Respiratory syncytial virus nonstructural protein 2 specifically inhibits type I interferon signal transduction

Virology 344 (2006) 328 – 339 www.elsevier.com/locate/yviro

Respiratory syncytial virus nonstructural protein 2 specifically inhibits type I interferon signal transduction Murali Ramaswamy a, Lei Shi a, Steven M. Varga b, Sailen Barik c, Mark A. Behlke d, Dwight C. Look a,* b

a Department of Internal Medicine, University of Iowa Carver College of Medicine, 200 Hawkins Drive, Iowa City, IA 52242, USA Department of Microbiology, University of Iowa Carver College of Medicine, 3-532 Bowen Sciences Building, Iowa City, IA 52242, USA c Department of Biochemistry and Molecular Biology, University of South Alabama College of Medicine, 307 University Blvd., Room MSB 2370, Mobile, AL 36688, USA d Integrated DNA Technologies, 1710 Commercial Park, Coralville, IA 52241, USA

Received 20 June 2005; returned to author for revision 5 July 2005; accepted 7 September 2005 Available online 10 October 2005

Abstract Human respiratory syncytial virus (RSV) inhibits type I interferon-induced gene expression by decreasing expression of signal transducer and activator of transcription (Stat)2. To identify the RSV protein that mediates effects on Stat2, airway epithelial cells were infected with vaccinia virus vectors that express single RSV proteins. Expression of RSV nonstructural (NS)2 protein alone was sufficient to decrease Stat2 levels. Furthermore, decreasing RSV NS2 levels using RNA interference in respiratory epithelial cells inhibited the RSV-mediated decrease in Stat2 expression. Airway epithelial cells were also infected with equivalent inoculums of RSV without or with single gene deletions of NS1 or NS2. RSV infection without NS2 expression did not result in decreased Stat2 levels or loss of type I interferon-dependent signaling, indicating that NS2 expression is necessary for RSV effects on Stat2. Taken together, our results indicate that NS2 regulates Stat2 levels during RSV infection, thereby modulating viral effects on interferon-dependent gene expression. D 2005 Elsevier Inc. All rights reserved. Keywords: Airway epithelial cell; JAK-STAT pathway; RNA interference

Introduction Human respiratory syncytial virus (RSV) has generated intense research interest since its identification over 40 years 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 (Sigurs, 2001). RSV is an enveloped, nonsegmented, single-stranded, negative sense RNA virus in the subfamily Pneumovirinae of the family Paramyxoviridae (Collins et al., 2001). The RSV genome encodes 11 proteins, and shares several genes with other paramyxoviruses that include nucleocapsid proteins (N,

* Corresponding author. Fax: +1 319 353 6406. E-mail address: [email protected] (D.C. Look). 0042-6822/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.virol.2005.09.009

P, and L), surface proteins (F and G), and a matrix protein (M). Other viral proteins that RSV shares with paramyxoviruses have less well-defined functions and include SH and M2-1. Proteins in RSV that are not clearly homologous with other paramyxoviruses include M2-2, and the nonstructural proteins NS1 and NS2. RSV gains entry into host cell by cell-surface fusion, replicates in the cytoplasm, and acquires a lipid envelope from the host cell plasma membrane by budding. Epithelial cells are the primary site of viral replication in the airway, with RSV entry and exit primarily through the apical surface (Zhang et al., 2002). 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 (Decker et al., 2002). RSV is a potent inducer of type I interferon release from infected airway epithelial cells (Garofalo et al., 1996; Jamaluddin et al., 2001; Ramaswamy et al., 2004). Type I interferons (IFN-a and IFN-

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h) mediate host cell effects by binding to a specific receptor complex linked to a Janus family kinase-signal transducer and activator of transcription (JAK-STAT) signaling cascade (Taniguchi and Takaoka, 2001). Activation of the type I interferon-driven pathway is triggered by engagement and multimerization of the IFN-a-receptor (IFNAR) by IFN-a or IFN-h, phosphorylation of IFNAR1-associated Tyk2 and IFNAR2-associated Jak1 tyrosine kinases, and then phosphorylation of IFNAR1 and IFNAR2 (Darnell et al., 1994; Platanias et al., 1994). Phosphorylation of the IFNAR1 chain of the IFN-a receptor results in recruitment, phosphorylation, and subsequent release of Stat2 and Stat1 from the receptor (Leung et al., 1995; Qureshi et al., 1996). Activated Stat2 and Stat1 associate with interferon regulatory factor (IRF)-9 to form the transcriptional activator complex interferon-stimulated gene factor 3 (ISGF3), which translocates to the nucleus and binds specific DNA recognition sequences to activate transcription of type I interferon-inducible genes (Horvath et al., 1996). Many genes are induced by type I interferon, but several, including MxA, 2V,5V-oligoadenylate synthase (2,5OAS), and protein kinase R (PKR), are more specific to type I interferon and have antiviral properties that are particularly important for establishment of a host cell antiviral state (Katze et al., 2002). 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 common theme for paramyxoviral subversion of interferon-dependent antiviral effects is through inhibition of interferon signal transduction. Although paramyxoviruses are genotypically related, these viruses have evolved a variety of different strategies to counteract the antiviral effects of interferons (Gotoh et al., 2001; Young et al., 2000). We have reported previously that RSV selectively inhibits the type I interferon JAK-STAT signaling pathway in airway epithelial cells resulting in reduced and delayed antiviral gene expression, but has little effect on type II interferon (IFNg)-induced signaling or gene expression (Ramaswamy et al., 2004). RSV effects on type I interferon signaling is mediated through degradation of Stat2 that is likely proteasomedependent. RSV lacks the C and V accessory proteins that mediate interferon resistance in many paramyxoviruses and the proteins produced by human RSV that modulate interferon signaling are being defined (Lo et al., 2005; Spann et al., 2004, 2005). In this report, we demonstrate that expression of the RSV NS2 protein is necessary and sufficient for inhibition of type I interferon-dependent signaling. Expression of NS2 resulted in decreased airway epithelial cell Stat2 expression with consequent blockade of type I interferon-dependent gene expression. Furthermore, decreased NS2 levels in RSV-infected cells using RNA interference or viral mutation inhibited RSV effects on Stat2. Our results support the concept that specific RSV proteins modulate immune response gene expression in airway epithelial cells, which may allow for evasion of the airway defense response and establishment of a productive infection.

