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

Abstract

Respiratory syncytial virus (RSV) nonstructural protein 1(NS1) attenuates type-I interferon (IFN) production during RSV infection; however the precise role of RSV NS1 protein in orchestrating the early host-virus interaction during infection is poorly understood. Since NS1 constitutes the first RSV gene transcribed and the production of IFN depends upon RLR (RIG-I-like receptor) signaling, we reasoned that NS1 may interfere with this signaling. Herein, we report that NS1 is localized to mitochondria and binds to mitochondrial antiviral signaling protein (MAVS). Live-cell imaging of rgRSV-infected A549 human epithelial cells showed that RSV replication and transcription occurs in proximity to mitochondria. NS1 localization to mitochondria was directly visualized by confocal microscopy using a cell-permeable chemical probe for His(6)-NS1. Further, NS1 colocalization with MAVS in A549 cells infected with RSV was shown by confocal laser microscopy and immuno-electron microscopy. NS1 protein is present in the mitochondrial fraction and co-immunoprecipitates with MAVS in total cell lysatesof A549 cells transfected with the plasmid pNS1-Flag. By immunoprecipitation with anti-RIG-I antibody, RSV NS1 was shown to associate with MAVS at an early stage of RSV infection, and to disrupt MAVS interaction with RIG-I (retinoic acid inducible gene) and the downstream IFN antiviral and inflammatory response. Together, these results demonstrate that NS1 binds to MAVS and that this binding inhibits the MAVS-RIG-I interaction required for IFN production.

Figure 1. Live cell imaging of Rg-RSV-infected cells.

(A). A549 cells were seeded in 35 mm glass-bottom fluorodishes (Fisher Scientific) and stained with CMXRos, live mitochondrial labeling kit (Molecular Probes) for 15 min at 37°C, washed and then fresh medium (DMEM with 2% FBS) was added to the cells. Cells were infected with rgRSV at MOI of 1 and visualized on a Perkin Elmer UltraVIEW®VoX 3D Live Cell Imaging System equipped with a Nikon T inverted microscope. The cells were maintained at 37°C with 5% CO₂ throughout the imaging (16 h post-infection). Images were taken at 0∶40, 3∶31, 5∶21, 6∶52, 8∶53, and 10∶43 hours post-infection. The last two images in the bottom panel show mitochondrial staining and GFP alone respectively at 10∶43 h p.i. The colocalization of GFP (green) with mitochondria (red) is shown as yellow. (B). The Pearson's and Manders' overlap coefficients are represented as the average of ten individual cells at 10∶43 hp.i.

Figure 2. Co-localization of His₆-NS1 with mitochondria in infected cells using Ni²⁺-NTA₂-BM.

(A) Structure of Ni²⁺-NTA₂-BM . (B) rA2-His₆-NS1 or rA2 were used to infect HEp-2 cells at a MOI of 2. At 10h p.i., infected cells were labeled with CMXRos (red), DRAQ5 (blue), and 50 μMNi²⁺-NTA₂-BM. Fluorescence was detected using an Olympus FV1000 MPE multiphoton laser scanning microscope. The cells were maintained at 37°C with 5% CO₂ throughout the imaging (Z min). Shown are micrographs of rA2-His₆-NS1-infected cells collected 10 h p.i (a–c)and rA2 RSV at 10 hp.i. (d–f) demonstrating site-specificity for Ni²⁺-NTA₂-BM. Excitation wavelengths of 405, 563 and 633 nm and differential interference contrast (DIC) were used to collect a series of 0.5 µm Z-stack images for each specimen and experiments were performed in triplicate. Images a and d show the mitochondria (red), b and e represent His₆-NS1 (green) and candf are the merged images of red and green along with the blue nuclear stain DAPI and DIC. (C). Thresholds for the Z-stack images collected in (B) were acquired using RenyiEntropy AutoThreshold ImageJ plugin (Landini) and Pearson's and Mander's coefficients derived with JACoP plugin (Bolte & Cordelieres). The Mander's coefficient M2 indicates that ∼70% of the His₆-NS1 detected overlaps with CMXRos-stained mitochondria at 10 hrs p.i. in rA2-His₆-NS1-infected cells.

Figure 3. Colocalization of NS1 with MAVS by using immunogold electron microscopy.

A549 cells were mock-infected or infected with 0.5 MOI rA2-His₆-NS1 for 1 h. Cells were harvested at 12 and 24 h post-infection and processed for immunogoldelectron microscopy. Thin sections on nickel grids were treated with rabbit anti-MAVS antibody (1∶100 dilution) for 3 h at room temperature, washed and then stained with 15 nm gold-labeled anti-rabbit secondary antibody. The grids were washed thoroughly and treated with a 1∶20 dilution of 5 nm gold-Ni-NTA (Molecular Probes) that binds to His tags. Cells infected with rA2-His₆-NS1 were stained for His-NS1 with 5 nm gold (red arrow) and for MAVS protein on mitochondria with 15 nm gold (black arrows). (a) Ultrathin sections of uninfected cells showing the 15 nm gold staining of MAVS protein (black arrow). (b) Ultrathin sections of infected cells 12 h p.i. showing His₆ protein stained using 5 nm gold-Ni-NTA. The inset shows His₆-NS1 localized on the mitochondria. (c & d) Sections of cells 12 and 24 h p.i. showing colocalization of NS1 with the MAVS protein. Insets (1 & 2) show colocalization of NS1 (red arrow) and MAVS (black arrow).

