Respiratory syncytial virus NS1 protein colocalizes with mitochondrial antiviral signaling protein MAVS following infection.

Cell culture Human embryonic lung fibroblast HEp-2

(CCL-23) cells, human alveolar epithelial A549 (CCL-185) cells and African green monkey kidney Vero (CCL-81) cells were all purchased from the American Type Culture Collection (ATCC) and were cultured in standard Dulbecco's MEM (DMEM) containing 5% heat-inactivated fetal bovine serum, L-glutamine, 100 IU/ml penicillin and 100 µg/ml streptomycin sulfate. Viruses Recombinant RSV strains rA2, rA2ΔNS1, rA2ΔNS2 and rA2-His 6 -NS1 have been previously described [25] . RgRSV, which expresses green fluorescent protein, was a gift from Dr. Mark Peeples. Viruses rA2-His 6 -NS1 and rA2 RSV were grown in HEp-2 cells and harvested when cytopathic effects became visible (2–3 days). RSV-infected cells were subjected to a single round of freeze-thaw cycles and viral supernatant was clarified by centrifugation at 3200 rpmat 4°C for 10 min. Viral titers were obtained through plaque assay with 0.8% methylcellulose overlay and immunostaining with monoclonal murine anti-RSV F antibody, followed by a horseradish peroxidase-conjugated secondary antibody and visualization with 4CN substrate (Kirkegaard and Perry Laboratories). The deletion mutant rA2ΔNS1 was cultured in Vero cells and viral titer was also determined in the same cells. For microscopy studies, A549 and HEp-2 cells were seeded to sub-confluency on glass-bottom culture dishes 24 hrs before infection. On the day of infection, growth media was replaced with Opti-MEM (Invitrogen) containing the appropriate amount of virus suspension for obtaining the indicated multiplicity of infection (MOI).

Confocal microscopy

A549 cells were infected with rgRSV and imaged from the start of infection for 16 h using a Nikon T inverted microscope running the Perkin Elmer UltraVIEW®VoX 3D Live Cell Imaging System. In a different experiment, A549 cells were infected with rA2 or rA2ΔNS1 and 24 h later the cells were stained with the Image-iT Live Mitochondrial and Nuclear Labeling Kit (Molecular Probes, Invitrogen), using the mitochondrial stain CMXRos, according to the manufacturer's protocol. Cells were fixed with 4% paraformaldehyde, permeabilized with cold 0.2% Triton X-100 in PBS for 10 min, washed twice with PBS and incubated with primary rabbit IgG antibody conjugated to ZenonAlexa Fluor (Invitrogen). Alexa 488 was conjugated to anti-His 6 -NS1antibody and Alexa 647 was conjugated to anti-MAVS antibody (Bethyl Laboratories). Stained cells were viewed under a Leica TCS SP2 laser scanning confocal microscope. Fluorescent imaging of histidine-tagged protein using cell permeable Ni 2+ NTA-BM dye in live cells HEp-2 cells were seeded in 35 mm glass-bottom confocal culture dishes and infected with rA2-His 6 -NS1 at an MOI of 2 for 10 h. Ni 2+ -NTA 2 -dibromobimane (Ni 2+ -NTA 2 -BM) was prepared as previously described [26] , [27] and used to label rA2-His 6 -NS1-infected live HEp-2 cells 10 hrs post-infection. 50 µM NTA 2 -BM was complexed with a 10-fold molar excess of NiSO 4 and incubated with ammonium bicarbonate buffer-washed RSV-infected cells, together with a mitochondrial labeling probe, CMXRos and nuclear (DRAQ5, Cell Signaling) stain, following manufacturers' instructions. Infection with rA2 was used as a control to test target specificity. Fluorescence was visualized by laser confocal microscopy at wavelengths of 405, 563 and 633 nm. Cells were also photographed using differential interference contrast (DIC). A series of 0.5 µm Z-stack images was collected of each specimen and experiments were performed in triplicate. Semi-quantitative analysis with the JACoP provided Pearson's and Manders' overlap coefficients ( http://rsb.info.nih.gov/ij/plugins/track/jacop.html ).

