus 5 inhibit type I and II IFN signaling by decreasing levels of Stat1 (10, 34, 35, 44). Sendai virus inhibits both type I and II IFN signaling through blockade of IFN-dependent tyrosine phosphorylation of Stat1 and Stat2 and production of a protein that binds Stat1 (45). The newly recognized Nipah virus produces a protein that binds Stat1 and Stat2 and inhibits activation (46). Thus, a common theme for paramyxoviral subversion of IFNdependent antiviral effects is through inhibition of IFN signal transduction. Although RSV is resistant to the antiviral effects of type I IFN, previous studies directed at understanding the basis for this effect generated conflicting results. RSV appears to have the capacity to specifically bypass the effects of type I IFNinduced MxA and other undefined antiviral proteins (9). In addition, human RSV that lack viral NS1 and NS2 nonstructural proteins are attenuated, and studies with bovine RSV indicate that these viral proteins cooperatively inhibit type I IFN-induced antiviral effects (47, 48). However, some reports that assessed IFN signaling have suggested that human RSV has minimal effects on the function of IFN-dependent JAK-STAT pathways. Atreya and Kulkarni reported that A549 respiratory epithelial cells infected with RSV strain A2 maintained IFN-mediated MxA expression and inhibition of human parainfluenza virus type 3 replication when epithelial cells were treated with type I IFN prior to infection (9). Based on our results, IFN pretreatment of epithelial cells in their experiments would not allow time for expression of viral mechanisms that inhibit type I IFNdependent signaling and gene expression. Young and colleagues reported that type I IFN-mediated promoter activation, ISGF3 formation, and Stat1 and Stat2 levels were not decreased after 8–16 h of infection with RSV strain A2 in 2fTGH human diploid fibroblast cells (10). However, we have found that fibroblasts and other nonepithelial cells require longer durations of infection to manifest RSV effects on type I IFN signaling (results not shown). Bossert and colleagues recently reported that MDBK bovine kidney epithelial cells infected with bovine RSV strain A51908 maintained IFN-mediated MxA expression (49). For these experiments, cells were infected at m.o.i. 0.2, and this viral inoculum may be too low for detection of RSV effects on type I IFN-dependent gene expression. Our results do correlate with the observation that prolonged RSV infection can cause lowlevel activation of type I IFN-inducible genes (32). Thus, differences between our studies and other reports may be due to differences in experimental systems including cell culture conditions, viral strain, infection inoculum, timing of infection relative to IFN treatment, or use of airway epithelial cells (the natural host cell for this virus). Our model system is designed to test conditions during initial viral infection of human airway epithelial cells prior to IFN generation. We speculate that RSV evolution was driven to generate mechanisms to decrease Stat2 levels because high airway levels of type I IFN that are commonly induced by the virus could inhibit viral replication (5, 7). In contrast, it is possible that RSV evolution did not generate mechanisms to inhibit type II IFN

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signaling because IFN-␥ levels in the airway are often below the level required to inhibit viral gene expression during infection (50, 51). RSV is similar to other paramyxoviruses in inhibition of type I IFN by interaction with a STAT protein, despite its lack of P gene–encoded C or V proteins that inhibit IFN signaling in other paramyxoviruses. Induction of decreased Stat2 expression by RSV appears most similar to proteasome-dependent degradation of Stat2 that is mediated by the V protein of human parainfluenza virus type 2 (34), but the RSV protein(s) that mediate this effect are not known. A better understanding of viral mechanisms for altering IFN-dependent immune responses may allow for the development of therapeutic strategies that block this viral capacity during infection of airway epithelium. Acknowledgments: The authors gratefully acknowledge the University of Iowa Center for Gene Therapy and Genentech for generous gifts of cells or reagents, and thank S. Brody, M. Welsh, and J. Zabner for helpful discussion. This research was supported by grants from the National Institutes of Health, Environmental Protection Agency, Veterans Administration, Cystic Fibrosis Foundation, and American Lung Association.

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