Figure 4. Co-localization of NS1 with MAVS in mitochondria during RSV infection.

A549 cells were infected with rA2 or rA2ΔNS2 (0.1 MOI).Twelve hours after infection, cells were stained with the mitochondrial stain CMXRos (red). Cells were then fixed and stained with Alexa 488-conjugated anti-NS1 antibody (green) and Alexa 647-conjugated anti-MAVS antibody (purple). Stained cells were viewed under the Leica TCS SP2 laser scanning confocal microscope (A) Images (400× magnification) show individual staining colors, and merged images of NS1-mitochondria and NS1-MAVS are also shown. (B) The images are a 5× enlargement of a 400× image. The top panel shows the cells infected with rA2 and the bottom panel the cells infected with rA2ΔNS2. The areas marked by white circles show the presence of NS1 in mitochondria and colocalization with MAVS. The first pair of images are merges of NS1 and mitochondria. The second pair show NS1 merged with MAVS and the last pair shows all three. (C). Correlation analysis of the co-localization of NS1 with mitochondria in rA2 infected cells. (D). Correlation analysis of the colocalization of NS1 with mitochondria in rA2ΔNS2 infected cells.Pearson's coefficient for MAVS and NS1 is shown along with Manders' coefficients (M1 & M2), which represent the fraction of the mitochondrial red overlapping with NS1 green and the fraction of NS1 green overlapping with the mitochondrial red, respectively.

Figure 5. MAVS co-immunoprecipitates with NS1 in epithelial cells infected with rA2- RSV or rA2.

(A) HEp-2 cells were eithermock-infectedor infected with rA2 or rA2ΔNS1or rA2ΔNS2 at an MOI of 1. At 24 h p.i., total cell extracts were immunoprecipitated with anti-MAVS followed by western blot analysis using anti-NS1 or anti-MAVS antibodies. (B) Whole cell lysates from mock or rA2 infected HEp-2 cells were immunoblotted for RSV F protein using anti RSV F antibody (Chemicon MAB8262) (right panel). The lysates from mock or rA2 or rA2ΔNS1 were co-immunoprecipitated with anti-MAVS antibody and probed for MAVS and RSV F protein (left panel).

Figure 6. RSV NS1 is present on the mitochondria and co-immunoprecipitates with MAVS in NS1-transfected cells.

(A). A549 cells were transfected with pNS1-Flag (NS1) or pFlag (Vec). Mitochondria were isolated and the proteins were analyzed by western blot for Flag and Cox-IV (B). Total cell extracts were immunoprecipitated with anti-MAVS antibody and analyzed by western blotting with anti-Flag and anti-MAVS antibodies. (C). A549 cells transfected with pNS1-Flag were stained for Flag (green, a), mitochondria (CMXRos, red, b), and MAVS (purple, c) and visualized using the Leica TCS SP2 laser scanning confocal microscope. The squares indand e show co-localization of NS1 with mitochondria and MAVS respectively, and the box in f shows co-localization of NS1, MAVS and mitochondria. The insets in d,e, and f show the enlargement of the indicated areas.

Figure 7. NS1 protein prevents RIG-I from binding to MAVS.

(A) A549 cells were mock-infected or infected with rA2 or rA2ΔNS1 (MOI = 1). Whole-cell lysates were subjected to western blot analysis with anti-RIG-I, anti-MAVS, and anti-NS1antibodies (top panel). The same lysates were immunoprecipitated with anti-RIG-I antibody and the protein complexes were immunoblotted with the indicated antibodies (bottom panel). (B)The bar graph represents the densitometric quantification of the band intensities as the ratio of MAVS to RIG-I. (C) A549 cells were transfected with the indicated plasmids (12 µg of either pVAX or pNS1) and incubated with 0.2 ng/ml poly (I:C). Cytoplasmic extracts from cells 12 and 24 h after transfection were immunoprecipitated with anti-MAVS and immunoblotted with anti-RIG-I antibody. (D) Increasing amounts of pNS1 were co-transfected with constant amounts (6 µg) of pMAVS and pRIG-I, along with 0.2 ng/ml poly (I:C). Competitive inhibition of the interaction of MAVS with RIG-I was tested using IP, as described in (C). Transfection with pVAX was used to keep the total amount of transfected DNA constant.