3-Nitrotyrosine attenuates respiratory syncytial virus infection in human bronchial epithelial cell line

Yuh-Chin T. Huang,1 Zhuowei Li,2 Luisa E. Brighton,2 Johnny L. Carson,2 Susanne Becker,1 and Joleen M. Soukup1

1National Health and Environmental Effects Research Laboratory, Office of Research and Development, Environmental Protection Agency, Research Triangle Park, and 2Center for Environmental Medicine, Asthma and Lung Biology, The University of North Carolina, Chapel Hill, North Carolina

Submitted 8 October 2004 ; accepted in final form 9 January 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
3-Nitrotyrosine (NO2Tyr), an L-tyrosine derivative during nitrative stress, can substitute the COOH-terminal tyrosine of {alpha}-tubulin, posttranslationally altering microtubular functions. Because infection of the cells by respiratory syncytial virus (RSV) may require intact microtubules, we tested the hypothesis that NO2Tyr would inhibit RSV infection and intracellular signaling via nitrotyrosination of {alpha}-tubulin. A human bronchial epithelial cell line (BEAS-2B) was incubated with RSV with or without NO2Tyr. The release of chemokines and viral particles and activation of interferon regulatory factor-3 (IRF-3) were measured. Incubation with NO2Tyr increased nitrotyrosinated {alpha}-tubulin, and NO2Tyr colocalized with microtubules. RSV-infected cells released viral particles, RANTES, and IL-8 in a time- and dose-dependent manner, and intracellular RSV proteins coprecipitated with {alpha}-tubulin. NO2Tyr attenuated the RSV-induced release of RANTES, IL-8, and viral particles by 50–90% and decreased {alpha}-tubulin-associated RSV proteins. 3-Chlorotyrosine, another L-tyrosine derivative, had no effects. NO2Tyr also inhibited the RSV-induced shift of the unphosphorylated form I of IRF-3 to the phosphorylated form II. Pre-exposure of the cells to NO2 (0.15 ppm, 4 h), which produced diffuse protein tyrosine nitration, did not affect RSV-induced release of RANTES, IL-8, or viral particles. NO2Tyr did not affect the potential of viral spreading to the neighboring cells since the RSV titers were not decreased when the uninfected cells were cocultured with the preinfected cells in NO2Tyr-containing medium. These results indicate that NO2Tyr, by replacing the COOH-terminal tyrosine of {alpha}-tubulin, attenuated RSV infection, and the inhibition appeared to occur at the early stages of RSV infection.

RANTES; microtubules; tubulin; interferon regulatory factor; interleukin-8


IN VITRO AND IN VIVO STUDIES have indicated that several nitrating species, including nitrogen dioxide, peroxynitrite, and nitrous acid, can be produced during inflammation. This nitrative stress is evidenced by the presence of nitrated protein tyrosine in the tissues (9, 43) and by increased free 3-nitrotyrosine (NO2Tyr) in biological fluids (13, 23). Experimental evidence has indicated that nitration of tyrosine residues in proteins may alter protein function and protein turnover and modify signal transduction processes (24). The biological effects of free NO2Tyr, however, are less clear. NO2Tyr inhibited acetylcholine-induced relaxation of rat thoracic aorta (29), and NO2Tyr produced behavior abnormalities in mice associated with reduction in striatal tyrosine hydroxylase content (30). NO2Tyr can also be incorporated into {alpha}-tubulin, causing microtubular dysfunction in A549 cells (10) and preventing morphological differentiation of myogenic cells (7). The latter mechanism can potentially affect intracellular signaling processes that are dependent on microtubules (MT) (20).

As a cytoskeletal component, MT cooperate with actin filaments functionally in a wide variety of processes, e.g., vesicle and organelle transport, directed cell migration, and nuclear migration, that may be important during viral infection (4, 17). MT-dependent motilities allow cytosolic adenovirus to be directed toward the MT-organizing center near the nucleus (46). Dynein, a minus-end-directed MT motor, coprecipitates with cytosolic herpes simplex virus capsids (44). Movement of endosomal reovirus can be blocked by colchicine or nocodazole, inhibitors for MT polymerization (16).

