Respiratory syncytial virus NS1 inhibits anti-viral Interferon-α-induced JAK/STAT signaling, by limiting the nuclear translocation of STAT1

Abstract

Human respiratory viruses are the most prevalent cause of disease in humans, with the highly infectious RSV being the leading cause of infant bronchiolitis and viral pneumonia. Responses to type I IFNs are the primary defense against viral infection. However, RSV proteins have been shown to antagonize type I IFN-mediated antiviral innate immunity, specifically dampening intracellular IFN signaling. Respiratory epithelial cells are the main target for RSV infection. In this study, we found RSV-NS1 interfered with the IFN-α JAK/STAT signaling pathway of epithelial cells. RSV-NS1 expression significantly enhanced IFN-α-mediated phosphorylation of STAT1, but not pSTAT2; and neither STAT1 nor STAT2 total protein levels were affected by RSV-NS1. However, expression of RSV-NS1 significantly reduced ISRE and GAS promoter activity and anti-viral IRG expression. Further mechanistic studies demonstrated RSV-NS1 bound STAT1, with protein modeling indicating a possible interaction site between STAT1 and RSV-NS1. Nuclear translocation of STAT1 was reduced in the presence of RSV-NS1. Additionally, STAT1's interaction with the nuclear transport adapter protein, KPNA1, was also reduced, suggesting a mechanism by which RSV blocks STAT1 nuclear translocation. Indeed, reducing STAT1's access to the nucleus may explain RSV's suppression of IFN JAK/STAT promoter activation and antiviral gene induction. Taken together these results describe a novel mechanism by which RSV controls antiviral IFN-α JAK/STAT responses, which enhances our understanding of RSV's respiratory disease progression.

Keywords: Interferon; JAK/STAT signaling; RSV; nuclear translocation; viral immune evasion.

Figure 1

RSV-NS1 suppresses Type I IFN promoter activity and IRG expression in the BEAS 2b epithelial cell line. BEAS 2b cells were transfected EV, RSV-NS1 or RSV-NS2, along with ISRE-Luc or GAS-Luc and TK Renilla. At 24h post-transfection, cells were stimulated by 1000U/mL of IFN-α. Dual luciferase activity was measured 18h post-IFN-α treatment for levels of (A) ISRE and (B) GAS (n=4). BEAS 2b cells were transfected with EV, NS1 or NS2 for 24h or 48h. Cells were extracted for total RNA, before analyzing mRNA levels of (C) MxA, (D) UPS18, (E) PKR and (F) ISG15 (n=3). Gene expression was calculated relative to RPS15 and compared to EV controls, which were normalized to 1. All data is shown as mean ± SD. Significance was determined by unpaired t-test *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

Figure 2

RSV-NS1 enhances IFN-α-induced STAT1 phosphorylation in the BEAS 2b epithelial cell line. BEAS 2b cells were transfected with EV, RSV-NS1 or RSV-NS2 for 24h and treated with 1000IU IFN-α for 20min. Cell lysates were collected and levels of (A) pSTAT1, (C) STAT1, (E) pSTAT2 and (G) STAT2 were measured by western blotting (n=5). Densitometry of (B) pSTAT1, (D) STAT1, (F) pSTAT2 and (H) STAT2 was performed using Image Lab software and values for STATs or phosphorylated STATs were calculated relative to β-actin and compared to the EV transfected untreated control, which was normalized to 1. (N.B pSTAT1 and STAT2 were probed in one membrane and therefore share the same β-actin, and the pSTAT2 and STAT1 were probed in one membrane and therefore share the same β-actin). Data is presented as mean ± SD. Statistics calculated by unpaired t test *p<0.05 and **p < 0.01.

Figure 3

Expression of RSV-NS1 limits nuclear translocation of STAT1. BEAS 2b cells were transfected RSV-NS1 or EV and stimulated with or without (UT) 1000IU IFN-α for 30min. (A) Cells were stained for RSV-NS1, STAT1 and DAPI, and visualized using confocal microscopy. Images are representative of three independent experiments. Scale measurement bar represents 25μm in length. (B) STAT1 is highlighted within the dashed box and shown at higher magnification. Scale measurement bar represents 10μm in length. Quantification of STAT1 intensity in the nucleus and cytoplasm was determined using IMARIS software and (C) the ratio of nuclear to cytoplasmic STAT1 intensity was determined. All data is shown as mean ± SD. Significance was determined by One-way ANOVA multiple comparison test (n=3) *p < 0.05 and **p < 0.01.

Figure 4

RSV-NS1 associates with STAT1, while KPNA1:STAT1 interaction is reduced. BEAS 2b cells were transfected with RSV-NS1 or EV for 24h. (A) Total RNA was extracted before analyzing mRNA levels of KPNA1, gene expression was calculated relative to the RPS15 and compared to EV, which was normalized to 1. BEAS 2b cells were transfected with RSV-NS1 or EV for 24h. (B) Protein was extracted before analyzing protein levels of KPNA1. Densitometry of (C) KPNA1 was performed using Image Lab software and values for KPNA1 were calculated relative to β-actin and compared to EV, which was normalized to 1 (n=4). (D) BEAS 2b cells were transfected with RSV-NS1 or EV for 24h. Lysates were immunoprecipitated (IPed) using a STAT1 antibody. IP and whole cell lysates (WCL) were subjected to Western blotting using STAT1, RSV-NS1 and β-actin antibodies. (E) BEAS 2b cells transfected with RSV-NS1 or EV for 24 h and stimulated with 1000IU IFN-α for 30min. Cells were lysed and immunoprecipitated using STAT1 antibody. IP and WCL were subject to Western blotting using STAT1, KPNA1, RSV-NS1 and β-actin. (F) Densitometric analysis was performed using Image Lab software and values of KPNA1 were calculated relative to STAT1 (IP) and compared to EV, which was normalized to 1 (n=4). Data is shown as mean ± SD. Significance was determined by unpaired t-test. *p < 0.05.

Figure 5

RSV-NS1 suggested binding to STAT1. Best protein-protein docked pose of STAT1 (PDB: 1YVL yellow) and RSV-NS1 (PDB: 5VJ2 blue) predicted by MOE 2022.02. (A) MOE Contact analysis results highlighting amino acid residues (red) involved in possible interactions between RSV-NS1 and STAT1. (B) Hydrogen bond interactions (dotted lines) between amino acids of RSV-NS1 (Ala42, Ala44) and STAT1 (Gln9, Tyr5). Common amino acid contacts (red) that may be responsible for the interactions between (C) STAT1 (PDB: 1YVL yellow) and (D) RSV-NS1 (PDB: 5VJ2 blue).