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Results RSV NS2 expression is sufficient to decrease epithelial cells Stat2 levels To begin identification of RSV proteins that modulate Stat2 levels in human airway epithelial cells, 9 of the 11 RSV proteins were individually expressed in human tracheobronchial epithelial (hTBE) cells using recombinant vaccinia viruses under conditions in which the majority of the cells in the sample were infected. Expression of RSV M and P protein had a slight effect on Stat2 expression, but RSV NS2 expression markedly decreased Stat2 levels as detected using immunoblot analysis of epithelial cells infected with vector for 24 h (Fig. 1A). Control vaccinia vector or expression of the other RSV proteins had little effect on Stat2 under these conditions. The possibility that decreased Stat2 levels were due to NS2-induced epithelial cell cytotoxicity was excluded by assessing epithelial cell uptake of ethidium homodimers, and these results indicated no significant cytotoxic effects of NS2 expression (Fig. 1B). Similar to wild-type RSV infection (Ramaswamy et al., 2004), Stat2 levels were not altered after expression of NS2 in hTBE cells that were pretreated with the cell-permeable nonspecific proteasome inhibitor MG-132 (Fig. 1C). These results suggest that RSV NS2 is sufficient to decrease Stat2 levels in airway epithelial cells, and through this effect modulates type I interferon signaling and gene expression. Therefore, subsequent experiments were directed at determining whether NS2 is required to decrease epithelial cell Stat2 levels during RSV infection. RNA interference of NS2 expression modulates RSV effects on Stat2 levels To manipulate RSV protein levels, we initially tested RNA interference strategies that require transfection of sequence-specific 21 or 25 –27 nucleotide double-stranded RNA molecules into A549 respiratory epithelial cells (Bitko and Barik, 2001; Kim et al., 2004). Experiments using vaccinia virus vectors to express RSV NS1 and NS2 indicated that a 21 nucleotide double-stranded small interfering (si)RNA significantly decreased NS2, but not NS1, mRNA levels (Fig. 2A). In contrast, multiple RNA interference strategies to decrease NS1 were tested, but for unclear reasons, all affected NS2 levels in addition to inhibiting NS1 expression. A 25 – 27 nucleotide double-stranded siRNA against NS1 gave the best specificity, as some NS2 protein was detectable after infection of epithelial cells with vaccinia virus expressing NS2 (Fig. 2B). The effect of NS2 expressed by vaccinia virus on Stat2 levels in A549 cells was inhibited using siRNA directed against NS2, but not NS1 or control siRNA (Fig. 2C). These preliminary results support the possibility that decreased Stat2 levels after RSV infection are mediated by NS2 expression. RNA interference was then tested on viral protein expression after wild-type RSV infection. We found that

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with potent and specific silencing of NS2 expression using the 21 nucleotide siRNA, RSV lost the capacity to inhibit Stat2 expression in epithelial cells (Fig. 3A). The reported lack of requirement for NS2 in expression of other viral proteins was confirmed when we observed that inhibition of expression of this viral protein had little effect on expression of RSV P (Fig. 3A) and other viral proteins (results not shown) after infection. Experiments using siRNA against NS1 were less informative because siRNA tested were less potent and specific, despite the use of two different strategies (Figs. 3B and C). Both RNA interference strategies against NS1 caused a decrease in NS2 expression that was similar, but more complete after RSV infection compared to vaccinia vector expression of RSV proteins. As expected, we observed that siRNA directed at the P gene resulted in a global decrease in RSV protein expression including NS2. Under all conditions, restoration of Stat2 levels in RSV-