Respiratory syncytial virus (RSV), a negative-stranded RNA virus, is a common viral pathogen for respiratory infection in both children and immunocompromised adults. It is estimated that up to 50% of children hospitalized with bronchiolitis and 25% of children with pneumonia are infected with RSV (21). RSV infection causes a predisposition to the development of hyperreactive airway disease (53) and precipitates recurrent attacks in asthmatic children. The mechanisms of RSV-induced airway disease are incompletely understood, but local inflammatory responses that generate various chemotactic chemokines, e.g., RANTES (regulated upon activation, normal T-cell expressed and secreted) and interleukin-8 (IL-8), appear to play a central role (35, 42, 49).

Lung epithelial cells are the main target of RSV. Infection of RSV is mediated, in part, by initial interaction between attachment proteins (F and G) and a heparan sulfate located on the cell surface (11). Once inside the cells, RSV replicates and assembles with cytoskeletal components. Cellular actin is found inside RSV (51). Transcription of the RSV genome RNA requires actin as well as an actin-modulatory protein, profilin (5, 6). The uptake of RSV by dendritic cells can be inhibited by agents that block caveolae (54), the internalization of which is dependent on an intact actin network (37).

The integral role of MT in intracellular viral processing and the close association of MT with actin in maintaining cytoskeletal function indicate that MT may play an important role in RSV infection. If so, NO2Tyr, which may irreversibly modify {alpha}-tubulin posttranslationally, may inhibit signal transduction during RSV infection. The present study tested this hypothesis in a human bronchial epithelial cell line.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Cell culture. The human bronchial epithelial cell line (BEAS-2B, subclone S6) was obtained from the laboratory of Dr. Curtis C. Harris and maintained in serum-free growth medium (KGM; Clonetics, San Diego, CA) in T-75 tissue culture flasks. The cells were used in experiments in their 62nd–73rd passage. Cells were plated at 1 x 105 cell/well of a 12-well tissue culture plate (Costar, Cambridge, MA) in 1 ml of KGM and incubated at 37°C and 5% CO2 for 72 h, when they were used in experiments.

Preparation of RSV. RSV (Long strain/lot 15D) was obtained from American Type Culture Collection (ATCC, Bethesda, MD) and was propagated in mycoplasma-free HEp2 cells (ATCC 23-CCL) as previously described (3). HEp2 supernatants containing infectious RSV were collected, and the virus was precipitated with 10% polyethylene glycol (Sigma, St. Louis, MO). The precipitate was dissolved in NTE (50 mM Tris·HCl, 150 mM NaCl, 1 mM EDTA), pH 7.4, and overlaid on a discontinuous 60, 45, and 30% sucrose gradient made up in NTE. After centrifugation for 90 min at 85,000 g in a Sorvall TH-641 rotor, the virus was collected from the 45–60% interface. This preparation contained ~1 x 107 plaque-forming units (PFU)/ml of RSV when tested for syncytia formation on HEp2 cells. The virus was snap frozen in liquid nitrogen and stored in small aliquots at –70°C until use.

Plaque assay. RSV in the medium and the cell lysate (PFU/ml) was determined by syncytia formation on HEp2 cells induced by serial 1:10 dilutions of the cell supernatants as has been described (36). After allowing the virus to adsorb to the cells for 2 h, we removed the supernatant dilutions and added 1 ml of overlay methylcellulose (1.2%, 6000cP, Sigma) in Dulbecco's modified Eagle's medium supplemented with 2% fetal bovine serum (Life Technologies, Gaithersburg, MD).

RSV infection. Confluent monolayer cultures of BEAS-2B in 12-well tissue culture plates were infected with RSV at a multiplicity of infection (MOI) of 0.1–1 PFU/cell (45). Virus was added to the cells for 2 h and then removed by a gentle wash with culture medium, followed by addition of 1 ml/well KGM for culture for up to 72 h.