Immunogold electron microscopy

A549 cells were grown to near confluence in T-75 flasks (USA Scientific), infected with 0.5 MOI of rA2-His 6 -NS1 and harvested at 12 and 24 h post-infection. The cells were pelleted and fixed in 1% paraformaldehyde, 0.5% glutaraldehyde, 0.05% sodium cacodylate, pH 7.1, for 10 min at 4°C, then in 2% paraformaldehyde, 2.5% glutaraldehyde, 0.05% sodium cacodylate, pH 7.1, for 30 min at 4°C. After washing three times (10 min each time) with 0.05 M sodium cacodylate, the cells were dehydrated with a series of ethanol concentrations (50%, 70%, 85%, 95%, and 3× with 100%) for 30 min for each step at 4°C. Cells were then infiltrated with ethanol∶LR White Resin using ratios of 1∶1 (2 h at 4°C), and 1∶3 (overnight at 4°C) followed by pure LR White (24 h at 4°C, with a resin change after 8 h). The cells were then embedded in gelatin capsules, and resin was polymerized at 4°C for 48 h under UV light. The blocks were sectioned at the Lisa Muma Weitz Advanced Microscopy & Cell Imaging Core Laboratory (University of South Florida) and the sections were laid on nickel grids. Gold labeling of the nickel grids was carried out as described previously [28] . Grids were treated with 25 µl of TBS-supplemented buffer (0.05 M Tris, 0.85% NaCl, pH 8.3–8.5, 0.5% normal goat serum, 0.5% normal pig serum and 0.5% BSA) with 3% nonfat dry milk for 2 h at room temperature. Grids with thin sections were incubated with 50 µl of anti-MAVS rabbit antibody (1∶100 dilution) diluted in TBS-supplemented buffer with 3% nonfat dry milk for 3 h at room temperature, washed and stained with the anti-rabbit antibody conjugated with 15 nm gold (Ted Pella, Inc.) for 1 h at room temperature. The grids were washed thoroughly and treated with 1∶20 dilution of 5 nm gold-Ni-NTA (Molecular Probes) that binds to His 6 tags.The grids were washed three times with TBS buffer, and incubated with 25 µl of 1∶100 dilution of goat anti-rabbit antibody conjugated with 10 nm gold particles (Ted Pella Inc.) for 1 h at room temperature. After stream washing and three washes with distilled water (10 min each), the grids were dried and stained with 2% aqueous uranyl acetate for 5 min and examined on a JOEL 1200 EX scanning/transmission electron microscope at 80 kV.

Plasmids

The plasmids used in the study were pNS1-Flag, pFlag vector control for the mitochondrial fraction isolation, pVAX, pNS1, pRIG-I (Invivogen) and pMAVS (Invivogen). Cells were transfected with the specified amounts of DNA using Lipofectamine 2000 (Invitrogen). Plasmid pNS1 was constructed by subcloning a codon-optimized RSVNS1 sequence with restriction enzyme digestion into the parental pVAX vector. pNS1-Flag was similarly constructed by restriction enzyme digest into pFlag vector, with Flag on the amino terminus of NS1. Constructs were verified with sequencing and molecular weight of pNS1-Flag was ∼17 kDa when probed with an anti-Flag antibody. Mitochondrial isolation 20×10 6 cells were either transfected with pNS1-Flag or pFlag vector alone, then the mitochondrial fraction was isolated using a mitochondrial isolation kit (ThermoFisher Scientific). The mitochondrial protein was estimated using the BCA protein assay (ThermoFisher Scientific) and subjected to western blot analysis as described below. Immunoprecipitation 30×10 6 cells were either transfected with the indicated plasmids or infected with RSV at the specified MOI. At specified times, the cells were lysed, and 500 µg aliquots of each protein sample were incubated with the indicated antibody and immunoprecipitated using protein A-agarose beads (Invitrogen). Immunoprecipitation (protocols from eBioScience) was performed using anti-Flag antibody (Sigma), anti-MAVS antibody (Bethyl Laboratories), or anti-RIG-I (Cell Signaling) antibodies and the antigen-antibody complexes were evaluated by western blot analysis.

Western blot analysis of proteins

Aliquots of the mitochondrial proteins (100 µg) or the immunoprecipitated complexes were boiled for 5 min in 2× protein denaturation buffer (2.3% SDS, 10% glycerol, 5% 2-mercaptoethanol, 62.5 mMTris-HCl and 0.01% bromophenol blue, pH 6.8) and were resolved by electrophoresis on 4–20% SDS-PAGE gels (Bio-Rad Laboratories). Proteins were transferred for 30 min with Towbin buffer (10 mMTris base, 96 mM glycine in 10% methanol) to nitrocellulose membranes (Bio-Rad). Blots were blocked for 30 min at room temperature with 5% nonfat dry milk and 0.1% Tween-20 in Tris-buffered saline (TBS). Blots were separately probed with rabbit anti-Flag antibody (Sigma), mouse anti-MAVS (Santa Cruz), anti-COX-IV (Cell Signaling), anti-RIG-I antibody (Cell Signaling) or polyclonal rabbit antiserum against recombinant His 6 -NS1 [25] , and finally, the protein bands were detected by ECL (ThermoFischer Scientific, SuperSignal West Pico).