infected cells correlated with loss of NS2 expression. Taken together, our RNA interference experiments were consistent with the possibility that NS2 is necessary for RSV-induced Stat2 degradation, but did not completely exclude a role for other viral genes. RSV is a potent inducer of IFN-h release from respiratory epithelial cells, although RSV effects on type I interferon signaling are identical in epithelial cells exposed to IFN-a or IFN-h (Jamaluddin et al., 2001; Ramaswamy et al., 2004; Spann et al., 2004). However, RNA interference may also activate type I interferon production and antiviral gene expression in some systems, and thus could potentially attenuate viral replication and RSV-induced decreases in Stat2 levels (Sledz et al., 2003). We were reassured that our RNA interference systems had minimal confounding effects by the observation that control siRNA did not cause a global decrease in viral gene expression (Fig. 3). In addition, transfection of siRNA molecules into A549 cells did not cause detectable IFN-h release, nor significantly affect virus-induced IFN-h release in RSV-infected cells (Fig. 4A). Because RSV is a relatively poor inducer of IFNa release from airway epithelial cells, we thought it was unlikely that other type I interferon release is activated by our RNA interference systems (Ramaswamy et al., 2004; Spann et al., 2004). However, to support this conclusion, interferon signaling pathway activation and gene expression were also assessed. Type I and type II interferon-driven transcription of antiviral genes in airway epithelial cells requires tyrosine phosphorylation of Stat1 that is mediated through specific JAK-STAT pathways (Darnell et al., 1994). In experiments using immunoblot analysis with phosphorylation-specific Stat1 antibodies to detect activation of this transcription factor, siRNA did not induce Stat1 activation 30 min or 24 h after transfection (Fig. 4B). This finding correlated with a lack of detectable increase in expression of the type I interferon-inducible genes MxA or PKR in epithelial cells exposed to siRNA. Fig. 1. RSV NS2 expression is sufficient to decrease Stat2 levels. (A) Stat2, RSV proteins (using polyclonal antiserum that recognizes multiple viral proteins), RSV NS1 and NS2 (using polyclonal antiserum that reacts more strongly with NS1 compared to NS2), and h-actin (to verify equivalent protein isolation and loading) protein levels in hTBE cells were assessed using immunoblot analysis. Extracts for analysis were from cell monolayers that were left uninfected or were infected at MOI 10 for 24 h with vaccinia virus vectors that express control or individual RSV proteins. The positions of Stat2, NS1, NS2, and h-actin are indicated by arrows, and detection of NS1 and NS2 required two different exposures of the immunoblot membrane. (B) 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. Cells were left uninfected or infected with vaccinia virus vectors at MOI 10 for 24 h. Values were calculated as live cells/total cells, each condition represents the mean T SD for 3 microscopic fields (90 – 200 cells/ field), and a statistically significant difference between levels in any condition was not detected. (C) Stat2, and RSV NS1 and NS2 protein levels in hTBE cells were assessed using immunoblot analysis of extracts from cell monolayers that were pretreated with control DMSO or 2.1 AM MG-132, and then were left uninfected or were infected at MOI 10 for 24 h with vaccinia virus vectors that express control or individual RSV proteins. Ponceau S staining of the membrane was used to verify equivalent protein isolation and loading.

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RSV with deletion of the NS2 or NS1 genes were also tested for effects on Stat2 expression (Jin et al., 2000). For these experiments, hTBE cells were infected under conditions that resulted in infection of the majority of cells in the samples as verified by immunofluorescence staining for viral protein expression. Effects of NS gene deletion on viral gene mRNA levels were assessed using real-time RT-PCR analysis, and the results confirmed selective loss of NS1 or NS2 mRNA expression (Fig. 5A). Furthermore, there were decreased RSV M gene (Fig. 5A) and P gene (Fig. 5B, and results not shown) expression in cells infected with NS1- or NS2deficient RSV compared to wild-type virus. These results correlate with a previous report indicating restricted replication of RSV deficient in either NS gene in respiratory epithelial cells (Spann et al., 2004). However, a key finding was that infection with either wild-type RSV or RSV deficient in NS1 expression resulted in decreased epithelial cell levels of Stat2 (Fig. 5B). In contrast, RSV without NS2 expression lost the capacity to alter Stat2 levels. Moreover, RSV that expressed NS2 inhibited Stat1 phosphorylation in response to IFN-a, while RSV deficient in NS2 lost this capacity (Fig. 5C). The results correlate with our results using RNA interference and indicate that NS2 expression is necessary for RSV inhibition of Stat2 expression and Stat1 activation in airway epithelial cells. RSV that express NS2 specifically affect Stat2 expression and antiviral gene expression Fig. 2. RNA interference of NS2 expression modulates NS2 effects on Stat2 levels. (A) RSV NS1 and NS2 mRNA levels in A549 respiratory epithelial cells were determined using real-time RT-PCR analysis of samples from cell monolayers that were initially left untreated or were transfected for 4 h with control siRNA or siRNA against NS1 (using a 21 nucleotide siRNA design) (Bitko and Barik, 2001) or NS2 (using a 25 – 27 nucleotide siRNA design) (Kim et al., 2004). Cell monolayers were then infected at MOI 10 for 24 h with vaccinia virus vectors that express RSV NS1 (solid bars) or NS2 (open bars). NS1 levels in cell samples infected with vector that expresses NS1, and NS2 levels in cell samples infected with vector that expresses NS2 are presented, and NS1 and NS2 mRNA was not detected in samples infected with vectors that express the opposite RSV protein. Values are expressed as mean mRNA level compared to control hypoxanthine phosphoribosyltransferase (HPRT) mRNA level T SD for duplicate samples, and a significant difference in mRNA levels compared to cells treated with the corresponding control siRNA is indicated by an asterisk. (B) NS2, NS1, and h-actin protein levels in A549 cells were assessed using immunoblot analysis of extracts from cell monolayers that were initially transfected with siRNA against NS1 or NS2 as in panel A. Cell monolayers were then left uninfected or infected at MOI 10 for 24 h with vaccinia virus vectors that express RSV NS1 or NS2. (C) Stat2 and h-actin protein levels in A549 cells were assessed using immunoblot analysis of extracts from cell monolayers that were initially left untreated or were transfected with control siRNA or siRNA against NS1 or NS2 as in panel A. Cell monolayers were then left uninfected or infected at MOI 10 for 24 h with vaccinia virus vectors that express RSV NS1 or NS2.