In additional experiments, the uninfected BEAS-2B cells were cocultured with RSV-infected BEAS-2B cells to determine the potential of viral spreading to the neighboring cells in the presence or absence of NO2Tyr. BEAS-2B cells were incubated with RSV at 1.0 MOI for 2 h and then allowed to grow in the culture medium for 24 h. These preinfected cells were washed with fresh medium, removed by trypsinization, and overlaid on the uninfected cells, which had been incubated with or without NO2Tyr for 24 h according to the protocol below. These preinfected cells provided an intracellular source of RSV that would infect the bottom layer of cells via cell-cell spreading. After 2 h of coculture, these preinfected cells were removed by a gentle wash with culture medium, and the bottom cells were replenished with fresh medium without or without NO2Tyr and maintained for an additional 72 h.

RANTES and IL-8 measurements. RANTES and IL-8 in the medium were measured with ELISA kits purchased from R&D Systems (Minneapolis, MN) according to the manufacturer's directions. The detection limit for the kits was 10 pg/ml.

Incubation with NO2Tyr and 3-chlorotyrosine. KGM containing NO2Tyr was added to BEAS-2B culture plates 24 h before incubation of cells with RSV. Preliminary experiments showed that it took at least 24 h before an increase in nitrotyrosinated {alpha}-tubulin was detectable by Western blot. Giving NO2Tyr 24 h before RSV would allow time for the cells to incorporate NO2Tyr into {alpha}-tubulin. In experiments with 3-chlorotyrosine, 3-chlorotyrosine was also added to the medium 24 h before RSV infection. At the end of 24 h, the medium was removed and RSV was added. After 2 h of incubation, the cells were washed several times with medium to remove RSV, and fresh medium containing either NO2Tyr or 3-chlorotyrosine was added. The cells were cultured for up to 72 h. KGM contains ~15 µM of L-tyrosine.

NO2 exposure. Cells cultured at an air-surface interface were exposed to NO2 for 4 h in a CO2-water-jacketed incubator (IR Autoflow; NuAire, Plymouth, MN). NO2 was delivered to the incubator from a source tank containing 5,000 ppm of NO2 (Air Products and Chemicals, Allentown, PA). The NO2 concentration in the incubator was monitored continuously with a chemiluminescence NO-NO2-NOx analyzer (model 42; Thermo Environmental Instruments, Franklin, MA). The concentration of NO2 in the incubator was within 5% of the desired final concentration. After NO2 exposure, the cells were then moved to regular incubator without NO2 for RSV infection.

Western blotting and immunoprecipitation. Cells were harvested and lysed with a 23-gauge needle in radioimmunoprecipitation assay (RIPA) lysis buffer, which consists of 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS in PBS, pH 7.4, and protease inhibitors (1 mM vanadyl sulfate, 0.5 mg/ml aprotinin, 0.5 mg/ml E-64, 0.5 mg/ml pepstatin, 0.5 mg/ml bestatin, 10 mg/ml chymostatin, and 0.1 mg/ml leupeptin). The lysate was centrifuged at 10,000 g for 10 min. The supernatant was decanted, and an aliquot was stored at –20°C. Protein concentrations were measured in all samples by the method of Bradford (Bio-Rad Protein Assay; Bio-Rad Laboratories, Hercules, CA).

For immunoprecipitation, cell homogenates in 400 µl of RIPA buffer were precleared with 5 µl of protein A-agarose (Santa Cruz Biotechnology, Santa Cruz, CA) for 30 min at 4°C. After a brief centrifugation at 10,000 g for 1 min, supernatant was transferred to a fresh tube and then incubated with primary antibody against a target protein by end to end rotation for 2 h at 4°C. To precipitate the antigen-antibody complexes, 20 µl of resuspended protein A-agarose were added to every tube. After an overnight mixing at 4°C, pellets of immune complexes were collected by centrifugation, washed twice with lysis buffer and once with cold PBS, and finally resuspended in 30 µl of sample loading buffer and boiled for 5 min before being subjected to SDS-PAGE and Western blotting. Prestained molecular-weight markers were run on adjacent lanes. Proteins were then electroblotted onto nitrocellulose. The blots were blocked with nonfat milk, washed, and incubated overnight with a primary antibody and a secondary horseradish peroxidase-conjugated antibody. Protein bands were detected using enhanced chemiluminescence reagents and film according to the manufacturer's instructions (Amersham Pharmacia Biotech, Piscataway, NJ). The primary antibodies used in this study were monoclonal antibody against human {alpha}-tubulin (clone DM1A, Sigma), goat anti-RSV antibody (United States Biological, Swampscott, MA), polyclonal nitrotyrosine antibody (Upstate Biotechnology, Lake Placid, NY), and monoclonal antibody against human interferon regulatory factor-3 (IRF-3) (catalog no. sc-15991, Santa Cruz Biotechnology).

