Sastre, Mount Sinai School of Medicine) and inserted into the pCAGGS vector [48,49,50]. The b-lactamase gene was PCR amplified from pcDNA3.1 and fused N-terminally to M1 within pCAGGS to create a modified b-lactamase-M1 fusion. In the modified b-lactamase, the first 24 amino acids were excluded to remove a secretion signal and His 24 was substituted with Asp to create an optimal Kozak consensus sequence. The pcDNA3.1-b- lactamase construct has been described previously [51]. The influenza A/Puerto Rico/8/34/Mount Sinai (H1N1) HA (pCAGGS-HA) and (pDZ-NA) over-expression vectors were generously provided by Dr. Aldolfo Garcia-Sastre and have been described previously [48,49,50,52]. Production of VLPs. To generate influenza A/PR/8/H1N1 b-lactamaseM1VLPs (PR8 b-lacM1 VLPs), 293T cells were co- transfected with 3 mg each of pCAGGS-HA, pDZ-NA, and pCAGGS-b-lacM1 PR8 using FuGENEH HD (Roche Applied Science, Indianapolis, IN) according to manufacturer’s instructions. The supernatant containing the VLPs were collect 48 h post-transfection and clarified of floating cell debris by centrifugation at 3,000 rpm for 10 min. The VLPs were concentrated once by low-speed centrifugation through an Amicon Ultra 100 kD centrifuge filter unit (Millipore; Billerica, MA), and the retentates were aliquoted and stored at 280uC. VLP entry assay. The VLP entry assay was performed as previously described [6] with modifications. Briefly, target cells were exposed to filtered air or 0.4 ppm ozone for 4 h. 24 h post- exposure, VLPs were added to the apical surface in a total volume of 100 ml and incubated at 37uC for 3 h. The cells were washed twice with HBSS to remove unbound virus and infected cells were detected by using GeneBLAzerTM FRET in vivo cell-based assay system substrate CCF2-AM according to manufacturer’s recommendations (Invitrogen). Samples were lysed in HBSS by freeze-thaw method repeatedly treated for 3 cycles (frozen in liquid nitrogen for 3 min and thawed in a 65uC water bath for 3 min). Viral entry was quantified by using the POLARstar OPTIMA plate reader (BMG LABTECH, Inc.). All experiments were performed in triplicate in three independent experiments. Infection with Influenza A We used influenza A/Bangkok/1/79 H3N2 serotype (which was kindly provided by Dr. Melinda Beck, University of North Carolina). We also obtained influenza A/Malaya/302/1954 H1N1 serotype from the American Type Culture Collection (ATCC, Manassas, VA) for Western blot analysis of the cleavage products of the viral HA protein. Virus was propagated in 10-day- old embryonated hen’s eggs. The virus was collected in the allantoic fluid and titered by 50% tissue culture infectious dose in Madin-Darby canine kidney cells and hemagglutination as described before [53]. Stock virus was aliquoted and stored at 280uC until use. Unless otherwise indicated, for infection about 56105 cells were infected with approximately 128 hemagglutina- tion units (HAU) of influenza A, which resulted in approximately 10% of the cells being infected with influenza 24 h post-infection. Total RNA, total protein, basolateral supernatants, and apical washes were collected 24 h post-infection. Immunoblotting Apical surface liquid was collected at 24 h post-exposure by washing the apical surface of the cells with HBSS and total protein concentrations were determined by Bradford protein assay (Bio- Rad). Cell lysates were prepared at 24 h post-infection in Passive Lysis Buffer (Promega, Madison, WI) with a protease inhibitor mixture (Cocktail Set III; Calbiochem, San Diego, CA) as well as phosphatase inhibitors (0.5 mM NaVO4, 1 mM b-glyceropho- Ozone Exposure on Influenza Infection phate) on ice for 30 min. After centrifugation, total protein concentrations were determined by Bradford protein assay (Bio- Rad). Both the apical supernatants and cellular lysates were subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Schleicher & Schuell Biosciences, Keene, NH). Proteins were detected using specific antibodies to HAT, TMPRSS2, and SPLI (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) or Influenza A virus hemagglutinin H1 antibody (1:1,000; Abcam, Cambridge, MA). b-actin (1:2,000; US Biological, Swampscott, MA), which was used as a loading control. Antigen-antibody complexes were stained with anti-rabbit or anti-mouse, horseradish peroxidase (HRP)-conjugated antibody (1:2000, Santa Cruz Biotechnology) and detected with SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL). Densitometry was performed using a Fujifilm LAS-3000 imager (Fuji Film Global Tokyo, Japan). Influenza HA cleavage Apical surface liquid was collected by washing the apical surface of the cells with HBSS and total protein concentrations were determined by Bradford protein assay (Bio-Rad). 50 mg of total protein were immunoprecipated overnight at 4uC with 200 ng of anti-TMPRSS2, anti-HAT, or IgG isotype as a control, followed by 1 h incubation with 50 ml of protein G-agrose beads (Pierce, Rockford, IL). Cleared supernatants were incubated with 50 ul of Influenza A/Malaya/302/1954 H1N1 for 30 min at 32uC. Virus was subjected to 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to nitrocellulose (Schleicher & Schuell Biosciences, Keene, NH). Proteins were detected using specific antibodies to HAT, TMPRSS2, and SPLI (1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA) or Influenza A virus hemagglutinin H1 antibody (1:1,000; Abcam, Cambridge, MA). Influenza virus titer Influenza virus titers in apical washes were determined by 50% tissue-culture infectious dose (TCID50) in Madin Darby canine kidney (MDCK) cells and evaluated using agglutination of red blood cells as an indicator according to a modified protocol described before [54]. Briefly, MDCK cells grown in round- bottom 96-well plates were inoculated with virus-containing samples diluted in serum-free DMEM containing 20 mg/ml trypsin using log10 dilutions. After 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. Wells exhibiting hemagglutination were considered positive and virus titers were expressed as log TCID50. Protease activity assay A modification to the above protocol was performed to test secreted protease activity. In these experiments, NECs were exposed to either 0.4ppm of ozone or air for 4 h. 24 h post- exposure, apical supernatants were collected and protein concen- trations were determined by Bradford protein assay. 100 mg of protein within the apical supernatant, which contained the endogenous secreted proteases, was incubated with 80 ml of virus for 30 min at room temperature. MDCK cells grown in round- bottom 96-well plates were inoculated with the above samples diluted in serum-free DMEM or serum-free DMEM containing 20 mg/ml trypsin, as a positive control, using 10-fold dilutions. After 3 days incubation, a suspension of human erythrocytes (0.5%) was added to each well. Wells exhibiting hemagglutination were considered positive and virus titers were expressed as log TCID50. 10 mM Phenylmethyl sulfonyl floride (PMSF) (Sigma- PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e35108