NS2 expression is necessary for RSV to inhibit type I interferon signaling Based on the lack of definitive evidence in our RNA interference experiments that excluded a role for viral proteins other than NS2 in RSV modulation of Stat2 levels,

Modulation of Stat2 levels is an important mechanism for inhibition of type I interferon signaling in airway epithelial cell infected with RSV, resulting in intact upstream signaling events, but loss of Stat2-dependent functions (Ramaswamy et al., 2004). Based on our findings concerning RSV NS2 effects on Stat2 levels, we performed additional experiments directed at assessment of mutant virus effects on upstream and downstream components in the signaling cascade. We confirmed that wild-type and NS1-deficient RSV were similar in their ability to inhibit IFN-a-induced Stat2 expression and phosphorylation after as little as 6 – 12 h of infection, and this effect correlated with expression of NS2 (Fig. 6A). In contrast, NS2-deficient RSV had little effect on IFN-a-dependent phosphorylation of Stat2. Immunofluorescence staining for viral proteins indicated that the multiplicity of infection (MOI) of 15 based on plaque titration assay using Vero cells and used for these experiments resulted in infection of 88– 93 T 2% of hTBE cells by wild-type and mutant RSV. Thus, incomplete loss of Stat2 expression and antiviral gene induction in many of our experiments likely reflects incomplete infection of all cells, as well as the relatively early time points after inoculation that were examined. Epithelial signaling events upstream of Stat2 appeared to be maintained with all RSV mutants tested because infection of hTBE cells with wild-type, NS1-deficient, or NS2-deficient RSV had little effects on IFN-a-induced phosphorylation of Tyk2 (Fig. 6B). Similarly, no effects of wild-type or mutant RSV on type II interferon (IFN-g) activation of Stat1 were demonstrated,

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Fig. 3. RNA interference of NS2 expression modulates RSV effects on Stat2 levels. (A) Stat2, and RSV NS2, NS1, and P protein levels in A549 respiratory epithelial cells were assessed using immunoblot analysis of extracts from cell monolayers that were initially left untreated or were transfected with control siRNA or siRNA against NS2 (using a 21 nucleotide siRNA design) (Bitko and Barik, 2001), and then were left uninfected or were infected with RSV at MOI 15 for 24 h. (B) Stat2, and RSV NS2, NS1, and P protein levels in A549 cells were assessed using immunoblot analysis of extracts from cell monolayers that were initially left untreated or were transfected with control siRNA or siRNA against NS1 (using a 25 – 27 nucleotide siRNA design) (Kim et al., 2004), and then were left uninfected or were infected with RSV at MOI 15 for 24 h. (C) Stat2, and RSV NS2, NS1, and P protein levels in A549 cells were assessed using immunoblot analysis of extracts from cell monolayers that were initially left untreated or were transfected with siRNA against NS1 or P (using a similar 21 nucleotide siRNA design as in panel A) (Bitko and Barik, 2001), and then were left uninfected or were infected with RSV at MOI 15 for 24 h. In panels A – C, staining of proteins on the membrane with Ponceau S dye was used to verify equivalency of protein isolation and loading.

thereby verifying the specificity of RSV effects on type I interferon signaling (Fig. 6C). To assess viral effects on interferon-dependent gene expression, we tested viral effects on Vero cells that do not release IFN-a or h, thereby allowing exogenous administration of type I interferons with precise control over the timing and levels of interferon exposure during experiments. Both wild-type and NS1-deficient RSV significantly inhibited IFN-a-induction of MxA expression in Vero cells, while NS2-deficient RSV had much less effect (Fig. 7A). We were unable to confirm effects on MxA protein levels in Vero cells using immunoblot analysis, which is likely due to the relatively low level of expression in these cells, even with interferon treatment. However, wild-type and NS1deficient RSV inhibited IFN-a-induction of PKR protein

expression in Vero cells, while infection with NS2-deficient RSV had minimal effects on PKR levels after IFN-a treatment compared to uninfected control (Fig. 7B). These results correlate RSV NS2 regulation of type I interferondependent signaling with antiviral gene expression. The effects of mutant viruses on antiviral gene expression were also tested in hTBE cells using quantitative real-time RTPCR analysis. Unlike Vero cells, human respiratory epithelial cells are competent for type I interferon release, with increased levels starting to appear after 12 – 14 h of infection (Ramaswamy et al., 2004; Spann et al., 2004; and results not shown). MxA mRNA expression was induced after wild-type RSV infection of hTBE cells, and this effect was markedly decreased in cells infected with NS1-deficient RSV (Fig. 7C). In contrast, NS2-deficient RSV infection induced this

Fig. 4. RNA interference effects on the epithelial cell type I interferon system. (A) IFN-h release from hTBE cells was determined using an enzyme-linked immunoassay with cell monolayers that were initially left untreated or were transfected with the indicated concentration of control siRNA, and then were left uninfected or were infected with RSV at MOI 15 for 24 h. Values are expressed as mean T SD (n = 3), and ND = none detected. (B) MxA, PKR, and phosphorylated and total Stat1 protein levels in A549 cells were assessed using immunoblot analysis of extracts from cell monolayers that were left untreated or were transfected with 100 nM control siRNA or siRNA against NS1 or NS2 followed by incubation in media for 0.5 or 24 h. To assure cell response to type I interferon, some samples were incubated with 1000 units/ml of IFN-a for the same durations to allow for comparison.