Immunocytochemistry. Double immunostaining was performed to colocalize RSV with {alpha}-tubulin. BEAS-2B grown on slides were fixed with 4% paraformaldehyde and blocked with 5% donkey serum in 3% BSA/Tris-buffered saline (TBS). The fixed cells were probed with a polyclonal anti-RSV antibody and a monoclonal antibody against {alpha}-tubulin overnight at 4°C followed by incubation with appropriate FITC and rhodamine-conjugated secondary antibodies for 30 min. Images were examined with a fluorescence microscope equipped with FITC and rhodamine filters. Photos were taken with a digital camera system (Nikon Microphot-SA) and imaging software (ACT-1, version 2.10; Nikon, Tokyo, Japan).

Confocal laser scanning microscopy. Culture wafers were washed with TBS and blocked with 20% serum. Cultures were incubated overnight at 4°C with a rabbit antinitrotyrosine antibody diluted 1:50 and mouse anti-{alpha}-tubulin antibody diluted 1:100. Cultures were washed in TBS following incubation, and FITC-anti-mouse and rhodamine-anti-rabbit secondary antibodies (Molecular Probes, Eugene, OR) were applied to the surface of the culture and incubated for 1 h. Wafers were then washed in TBS, positioned on a glass microslide, and mounted with Vector 4',6-diamidino-2-phenylindole dihydrochloride mounting medium. Wafers were viewed under a Leica SP2 laser scanning confocal microscope (Leica Microsystems, Bannockburn, IL). Filters and lasers were set up for viewing of FITC (488 nm) and rhodamine (594 nm) in sequential scanning.

Statistical analysis. All data are expressed as means ± SE. Time series data were analyzed by repeated-measure analysis of variance. For comparison of two groups at different time points and doses, the unpaired t-test was used and adjusted for multiple comparisons by the Bonferroni's correction. The statistical analysis was performed using StatView (version 5.0.1, SAS, Cary NC). A P value of <0.05 was considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Nitrotyrosination of {alpha}-tubulin. We first determined whether the human bronchial epithelial cell line could incorporate NO2Tyr into {alpha}-tubulin. Figure 1, A and B, shows time-dependent increases in nitrotyrosinated {alpha}-tubulin in bronchial epithelial cells incubated with 100 µM NO2Tyr. Figure 1, C and D, shows nitrotyrosination of {alpha}-tubulin in cells treated with NO2Tyr for 48 h. The quantity of nitrotyrosinated {alpha}-tubulin was more for 100 µM than 10 µM NO2Tyr. At 100 µM NO2Tyr, which would give an NO2Tyr-L-tyrosine ratio in the medium of 7:1, the increase in nitrotyrosinated {alpha}-tubulin was ~500%. Confocal microscopy showed NO2Tyr was taken up by the cells and colocalized with MT (Fig. 2). There was also staining in the nucleus. Cells incubated with up to 1 mM NO2Tyr-containing medium for 72 h showed no LDH release (data not shown).



