Human metapneumovirus inhibits IFN-β signaling by downregulating Jak1 and Tyk2 cellular levels - PubMed (original) (raw)

Human metapneumovirus inhibits IFN-β signaling by downregulating Jak1 and Tyk2 cellular levels

Junping Ren et al. PLoS One. 2011.

Abstract

Human metapneumovirus (hMPV), a leading cause of respiratory tract infections in infants, inhibits type I interferon (IFN) signaling by an unidentified mechanism. In this study, we showed that infection of airway epithelial cells with hMPV decreased cellular level of Janus tyrosine kinase (Jak1) and tyrosine kinase 2 (Tyk2), due to enhanced proteosomal degradation and reduced gene transcription. In addition, hMPV infection also reduced the surface expression of type I IFN receptor (IFNAR). These inhibitory mechanisms are different from the ones employed by respiratory syncytial virus (RSV), which does not affect Jak1, Tyk2 or IFNAR expression, but degrades downstream signal transducer and activator of transcription proteins 2 (STAT2), although both viruses are pneumoviruses belonging to the Paramyxoviridae family. Our study identifies a novel mechanism by which hMPV inhibits STAT1 and 2 activation, ultimately leading to viral evasion of host IFN responses.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1. Induction of type I IFN signaling by hMPV infection.

(A) A549 cells were transfected with a luciferase reporter plasmid containing five repeats of ISRE-binding sequence from ISG54 (pISRE-luc) for 30 h. Cells were then mock infected, or infected with live or UV-inactivated hMPV, at an MOI of 0.5, and harvested at various times p.i. to measure luciferase activity. IFN-β (1000 units/ml) treatment was used as a positive control. (B) Fibroblast cells 2fTGH and mutants lacking Jak1 (U4A), Tyk2 (U1A), STAT1 (U3A) or STAT2 (U6A) were transfected with pISRE-Luc for 30 h, followed by mock infection or infection with hMPV, at an MOI of 3, for 15 h. Cells were then lysed to measure luciferase activity. (C) pISRE-luc-transfected 2fTGH and U3A cells were either mock treated or treated with IFN-β for 15 h, followed by luciferase activity measurement. In all experiments, luciferase was normalized to β-galactosidase reporter activity. Data are representative of three independent experiments and are expressed as mean ± standard error (SE) of normalized luciferase activity. *, P<0.05, relative to mock-infected/treated cells.

Figure 2. IFN-β-dependent signaling is inhibited by hMPV infection.

A549 cells were mock- or hMPV-infected at an MOI of 0.5 for 24 h, and then transfected with pISRE-luc, followed by mock- or IFN-β treatment for an additional 15 h. For each plate luciferase was normalized to β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean ± SE of normalized luciferase activity.

Figure 3. hMPV infection inhibits STAT1 and 2 phosphorylation.

A) A549 cells were mock- or hMPV-infected at an MOI of 0.5, for 6 or 15 h, and then mock- or IFN-β-treatment for one additional hour. Total cells were harvested, followed by Western blot to determine the abundance of phosphorylated and non-phosphorylated STAT1. Membranes were stripped and reprobed for β-actin to document equal loading of the samples. Expression ratio of pSTAT1/STAT1 was normalized to β-actin. (B) A549 cells were infected with hMPV and/or treated with IFN-β as described in A. Nuclear fractions were prepared followed by Western blot using anti phospho-STAT1 (p-STAT1) and STAT1 antibodies. Membranes were stripped and reprobed for lamin B, as loading control. The nuclear translocated ratio of pSTAT1/STAT1 was normalized to lamin B. (C) Total cell lysate were prepared as described in A, followed by Western blot to determine the abundance of phosphorylated and non-phosphorylated STAT2. Membranes were stripped and reprobed for β-actin as a control for equal loading of the samples. Expression ratio of pSTAT2/STAT2 was normalized to β-actin. (D) A459 cell were mock- or RSV- infected, at an MOI of 0.5, for 6 or 15 h, and then mock- or IFN-β-treated for one additional hour. Total cell lysates were prepared, followed by Western blot to determine the abundance of phosphorylated and total STAT2. Membranes were stripped and reprobed for β-actin. STAT2 expression was normalized to β-actin. For (A), (B) and (C), *, P<0.05, relative to mock-infected and IFN-β-treated treated cells at 15 h. For (D), *, P<0.05, relative to mock-infected cells at 15 h p.i., according to their respective IFN-β treatment condition. The results are representative of two to three separate experiments. Densitometric analysis of Western blot band intensities was performed using VisionWorksLS image acquisition and analysis software from UVP (Upland, CA). Data are presented as mean ± SE of arbitrary units.

Figure 4. hMPV infection affects cellular levels of Jak1 and Tyk2.