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Discussion It has been proposed that a prerequisite for successful viral invasion and replication in host cells is a mechanism for avoiding effects of interferons (Barnard and McMillan, 1999; Sen, 2001). For RSV, the observation that type I interferon inefficiently inhibits viral replication in human epithelial cells fits this paradigm (Atreya and Kulkarni, 1999; Gotoh et al., 2001; Loveys et al., 2000; Young et al., 2000). Based on our experiments, we conclude that RSV uses the NS2 protein to subvert the antiviral effects of type I interferon after its release.

Fig. 5. NS2 expression is necessary for RSV to inhibit type I interferon signaling. (A) RSV NS1 (closed bar), NS2 (open bar), and M (gray bar) gene mRNA levels in hTBE cells were determined using real-time RT-PCR analysis of samples from cell monolayers that were left uninfected or were infected at MOI 15 for 24 h with wild-type RSVor RSV deficient in NS1 or NS2. Values are expressed as mRNA level compared to control HPRT mRNA level. (B) Stat2, RSV P, and h-actin protein levels in hTBE cells were assessed using immunoblot analysis of extracts from cell monolayers that were left uninfected or were infected at MOI 15 for 24 h with wild-type RSVor RSV deficient in NS1 or NS2. (C) Phosphorylated and total Stat1, RSV P, and h-actin protein levels in hTBE cells were assessed using immunoblot analysis of extracts from cell monolayers that were left uninfected or were infected at MOI 15 for 12 h with wild-type RSVor RSV deficient in NS1 or NS2, followed by incubation without or with 1000 units/ml of IFN-a for 30 min.

antiviral gene to a higher level than wild-type RSV. Finally, MxA expression in response to RSV infection with NS gene silencing using RNA interference was assessed in A549 cells. Epithelial cell infection with wild-type RSV combined with siRNA against NS2 resulted in a significant increase in MxA mRNA expression compared to RSV without gene silencing. These results indicate that RSV uses NS2 expression to inhibit type I interferon-dependent signaling and antiviral gene expression. Although other effects of NS genes cannot be excluded, RSV effects on type I interferon production and signaling appear to allow viral subversion of interferon-dependent antiviral effects.

Fig. 6. RSV that express NS2 specifically affect Stat2 expression. (A) Phosphorylated and total Stat2, RSV NS2, NS1, and P, and h-actin protein levels in hTBE cells were assessed using immunoblot analysis of extracts from cell monolayers that were left uninfected or were infected at MOI 15 for the indicated duration with wild-type RSV or RSV deficient in NS1 or NS2, followed by incubation without or with 1000 units/ml of IFN-a for 15 min. (B) Phosphorylated and total Tyk2 protein levels in hTBE cells were assessed using immunoblot analysis of extracts from cell monolayers that were left uninfected or were infected at MOI 15 for 24 h with wild-type RSV or RSV deficient in NS1 or NS2, followed by incubation without or with IFN-a for 15 min. (C) Phosphorylated and total Stat1 protein levels in hTBE cells were assessed using immunoblot analysis of extracts from cell monolayers that were left uninfected or were infected at MOI 15 for 18 h with wild-type RSV or RSV deficient in NS1 or NS2, followed by incubation without or with 1000 units/ml of IFN-a or 100 units/ml of IFN-g for 30 min.

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Experiments using vaccinia virus vectors to express individual RSV proteins suggested that NS2 protein expression is sufficient for inhibition of epithelial cell Stat2 expression. Subsequent studies in which specific viral protein expression

was decreased or lost using RNA interference systems or recombinant RSV with single gene deletions demonstrated a requirement for the NS2 protein in inhibition of Stat2 expression. Thus, RSV NS2 protein is both sufficient and necessary for inhibition of Stat2 expression, thereby decreasing type I interferon-dependant signaling and gene expression. Viruses use several targeted strategies to modify antiviral effects of interferons classified broadly into: (1) minimizing interferon production; (2) blocking interferon receptors; (3) inhibiting interferon signaling; and (4) bypassing antiviral protein effects (Ploegh, 1998; Sen, 2001). Use of viral ‘‘accessory’’ proteins to mediate interferon resistance has been well described in the Paramyxoviridae family of viruses to which RSV belongs. However, although paramyxoviruses are genotypically related, these viruses have evolved a variety of different strategies to counteract the antiviral effects of interferons (Andrejeva et al., 2002; Gotoh et al., 2001; Young et al., 2000). For example, human parainfluenza virus type 2 inhibits type I interferon signaling by decreasing host cell levels of Stat2 (as has been shown for RSV), while mumps virus and simian virus 5 inhibit type I and II interferon signaling by decreasing levels of Stat1 (Kubota et al., 2001; Parisien et al., 2001; Ulane and Horvath, 2002; Young et al., 2000). Sendai virus inhibits both type I and II interferon signaling through blockade of interferon-dependent tyrosine phosphorylation of Stat1 and Stat2 and production of a protein that binds Stat1 (Gotoh et al., 2003). The newly recognized Nipah virus produces a protein that binds and inhibits activation of Stat1 and Stat2 (Rodriquez et al., 2002). Thus, a common theme for paramyxoviral subversion of interferondependent antiviral effects is through inhibition of signal transduction by viral protein interaction with STAT molecules. RSV is similar to many viruses in its ability to modulate interferon-dependent immune defense through multiple mechanisms. Our finding that RSV inhibits Stat2 expression