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Fig. 1. Nitrotyrosination of {alpha}-tubulin in cells supplemented with 3-nitrotyrosine (NO2Tyr). Cells were incubated with NO2Tyr for up to 72 h. The cell lysates were immunoprecipitated with an {alpha}-tubulin antibody followed by immunoblotting with an antinitrotyrosine antibody. Representative Western blot (A) and the densitometry result (B) showing time-dependent increase in nitrotyrosination of {alpha}-tubulin (n = 3 independent experiments). NO2Tyr concentration was 100 µM; 250 µg of protein were used for immunoprecipitation. Representative Western blot (C) and the densitometry result (D) showing concentration-dependent increase in nitrotyrosination of {alpha}-tubulin (n = 3 independent experiments). The incubation time was 48 h; 80 µg of protein were used for immunoprecipitation. *P < 0.01 vs. 24 h or 0 µM NO2Tyr.

 


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Fig. 2. Confocal microscopy showing the uptake of NO2Tyr by bronchial epithelial cells (red) and colocalization with microtubules (green). The cells were incubated with 100 µM NO2Tyr for 48 h.

 
RSV infection. Bronchial epithelial cells incubated with RSV increased the release of RANTES and IL-8 in a time- and dose-dependent manner (Fig. 3, A and B). Increases in RANTES and IL-8 were ~10-fold and threefold respectively at 1.0 MOI after 72 h of incubation compared with the control. RSV titer in the medium also increased with time and with increasing MOI (Fig. 3C). Many intracellular RSV proteins coprecipitated with {alpha}-tubulin (Fig. 3D). Three major RSV proteins, N (also known as N-RNA template), P (the phosphoprotein), and M (the transcription elongation factor), could be identified based on their molecular weights and the migration patterns (12). RSV immunoreactivity also colocalized with {alpha}-tubulin (Fig. 4).



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Fig. 3. Dose- and time-dependent release of regulated upon activation, normal T-cell expressed and secreted (RANTES), IL-8, and respiratory syncytial virus (RSV) by bronchial epithelial cells after RSV infection. Cells were incubated with 0.1, 0.3, or 1.0 multiplicity of infection (MOI) of RSV for up to 72 h. A: changes ({Delta}) in RANTES; B: {Delta} in IL-8; C: increases in RSV titer in the medium (from uninfected cells cultured for 72 h); D: increases in the quantity of intracellular RSV proteins associated with {alpha}-tubulin. The approximate positions of N, P, and M proteins are indicated based on their molecular weights (MW) and migration patterns. *P < 0.001 vs. no virus, #P < 0.001 vs. 24 h; n = 4 independent experiments for each time point and each MOI. The Western blot was representative of 3 independent experiments.

 


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Fig. 4. Colocalization of RSV (green) with {alpha}-tubulin (red). Cells were incubated with RSV for 2 h at 1.0 MOI. RSV was then removed, and the cells were cultured for 48 h. At the end of the 48-h incubation, the cells were fixed and stained with anti-RSV antibody and {alpha}-tubulin antibody followed by an FITC-conjugated and a rhodamine-conjugated secondary antibodies, respectively. Bar = 10 µm.

 
Effects of NO2Tyr on RSV infection. NO2Tyr inhibited the RSV-induced release of RANTES and IL-8 (Fig. 5, A and B). The inhibition of RANTES and IL-8 was ~60 and 50%, respectively, at 72 h. Incubation of RSV with up to 1 mM NO2Tyr in the cell-free solution did not affect the viability of RSV, suggesting that NO2Tyr was not toxic to RSV (data not shown).



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Fig. 5. Effects of NO2Tyr on RANTES and IL-8 release and RSV titer. Cells were incubated with 100 µM NO2Tyr for 24 h before incubation with RSV. A: changes in RANTES with time, MOI = 1.0; B: changes in IL-8 with time, MOI = 1.0; C: changes in RSV titer, MOI = 1.0; D: dose-dependent inhibition of NO2Tyr, MOI = 1.0. The viral titer was measured 48 h after RSV incubation. *P < 0.01 vs. no nitrotyrosine; n = 4–5 independent experiments.

 
NO2Tyr also inhibited extracellular release of RSV postinfection (Fig. 5C), and the inhibition reached ~90% at 72 h. The inhibition was more prominent at 100 µM and 1 mM NO2Tyr (Fig. 5D).