A549 cells (A) or Vero cells (B) were mock- or hMPV-infected, at an MOI of 0.5, for 6 or 15 h, followed by mock or IFN-β treatment for 1 h. Cells were harvested to prepare total cell lysates. Abundance of total Jak1 and Tyk2 was analyzed by Western blot, followed by densitometric analysis of Western blot band intensities using VisionWorksLS image acquisition and analysis software from UVP (Upland, CA). The band intensities were then normalized to β-actin levels. Data are representative of two to three independent experiments and are expressed as mean ± SE of normalized gene expression.*, P<0.05, relative to mock-infected cells, according to their respective IFN-β treatment condition and time point of p.i. (C) A549 cells were mock- or RSV-infected, at an MOI of 0.5, for 6 or 15 h, followed by mock or IFN-β treatment for 1 h. Cells were harvested to prepare total cell lysates. Abundance of total Jak1 and Tyk2 was analyzed as described in A and B. Data are representative of two to three independent experiments.

Figure 5. hMPV-induced Jak1 and Tyk2 degradation is proteasome-dependent.

A549 cells were pretreated with 1 or 5 µM MG132 (A) or 10 µM lactacystin (B) for 1 h. Untreated cells were used as a control. After treatment, cells were mock-infected or infected with hMPV for 15 h and harvested to prepare total cell lysates. Jak1 and Tyk2 abundance was analyzed by Western blot. Membranes were also probed with anti-hMPV antibody to evaluate the effects of MG132 on viral replication. Membranes were stripped and reprobed with β-actin as a control for equal loading of the samples. *, P<0.05, relative to mock-infected cells, according to their respective MG132/lactacystin treatment conditions. (C) Total RNA was extracted from uninfected or infected A549 cells at 6, 15, and 24 h p.i., and used for Q-RT-PCR to determine changes in Jak1 and Tyk2 gene expression. 18S RNA was used as an internal control for normalization. Data are presented as fold changes of Jak1/Tyk2 mRNA levels in hMPV-infected cells compared to mock-infected. *, P<0.05, relative to mock-infected cells. (D) A549 cells were mock-infected or infected with hMPV in the presence of 5 µM MG132 for 6 h and harvested to prepare total cell lysate. Small aliquots were saved for Jak1 and Tyk2 abundance analysis by Western blot. Membranes were stripped and reprobed with β-actin as a control for equal loading of the samples. The rest of the samples were immunoprecipitated using an anti-polyubiquitin antibody, followed by Western blot using anti-Jak1 or anti-Tyk2 antibody. For all the experiments, data are representative of two to three independent experiments and are expressed as mean ± SE of normalized gene transcription or translation.

Figure 6. Surface expression of IFNAR1 subunits is reduced in hMPV-infected cells.

(A) A549 cells were mock-infected or infected with hMPV at an MOI of 0.1 or 0.5, for 6 or 15 h. Cells were either stained with anti-IFNAR1 and isotype control antibody. After 1 h incubation at 37°C, cells were washed with PBS containing 5% FBS, followed by another incubation with anti-mouse FITC-conjugated secondary antibody at 37°C. (B) A549 cells were stimulated with 1,000 U/ml of IFN-β for various periods of time as indicated. Antibody staining was done as described in (A). Samples were analyzed on a FACSCAN flow cytometer equipped with BD FACSDiva software. Analysis was performed using FlowJo software (version 7.2.2; Tree Star Ashland, OR). % of IFNRA positive cells of mock- and hMPV-infected samples was compared. *, P<0.05, relative to mock-infected and IFNRA antibody-stained cells. Data are representative of two to three independent experiments, and are expressed as mean ± SE of IFNRA surface expression. (C) A549 cells were mock- or hMPV-infected, at an MOI of 0.1 or 0.5, for 15 h, and harvested to prepare total cell lysates. IFNAR1 abundance was determined by Western blot. Membranes were stripped and reprobed for β-actin as a control for equal loading of the samples. Data are representative of two to three independent experiments.

Figure 7. Inhibition of Jak/STAT1 signaling pathway is virus replication dependent.

(A) A549 cells were mock-infected or infected with live hMPV or UV-inactivated hMPV for 15 h. Cells were then treated with medium or 500 U/ml of IFN-β for 1 h. Abundance of phosphorylated and total STAT1, Jak1 and Tyk2 in total cell lysates was then analyzed by Western blot. Membranes were stripped and reprobed with β-actin as a control for equal loading of the samples. (B) Densitometric analysis for A. densitometric analysis of band intensities was performed using VisionWorksLS Image Acquisition and Analysis Software from UVP (Upland, CA) according to the manufacturer's instruction. Data are representative of three independent experiments, and are expressed as mean ± SE of protein expression. *, P<0.05, relative to mock-infected cells, according to their respective IFN-β treatment conditions.

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Human metapneumovirus inhibits IFN-β signaling by downregulating Jak1 and Tyk2 cellular levels - PubMed (original) (raw)