Fig. 7. NS2 deletion alters antiviral gene expression in airway epithelial cells. (A) MxA mRNA levels in Vero cells were determined using real-time RTPCR analysis of samples from cell monolayers that were left uninfected or were infected at MOI 15 for 6 h with wild-type RSV or RSV deficient in NS1 or NS2, followed by incubation without or with 1000 units/ml of IFN-a for 12 h. (B) PKR, RSV P, and h-actin protein levels in Vero cells were assessed using immunoblot analysis of extracts from cell monolayers that were left uninfected or were infected at MOI 15 for 6 h with wild-type RSV or RSV deficient in NS1 or NS2, followed by incubation without or with 1000 units/ ml of IFN-a for 12 h. (C) MxA mRNA levels in hTBE cells were determined using real-time RT-PCR analysis of samples from cell monolayers that were left uninfected or were infected at MOI 15 for 24 h with wild-type RSV or RSV deficient in NS1 or NS2. (D) MxA mRNA levels in A549 cells were determined using real-time RT-PCR analysis of samples from cell monolayers that were initially left untreated or were transfected with control siRNA, or siRNA against NS1 (using a 25 – 27 nucleotide siRNA design) (Kim et al., 2004) or NS2 (using a 21 nucleotide siRNA design) (Bitko and Barik, 2001), and then were left uninfected or were infected with wild-type RSV at MOI 15 for 18 h. In panels A, C, and D, values are expressed as mean mRNA level compared to control HPRT mRNA level T SD (n = 2 – 3), and a significant difference in mRNA levels compared to cells left uninfected but interferontreated in panel A, cells infected with wild-type RSV in panel C, and cells treated with control siRNA and infected with RSV in panel D is indicated by an asterisk.

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and type I interferon responsiveness has been reproduced by other research groups (Lo et al., 2005). RSV also uses the NS1 and NS2 proteins to suppress the induction of type I and II interferons (Spann et al., 2004, 2005). Viral effects on interferon release are likely important because interferons have important effects on nearby uninfected cells, although RSV effects on type I interferon signaling likely decrease antiviral effects in infected cells. However, other reports suggest that human RSV has minimal effects on the function of interferondependent JAK-STAT pathways. Atreya and Kulkarni reported that A549 respiratory epithelial cells infected with RSV strain A2 maintained interferon-mediated MxA expression and inhibition of human parainfluenza virus type 3 replication when epithelial cells were treated with type I interferon prior to infection (Atreya and Kulkarni, 1999). Based on our results, interferon pretreatment of epithelial cells in their experiments would not allow time for expression of viral mechanisms that inhibit type I interferon-dependent signaling and gene expression. Young et al. (2000) reported that type I interferonmediated 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. However, we have found that human fibroblasts and other nonepithelial cells require longer durations of infection to manifest RSV effects on type I interferon signaling (results not shown). 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 interferon 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 interferon generation. There is growing evidence indicating that human RSV nonstructural proteins function to subvert interferon-dependent immunity. This is in light of the observation that RSV lacks proteins with significant sequence homology to the C and V accessory proteins that mediate interferon resistance in many paramyxoviruses. RSV NS1 and NS2 proteins are deemed ‘‘accessory proteins’’ due to their initial unknown functions, the ability of the virus to replicate when these genes are deleted, and the historical difficulty involved in detection of these proteins in virus replicating cell systems. However, replication of recombinant human RSV that lack NS1 and/or NS2 expression is attenuated in systems that produce type I interferon, including cultured cells, mice, and primates, but is restored when grown in interferon-deficient Vero cells (Jin et al., 2000, 2003; Teng and Collins, 1999; Teng et al., 2000; Whitehead et al., 1999). Furthermore, the NS1 and NS2 proteins of bovine RSV have been shown to suppress the induction of type I interferon and activation of the IFNmediated antiviral state, and attenuated growth of NS1- and NS2-deleted bovine RSV is also restored in Vero cells (Bossert and Conzelmann, 2002; Bossert et al., 2003; Schlender et al., 2000; Valarcher et al., 2003). The mechanism for this effect appears to be through suppression of the activation of IRF-3 by bovine RSV NS1 and NS2 (Bossert et al., 2003). Bovine RSV

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NS1 and NS2 proteins share a 69% and 84% amino acid identity with the respective proteins in human RSV subgroup A (Pastey and Samal, 1995). Thus, it is not surprising that human RSV inhibits type I interferon release through a similar effect of NS1 and NS2 on IRF-3 (Spann et al., 2004; Spann et al., 2005). RSV also appears to have the capacity to specifically bypass the effects of type I interferon-induced MxA and other undefined antiviral proteins (Atreya and Kulkarni, 1999). Thus, RSV uses a common set of proteins to affect multiple mechanisms that modulate interferon-dependent immunity, highlighting the importance of bypassing this system in order to allow efficient virus propagation. The use of primary human airway epithelial cells (the natural host cell in the airway) was emphasized in most of our experiments. However, the use of RNA interference presented a relatively novel approach to study the function of RSV NS genes. For these experiments, altered viral gene expression was required in the majority of cells in the sample in order to detect effects on Stat2. A549 respiratory epithelial cells displayed similar RSV effects on Stat2 expression, but provided better transfection efficiency than hTBE cells (results not shown). Specific inhibition of NS2 expression was accomplished with RNA interference and suggested an important role for this viral protein in modulation of type I interferon signaling. Although other reports indicate successful inhibition of NS1 expression using siRNA, the reasons for incomplete separation of NS1 siRNA effects from NS2 despite the use of multiple strategies in our experiments are unclear (Zhang et al., 2005). Thus, to more completely separate the effects of the two RSV nonstructural proteins, subsequent experiments were performed with recombinant virus deficient in NS gene expression. Previous reports indicated that deletion of RSV NS1 or NS2 impaired viral replication in airway epithelial cells in the presence of endogenous (or exogenous) type I interferon, but had less effect in Vero cells that do not release type I interferon in response to virus infection (Lo et al., 2005; Spann et al., 2004). This not only supported the possibility that NS genes played a role in interferon resistance, but also supported the possibility that the use of recombinant viruses would allow separation of functional effects of the two viral genes. For our experiments, Vero cells were used for initial confirmation of inhibition of type I interferon-dependent gene (MxA and PKR) expression during infections in which NS2 was expressed. Interestingly, subsequent studies with hTBE cells under conditions of endogenous interferon release indicated that MxA levels were higher after infection with NS2-deleted RSV, indicating higher sensitivity to interferonor noninterferon-dependent induction. Indeed, this occurred under conditions in which NS2-deleted RSV induces lower levels of IFN-h release (Spann et al., 2004). In contrast, with NS1-deleted RSV, the expression of MxA in hTBE cells was lower despite comparable or increased levels of type I interferon release compared to wild-type virus infection, suggesting that the presence of NS2 in NS1-deleted RSV still inhibits type I interferon-dependent signaling and subsequent gene expression (Spann et al., 2004). Similar augmentation of MxA expression was seen after A549 cell