NO2Tyr also decreased the amount of {alpha}-tubulin-associated RSV protein (Fig. 6). The inhibition for the N, P, and M proteins was ~30, 80, and 70%, respectively, at 48 h postinfection (Fig. 6). RSV proteins did not show tyrosine nitration, indicating that NO2Tyr was not incorporated by the virus during viral replication (data not shown).



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Fig. 6. Effects of NO2Tyr on {alpha}-tubulin-associated RSV proteins. Cells were incubated with 100 µM NO2Tyr for 24 h followed by RSV infection at 1.0 MOI. The cell lysates at 48 h postinfection were immunoprecipitated with an {alpha}-tubulin antibody followed by Western blotting with an RSV antibody. A: representative Western blot showing decrease in N, P, and M protein associated with {alpha}-tubulin ({alpha}-Tub). Densitometry results for N (B), P (C), and M (D) proteins, respectively. *P < 0.05 vs. no nitrotyrosine; n = 4 independent experiments.

 
Effects of 3-chlorotyrosine on RSV infection. To determine whether or not the inhibitory effects were specific for NO2Tyr, we performed additional experiments with 3-chlorotyrosine, another naturally occurring L-tyrosine derivative. 3-Chlorotyrosine had little effect on the release of RANTES or RSV (Table 1).


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Table 1. Effects of 3-chlorotyrosine on RSV infection

 
Effects of NO2 exposure on RSV infection. To determine whether or not the NO2Tyr effects were specifically due to nitrotyrosination of {alpha}-tubulin, we pre-exposed bronchial epithelial cells to NO2, which would induce nonselective nitration of protein and nonprotein tyrosine residues in the cells, before infection with RSV. As expected, exposure of BEAS cells to 0.15 ppm of NO2 for 4 h increased diffuse tyrosine nitration of many cellular proteins including {alpha}-tubulin by Western blotting, but pre-exposure to NO2 did not alter RSV-induced RANTES, IL-8, or viral release (Table 2).


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Table 2. Effects of NO2 exposure on RSV infection

 
Effects of NO2Tyr on IRF-3 activation. Because activation of IRF-3 occurs early during viral infection (41), we further investigated whether or not RSV activated IRF-3 and whether or not NO2Tyr inhibited RSV-induced IRF-3 activation. The uninfected cells expressed approximately equal amounts of form I and form II of IRF-3. Treatment of cell lysate with acid phosphatase decreased the intensity of form II (data not shown), indicating that form II is a phosphorylated form of IRF-3 (41). RSV infection induced a shift from form I to form II, while total IRF-3 remained unchanged (Fig. 7). NO2Tyr prevented the shift and returned the II/I ratio to a level similar to that of the uninfected cells.



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Fig. 7. Effects of NO2Tyr on RSV-induced activation of interferon regulatory factor-3 (IRF-3). Cells were preincubated with or without NO2Tyr (100 µM) followed by incubation with RSV at 1.0 MOI. Cells lysates (5 µg protein) at 48 h postinfection were then immunoblotted with an IRF-3 antibody. A representative Western blot (A) and the densitometry results (B) are shown; n = 4 independent experiments. *P < 0.05 vs. control; #P < 0.05 vs. RSV.

 
Effects of NO2Tyr on RSV spreading. To determine whether or not NO2Tyr affected viral spreading to neighboring cells after viral uptake, we preinfected a batch of cells with RSV for 24 h. These preinfected cells were then overlaid on the uninfected BEAS-2B cells, which had been cultured in medium with or without NO2Tyr (100 µM) for 24 h. NO2Tyr did not affect the RSV titer in the medium or in the cells in these coculture experiments (Table 3). Fusion and large syncytia formation still occurred in the presence of NO2Tyr.


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Table 3. Effects of NO2Tyr on RSV titers after the uninfected BEAS-2B cells were cocultured with cells preinfected with RSV

 
Effects of NO2Tyr on cytokine mixture-induced chemokine release. We further examined whether or not NO2Tyr inhibited chemokine release induced by nonviral stimuli. The cells were treated with a cytokine mixture consisting of TNF-{alpha} (10 ng/ml) and IFN-{gamma} (10 ng/ml) for 48 h with or without 100 µM NO2Tyr. NO2Tyr inhibited the release of RANTES and IL-8 induced by the cytokine mixture by ~50% (Fig. 8).



