Human metapneumovirus glycoprotein G inhibits innate immune responses

Abstract

Human metapneumovirus (hMPV) is a leading cause of acute respiratory tract infection in infants, as well as in the elderly and immunocompromised patients. No effective treatment or vaccine for hMPV is currently available. A recombinant hMPV lacking the G protein (rhMPV-Delta G) was recently developed as a potential vaccine candidate and shown to be attenuated in the respiratory tract of a rodent model of infection. The mechanism of its attenuation, as well as the role of G protein in modulation of hMPV-induced cellular responses in vitro, as well as in vivo, is currently unknown. In this study, we found that rhMPV-Delta G-infected airway epithelial cells produced higher levels of chemokines and type I interferon (IFN) compared to cells infected with rhMPV-WT. Infection of airway epithelial cells with rhMPV-Delta G enhanced activation of transcription factors belonging to the nuclear factor (NF)-kappaB and interferon regulatory factor (IRF) families, as revealed by increased nuclear translocation and/or phosphorylation of these transcription factors. Compared to rhMPV-WT, rhMPV-Delta G also increased IRF- and NF-kappaB-dependent gene transcription, which was reversely inhibited by G protein expression. Since RNA helicases have been shown to play a fundamental role in initiating viral-induced cellular signaling, we investigated whether retinoic induced gene (RIG)-I was the target of G protein inhibitory activity. We found that indeed G protein associated with RIG-I and inhibited RIG-I-dependent gene transcription, identifying an important mechanism by which hMPV affects innate immune responses. This is the first study investigating the role of hMPV G protein in cellular signaling and identifies G as an important virulence factor, as it inhibits the production of important immune and antiviral mediators by targeting RIG-I, a major intracellular viral RNA sensor.

Figure 1. Characterization of recombinant viruses.

(A) Verification of G protein deletion. Viral RNA extracted from purified viruses was subjected to RT-PCR using paired primers for G or SH genes. PCR products were then analyzed on a 1% agarose gel. Numbers on the left side represent molecular weight marker size expressed in kilobase (Kb). (B) F protein expression analysis in infected cells. A549 cells were infected with rhMPV-WT or rhMPV-ΔG, MOI of 2, and harvested to prepare total cell lysates at the indicated times. Equal amounts of protein were subjected to SDS-PAGE, followed by Western blot using a monoclonal antibody against hMPV F protein. The results are the representative of three independent experiments. Densitometric analysis of band intensity was performed using the histogram function of Adobe Photoshop.

Figure 2. Effect of G protein deletion on type I IFN secretion.

A549 cells were infected with rhMPV-WT or rhMPV-ΔG, at MOI of 2, and harvested at 6, 15 and 24 h p.i. to measure secretion of IFN-α and IFN-β in cell supernatants by ELISA. Data shown are representative of three independent experiments. *, P<0.05 relative to rhMPV-WT.

Figure 3. Effect of G protein deletion on cytokine and chemokine secretion.

A549 cells were infected with rhMPV-WT or rhMPV-ΔG, at MOI of 2, and harvested at 6, 15 and 24 h p.i. to measure secretion of cytokines, CXC chemokines and CC chemokines in cell supernatants by Bio-Plex. Data shown are representative of three independent experiments. *, P<0.05 relative to rhMPV-WT.

Figure 4. hMPV G protein modulates viral-induced IRF-3 activation.

A549 cells were cotransfected with a luciferase reporter plasmid containing either the human IFN-β promoter (A) or multimers of the RANTES ISRE site (B), and the expression plasmid containing hMPV G or F protein or the control vector (CV), and infected with rhMPV-WT or -ΔG, at MOI of 2. Cells were harvested at 15 h p.i. to measure luciferase activity. Uninfected plates served as controls. For each plate luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±standard error of normalized luciferase activity. *, P<0.05, relative to rhMPV-ΔG-infected-CV transfected A549 cells. (C) A549 cells were infected with rhMPV-WT or rhMPV-ΔG, at MOI of 2, for various lengths of time and harvested to prepare nuclear extracts. Equal amounts of protein from uninfected and infected cells were analyzed by Western blot using either an anti-Ser396 phospho-IRF-3 (pIRF-3) or regular anti-IRF-3 antibody. Membranes were stripped and reprobed for lamin b, as control for equal loading of the samples. Densitometric analysis of IRF band intensity, performed using the histogram function of Adobe Photoshop, is shown after normalization to lamin b.

Figure 5. hMPV G protein modulates viral-induced NF-κB activation.

(A) A549 cells were transfected with a luciferase reporter plasmid containing the human IL-8 promoter and infected with rhMPV-WT or -ΔG, at MOI of 2. Cells were harvested at 15 h p.i. to measure luciferase activity. Uninfected plates served as controls. For each plate luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±standard deviation of normalized luciferase activity. *, P<0.05 relative to rhMPV-WT. (B) A549 cells were transfected with a luciferase reporter plasmid containing multimers of the IL-8 NF-κB site together with G or F protein expression plasmid or thes empty vector and infected with rhMPV-WT or -ΔG, at MOI of 2. Cells were harvested at 15 h p.i. to measure luciferase activity. Uninfected plates served as controls. For each plate luciferase was normalized to the β-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±standard deviation of normalized luciferase activity. *, P<0.05 relative to rhMPV-ΔG-infected-CV transfected A549 cells. (C) A549 cells were infected with rhMPV-WT or rhMPV-ΔG, at MOI of 2, for various lengths of time and harvested to prepare nuclear extracts. Equal amounts of protein from uninfected and infected cells were analyzed by Western blot using either an anti-p50 or anti-p65 antibody. Membranes were stripped and reprobed for lamin b, as control for equal loading of the samples. Densitometric analysis of NF-κB band intensity, performed using the histogram function of Adobe Photoshop, is shown after normalization to lamin b.

Figure 6. Inhibition of RIG-I-mediated signaling by G protein.

(A) A549 cells were transfected with a luciferase reporter plasmid containing the human IFN-β promoter, plasmids encoding either RIG-I, MDA-5 or MAVS or their control vectors, and a plasmid expressing hMPV G or the empty vector (as indicated at the bottom of each column). Cells were harvested 30 h post-transfection to measure luciferase activity. For each plate luciferase was normalized to the â-galactosidase reporter activity. Data are representative of two independent experiments and are expressed as mean±SD of normalized luciferase activity. *, P<0.05 relative to pCAGGS+RIG-I group. BG: background; CV: control vector. (B) 293 cells were transfected with plasmids encoding Flag-tagged RIG-I and V5-tagged G or their control vectors. Total cell lysates were immunoprecipitated with anti-V5 antibody followed by Western blot using anti-Flag antibody to detect RIG-I. Reverse immunoprecipitation was also done, where RIG-I was immunoprecipitated using anti-Flag antibody and G protein was then detected using anti-V5 antibody. Membranes were stripped and reprobed to check for proper expression of G and RIG-I. (C) A549 cells were mock infected or infected with rhMPV-WT or -ΔG, MOI of 2, and harvested at 24 h p.i. to prepare total cell lysates. Samples were subjected to immunoprecipitation using anti-RIG-I antibody or control isotype. The immunoprecipitated complexes were then subjected to SDS-PAGE followed by Western blot using anti-hMPV antibody. Membrane was then stripped and reprobed with anti-RIG-I antibody to determine levels of immunoprecipitated RIG-I. Numbers on the left side represent molecular weight marker size expressed in kilodalton. Arrows indicate bands corresponding to hMPV G protein.