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infection with RSV in which NS2 gene expression was silenced by RNA interference. A difference in results between experiments using mutant RSV and RNA interference was seen under conditions with loss of NS1 expression, and this is likely due to nonspecific NS1 siRNA effects that decrease NS2 expression and thereby allow functional type I interferon signaling. Thus, it appears that both RSV nonstructural proteins have complex and interacting effects on epithelial cell antiviral defense, and may have effects on viral replication independent of interferon release or signaling. When RSV NS1 and NS2 are present together, they perform different and complementary functions that provide greater viral resistance to interferon-dependent antiviral systems. We speculate that RSV evolution was driven to generate a mechanism to decrease Stat2 levels and thereby inhibit interferon signaling because airway levels of type I interferon that are commonly induced by the virus could inhibit viral replication (Garofalo et al., 1996; Jamaluddin et al., 2001). Although RSV NS1 partially suppresses type I interferon production in infected cells, interferon may also be released by leukocytes and other cells during viral infection (Diaz et al., 1999; Legg et al., 2003; Preston et al., 1995; Salkind et al., 1991; Spann et al., 2004, 2005). Thus, RSV NS1 and NS2 are likely both required for viral subversion of the antiviral effects of type I interferon. A better understanding of viral mechanisms for altering interferon-dependent immune responses may allow for the development of therapeutic strategies that block this viral capacity during infection of airway epithelium. Materials and methods Cell isolation, culture, and stimulation Human tracheobronchial epithelial 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 a Laboratory of Carcinogenesis (LHC)-8e medium on plates coated with collagen/albumin for study up to passage 10 as described previously (Aldallal et al., 2002; Look et al., 1992). The A549 respiratory epithelial cell line (American Type Culture Collection CCL-185, Manassas, VA) was cultured in RPMI 1640 medium supplemented with 10% heat inactivated (56 -C for 1 h) FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin, and 0.25 AM amphotericin B as described previously (Frick et al., 2000). The Vero African green monkey kidney cell line (American Type Culture Collection CCL-81) was cultured in minimum essential medium (Eagle) supplemented with 10% heat inactivated FBS, 2 mM l-glutamine, 100 U/ml penicillin, 100 Ag/ml streptomycin, 0.1 mM nonessential amino acids, and 1 mM sodium pyruvate. Cells were treated with recombinant human IFN-a2a (PBL Biomedical Laboratories, Piscataway, NJ) at a concentration (1000 units/ml) and for a duration (0.25 –24 h) that result in maximal effects on type I interferon JAK-STAT pathway component phosphorylation and gene expression. Some cells were treated with recombinant human IFN-g (a gift

from Genentech, San Francisco, CA) at a concentration (100 units/ml) and for a duration (0.5 h) that result in maximal effects on Stat1 phosphorylation. 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 RSV with deletion of the NS1 or NS2 genes (gifts from H. Jin, MedImmune, Gaithersburg, MD) (Jin et al., 2000) and wild-type RSV strain A2 were purified from Vero cell lysates by pelleting using centrifugation at 100,000  g for 45 min at 4 -C. RSV were incubated with epithelial cells for 6 –24 h in culture media at 37 -C in 5% CO2 at an MOI of 15, based on plaque titration assay using Vero cells. Efficiency of epithelial cell infection was determined using immunofluorescence staining for RSV proteins as described previously (Ramaswamy et al., 2004). Recombinant vaccinia viruses expressing a specific RSV protein or control h-galactosidase (gifts from G. Wertz, University of Alabama at Birmingham) were prepared as described previously (Ball et al., 1986; Cherrie et al., 1992; Johnson et al., 1998; Wertz et al., 1987). Primary antibodies Rabbit polyclonal IgG 06-502 against total human Stat2, rabbit polyclonal IgG 07-224 against phosphorylated human Stat2, and rabbit polyclonal IgG 06-638 against total human Tyk2 were from Upstate Biotechnology (Lake Placid, NY); goat polyclonal IgG against human RSV proteins was from Biodesign International (Saco, ME); rabbit polyclonal antiserum against RSV NS1 that faintly recognizes NS2 was from H. Jin (MedImmune); mouse IgG2a mAb clone AC-74 against human h-actin was from Sigma-Aldrich (St. Louis, MO); chicken polyclonal IgY against human MxA was from BacLab (Muttenz, Switzerland); rabbit polyclonal IgG 3072 against human PKR, rabbit polyclonal IgG 9171 against phosphorylated human Stat1, rabbit polyclonal IgG 9172 against total human Stat1, and rabbit polyclonal IgG 9321 against phosphorylated human Tyk2 were from Cell Signaling Technology (Beverly, MA). Immunoblot analysis Cell protein levels were assessed by immunoblot analysis using specific antibodies against human JAK-STAT pathway components as described previously (Joseph and Look, 2001; Look et al., 1998; Ramaswamy et al., 2004). 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 SDS-PAGE in 5– 18% polyacrylamide. Resolved proteins were electrophoreti-