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Fig. 8. Effects of NO2Tyr on the release of RANTES (A) and IL-8 (B) induced by a cytokine mixture (10 ng/ml TNF-{alpha} and 10 ng/ml IFN-{gamma}). Cells preincubated with or without NO2Tyr (100 µM) for 24 h were treated with the cytokine mixture for 48 h. The control RANTES and IL-8 concentrations at 48 h were 13 ± 1 pg/ml and 33 ± 3 pg/ml, respectively. *P < 0.05 vs. no nitrotyrosine; n = 6–8 independent experiments.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
During RSV infection, the virus utilizes some cytoskeletal factors for transcription of its genomes. Purified viral nucleocapsids contain cellular actin, and viral transcription was optimized in the presence of actin and profilin, a cytoskeletal modulatory protein (5, 6). Different viruses also have been shown to exploit MT for genome transcription. {beta}-Tubulin is necessary for the synthesis of Sendai virus, vesicular stomatitis virus RNA (33, 47), and measles virus RNA (32). Our present study showed that RSV infection in human bronchial epithelial cells may also utilize MT since the RSV proteins coprecipitated with {alpha}-tubulin. How MT are utilized by RSV is unclear, but experimental evidence using other viruses has shown that the intracellular movement of these viruses between the nuclear and cell surface is facilitated by MT (16, 22, 44, 46).

NO2Tyr attenuated the RSV-induced release of RANTES, IL-8, and viral particles. These effects were associated with nitrotyrosination of {alpha}-tubulin. The inhibition was not observed with 3-chlorotyrosine, another L-tyrosine derivative, or diffuse protein tyrosine nitration produced by NO2 exposure. Thus these findings were consistent with the proposed mechanism for NO2Tyr on the posttranslational tyrosination-detyrosination at the COOH terminus of {alpha}-tubulin (10). Nitrotyrosinated {alpha}-tubulin is resistant to detyrosination catalyzed by carboxypeptidase (10, 25), whereas incorporation of 3-halotyrosine, e.g., 3-fluorotyrosine, is reversible (31). This may explain why 3-chlorotyrosine had no effects. We could not exclude other unidentified mechanisms of NO2Tyr since NO2Tyr immunoreactivity was also seen in the nucleus. Incorporation of nitrotyrosinated {alpha}-tubulin increases chemical heterogeneity of MT and alters electric charges on MT surface. MT containing nitrotyrosinated {alpha}-tubulin may no longer provide a suitable platform on which translational machinery and signal transduction take place. This hypothesis was further supported by the inhibitory effects of NO2Tyr on the release of RANTES and IL-8 induced by a nonviral stimulus, TNF-{alpha}/IFN-{gamma} mixture. NO2 exposure induced tyrosine nitration of cellular proteins including {alpha}-tubulin (data not shown), but its lack of effects on RSV infection may be due to nonspecific nitration of tyrosine residues in other cellular proteins. The nitrated proteins may be degraded more rapidly with subsequent activation of new protein synthesis (24).

The role of MT in regulating signal transduction has been recognized (20). A large number of signal pathways are closely associated with MT. For example, I{kappa}B, a negative regulator of NF-{kappa}B, has been shown to interact with dynein light chain (8), and MT depolymerization leads to I{kappa}B destruction, allowing NF-{kappa}B to bind to DNA and stimulate transcription of RANTES (39, 50). In A549 cells, nitrotyrosination of {alpha}-tubulin decreases affinity of dynein heavy chain to {alpha}-tubulin, which might affect the MT minus-end-directed vesicular transport (10). In endothelial cells, MT may be important in increasing permeability induced by TNF-{alpha}-activated p38 mitogen-activated protein kinase (38). In epithelial cells, adenomatous polyposis coli protein is localized to clusters at peripheral membrane sites near the end of MT, indicating that MT may be involved in polarizing Wnt signal transduction so that it is active in only part of the cell (34). In myoblasts, NO2Tyr treatment prevents synthesis or activation of many markers of myogenesis, including muscle-specific myosin, {alpha}-actin, integrin-{alpha}, and myogenin (7).