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cally transferred to nitrocellulose membranes (Hybond, Amersham Biosciences, Piscataway, NJ), exposed to 5% nonfat milk in Tris-buffered saline with 0.1% Tween-20 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 Restorei buffer (Pierce, Rockford, IL) for 15 min at 37 -C. 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). RNA interference Sequence-specific 21 nucleotide double-stranded RNA molecules that target RSV mRNA sequences were synthesized (Dharmacon, Lafayette, CO) and transfected into A549 cells at a concentration of 100 nM using Lipofectamine2000 (Invitrogen, Carlsbad, CA) as described previously (Bitko and Barik, 2001). The following dsRNA sequences with 3V-extensions consisting of two 2V-deoxythymidines were used: (1) RSV NS1 gene forward 5V-GUGAUUCAACAAUGACCAATT-3V and reverse 5V-UUGGUCAUUGUUGAAUCACTT-3V; (2) RSV NS2 gene forward 5V-GACAUGAGACCGUUGUCACTT-3V and reverse 5V-GUGACAACGGUCUCAUGUCTT-3V; and (3) RSV P gene forward 5V-CGAUAAUAUAACUGCAAGATT-3V and reverse 5V-UCUUGCAGUUAUAUUAUCGTT-3V. Sequence-specific 25 – 27 nucleotide double-stranded RNA molecules that target RSV mRNA sequences were synthesized (Integrated DNA Technologies, Coralville, IA) and transfected into A549 cells at a concentration of 25 nM using Lipofectamine2000 as described previously (Kim et al., 2004). The following dsRNA sequences with the forward sequence containing a 5V-phosphorylation and two 2V-deoxynucleotides at the 3V-end and the reverse sequence containing a two nucleotide 3V-overhang were used: RSV NS1 gene forward 5VGGCAGCAAUUCAUUGAGUAUGAUAA-3V and reverse 5VUUAUCAUACUCAAUGAAUUGCUGCCCA-3V. Scrambled sequences were used as controls.

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Fluorescence Thermocycler (Bio-Rad, Hercules, CA) with SYBR Green I DNA dye (Molecular Probes, Eugene, OR), and Platinum Taq DNA Polymerase (iQ SYBR Green Supermix kit, BioRad). PCR conditions included denaturation at 95 -C 3 min, then for 35 cycles of 94 -C 30 s, 60 -C 30 s, and 72 -C 30 s, followed by 5 min at 72 -C for elongation. The following primers were used: (1) RSV NS1 gene forward 5VAGAGATGGGCAGCAATTCAT-3V and reverse 5V-CCATTAGGTTGAGAGCAATGTG-3V; (2) RSV NS2 gene forward 5V-ATGGACACAACCCACAATGA-3V and reverse 5VTGCCAATGCATTCTAAGAACC-3V; (3) RSV M gene forward 5V-TCACGAAGGCTCCACATACA-3V and reverse 5VGCCTTGATTTCACAGGGTGT-3V; (4) MxA gene forward 5V-GAAGCCCTGCAGAGAGAGAA-3V and reverse 5V-AACTCGTGTCGGAGTCTGGT-3V; and (5) human hypoxanthine phosphoribosyltransferase (HPRT) forward 5V-CCTCATGGA CTGATTATGGAC-3V and reverse 5V- C A G ATTCAACTTGCGCTCATC-3V. Fluorescence data were captured during the 10 s dwell to ensure that primer dimers were not contributing to the fluorescence signal, and specificity of the amplification was confirmed using melting curve analysis. Data were collected and recorded by iCycler iQ software (Bio-Rad) and initially determined as a function of threshold cycle (C t). Levels of mRNA are expressed relative to control HPRT levels, calculated as 2DCt. Enzyme-linked immunoassay IFN-h concentrations in cell culture media were determined using a highly selective, commercial sandwich ELISA kit (PBL Biomedical Laboratories, Piscataway, NJ). Statistical analysis Numerical data were analyzed for statistical significance using a one-way ANOVA for a factorial experimental design. The multicomparison significance level for the one-way analysis of variance was 0.05. If significance was achieved by one-way analysis, post-ANOVA comparison of means was performed using Scheffe F tests (Zar, 1984). Acknowledgments The authors gratefully acknowledge H. Jin, G. Wertz, Genentech, Integrated DNA Technologies, MedImmune, and the University of Iowa Center for Gene Therapy for generous gifts of cells, viruses, and reagents, and thank G. Hunninghake for helpful discussion. This research was supported by grants from the National Institutes of Health and Parker B. Francis Foundation.

Real-time reverse transcription PCR mRNA analysis

References

Total cellular RNA was isolated using a commercial spin column isolation kit (Stratagene, La Jolla, CA), and 0.5 Ag reverse transcribed using a RT-PCR kit (Ambion, Austin, TX). An aliquot of cDNA was subjected to PCR using an iCycler iQ

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