Nitrotyrosination of {alpha}-tubulin appeared to affect RSV infection at its early stages. First, our study showed that RSV activated an early transcription factor, IRF-3, during viral infection by shifting the unphosphorylated form I to the phosphorylated form II, and this shift was inhibited by NO2Tyr. IRF-3 belongs to a family of nine IRFs that are central to host defense against extracellular pathogens (48). During viral infection, multiple activated forms of IRF-3, including the unphosphorylated form II produced by phosphorylation of the NH2-terminal Ser residue (41) and forms III–V produced by phosphorylation of the COOH-terminal Ser-Thr clusters (27, 28), may be detected as early as 4 h postinfection. Second, when the uninfected cells were cocultured with the cells in which RSV infection had been established, NO2Tyr had no effect, indicating that the potential for viral spreading to the neighboring cells was unaffected. Early processes during RSV infection that may be affected by NO2Tyr may include viral uptake, intracellular transport by MT, and viral transcription. Many of these functions may be linked to RhoA, a small GTP binding protein in the Ras superfamily that can be activated early during RSV infection and influences a variety of biological functions, including organization of actin stress fibers, production of cytokines, and interaction with the F glycoprotein of RSV (2, 18)

In the original study on the antiviral effects of IFN-{gamma}, the inhibition of replication of ectromelia, vaccinia, and herpes simplex-1 viruses in mouse macrophages correlated positively with the endogenous production of nitric oxide (NO) (26). Numerous studies have since extended these findings to NO produced from different sources against many different viruses. For example, exogenous NO released from a donor chemical inhibits rhinovirus replication and the production of IL-8 and IL-6 in a human epithelial cell line (40). Endogenous NO produced by nitric oxide synthase-transfected HEp2 cells correlated inversely with the titer of RSV (1). Because NO is an important source of nitrogen for the formation of nitrating species, our results may provide a potential mechanism for the inhibitory effects of NO on viral infections, especially when the microenvironment also is favorable for the generation of other essential components of nitrative stress, such as during inflammation (myeloperoxidase, eosinophil peroxidase) (52, 55).

The concentration of exogenous NO2Tyr used in our study was relatively high compared with the endogenous concentration detected in the body fluids. Under physiological conditions, the concentration of endogenous free NO2Tyr in the plasma is <100 nM (14, 23) but can increase by 80- to 100-fold in the joint fluid of rheumatoid arthritis and up to 100 µM in the plasma of patients with septic shock and renal failure (15). The level of NO2Tyr in these biological fluids, however, may not represent the local concentration in close proximity to the site of production of nitrating species (19). Thus protein and nonprotein tyrosine residues at the vicinity of injury site and within specific cell types may encounter much higher local concentration of nitrating species and have a higher probability of being nitrated. Our study showed for the first time that free NO2Tyr, generally considered a biomarker of nitrative stress, attenuates RSV infection and signaling at high concentrations. These effects may be related to the formation of nityrosinated {alpha}-tubulin altering the MT properties. These results indicate that exogenous NO2Tyr may be used as an immunomodulatory agent if given at early stages of RSV infection.


    DISCLOSURES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
The research described in this article has been reviewed by the Health Effects and Environmental Research Laboratory, United States Environmental Protection Agency and has been approved for publication. Approval does not signify that the contents necessarily reflect the views and policies of the Agency, nor does mention of the trade names or commercial products constitute endorsement or recommendation for use.


    ACKNOWLEDGMENTS
 
The authors thank Lisa Dailey for assistance in cell culture and Dr. Raymond Pickles at Cystic Fibrosis Center of the University of North Carolina for reading the manuscript.


    FOOTNOTES
 

Address for reprint requests and other correspondence: Y.-C. T. Huang, CB 7315, 104 Mason Farm Rd., Chapel Hill, NC 27599 (E-mail: huang.tony{at}epa.gov)

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    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 

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