Surface expression of the hRSV nucleoprotein impairs immunological synapse formation with T cells

Abstract

Human respiratory syncytial virus (hRSV) is the leading cause of bronchiolitis and pneumonia in young children worldwide. The recurrent hRSV outbreaks and reinfections are the cause of a significant public health burden and associate with an inefficient antiviral immunity, even after disease resolution. Although several mouse- and human cell-based studies have shown that hRSV infection prevents naïve T-cell activation by antigen-presenting cells, the mechanism underlying such inhibition remains unknown. Here, we show that the hRSV nucleoprotein (N) could be at least partially responsible for inhibiting T-cell activation during infection by this virus. Early after infection, the N protein was expressed on the surface of epithelial and dendritic cells, after interacting with trans-Golgi and lysosomal compartments. Further, experiments on supported lipid bilayers loaded with peptide-MHC (pMHC) complexes showed that surface-anchored N protein prevented immunological synapse assembly by naive CD4(+) T cells and, to a lesser extent, by antigen-experienced T-cell blasts. Synapse assembly inhibition was in part due to reduced T-cell receptor (TCR) signaling and pMHC clustering at the T-cell-bilayer interface, suggesting that N protein interferes with pMHC-TCR interactions. Moreover, N protein colocalized with the TCR independently of pMHC, consistent with a possible interaction with TCR complex components. Based on these data, we conclude that hRSV N protein expression at the surface of infected cells inhibits T-cell activation. Our study defines this protein as a major virulence factor that contributes to impairing acquired immunity and enhances susceptibility to reinfection by hRSV.

Keywords: T lymphocyte priming; cSMAC; nucleocapsid protein; pSMAC.

Conflict of interest statement

Conflict of interest statement: P.F.C., R.S.G., S.M.B., and A.M.K. have a pending patent request regarding the anti-N-hRSV 1E9/D1 and 8E4/A7 clones and their applications for diagnosis.

Figures

Fig. 1.

The hRSV N localizes on the surface of infected cells. (A) Flow cytometry analyses of mouse DCs inoculated with mock (noninfectious supernatant), UV-inactivated hRSV or infectious hRSV (MOI = 1) and stained at 24 hpi against the hRSV F (Left), G (Center Left), and N (Center Right) proteins. Mean values ± SEM are shown (n = 3). (Right) Histogram overlays representative for the F, G, and N flow cytometry analyses (blue, mock; gray, UV-hRSV; red, hRSV). (B) Histogram overlays of flow cytometry analyses for surface expression of hRSV F, G, and N proteins as in A. HEp-2 cells inoculated with mock (blue) and infectious hRSV13018-8 (red) (MOI = 1) were analyzed at 24 hpi. (C and D) Laser confocal microphotographs of mock or hRSV13018-8-infected mouse DCs and HEp-2 cells stained at 24 hpi using nonpermeabilizing conditions. In B−D, data are representative of at least three independent experiments. **P < 0.005; ***P < 0.001.

Fig. 2.

The hRSV N reaches the cell surface early during the replication cycle. (A) Contour plots of HEp-2 cells uninfected or inoculated (MOI = 1) either with UV-inactivated hRSVGFP (UV-hRSVGFP), infectious hRSVGFP, anti-F-neutralized hRSVGFP (IC-hRSVGFP) or hRSV incubated with a nonneutralizing, anti-N hRSV antibody (α-N-hRSVGFP). HEp-2 cells were harvested and stained 48 hpi with propidium iodide (for dead-cell exclusion) and an anti-N-hRSV antibody (clone 1E9/D1). (B) Kinetics (1−72 hpi) of surface N protein expression in hRSVGFP-infected HEp-2 cells. (C) (Left) Quantitative real-time RT-PCR for the detection of N transcripts in four different HEp-2 subpopulations, which were purified by FACS under a four-way purity precision schema (>90% purity) as N+/GFPneg, N+/GFPlo, Nlo/GFP+, and Nneg/GFPneg. Uninfected cells and cells inoculated with UV-hRSVGFP were used as controls. (Right) Representative purity controls for the subpopulations evaluated by qRT-PCR. (D) Nucleoprotein ELISA of cell-cleared supernatants obtained at 48 hpi from control and hRSV-infected HEp-2 and DCs. Recombinant N-hRSV was used as a standard for quantification. (E and F) Virus adsorption assays measuring surface N- and F-hRSV expression in HEp-2 cells incubated for 1 h with either untreated hRSV or hRSV pretreated with UV, anti-F, heparin (400 UI/mL), or anti-F/heparin. (G) Representative plots showing double staining for the hRSV F and N proteins in HEp-2 cells inoculated for 1 h with hRSV13018-8. (H) TEM analyses of HEp-2 cells inoculated for 1 h with hRSVGFP and then prepared as described in SI Material and Methods (scale bar: 300 nm; V, virus) (n = 2). In A−F, mean values ± SEM are shown (n = 3). For statistical analyses, one-way ANOVA and Dunnett´s multiple comparison test were used to compare all treatments against uninfected cells (B−D) or to hRSV-untreated, inoculated cells (1 hpi, E and F). *P < 0.05; ***P < 0.001. n.s., nonsignificant.

Fig. 3.

Brefeldin A reduces surface nucleoprotein expression and increases N protein accumulation within the Golgi compartment. (A) Flow cytometry analyses of BFA- and WM-inhibition assays. At 24 hpi, control and hRSV-infected (MOI = 5) HEp-2 cells were incubated for 5 h with either BFA (10 μg/mL) or WM (500 nM), washed twice, and analyzed by flow cytometry for expression of surface hRSV N. One group for each drug (along its vehicle control) was washed and left in culture to determine N expression in the cell surface at 48 hpi (n = 3). (B) HEp-2 cells were coinfected with hRSV (MOI = 5) and a baculovirus encoding a fusion RFP-GALNT2 (particles per cell = 30). At 24 hpi, HEp-2 cells were incubated for 5 h with BFA (10 μg/mL), DMSO (vehicle), or media (untreated) and then fixed with 4% paraformaldehyde (PFA) in PBS, permeabilized with 0.2% Triton X-100 in PBS, and stained using a directly conjugated anti-N 1E9/D1 Alexa Fluor 647 (depicted in green) (n = 3). Open arrowheads (white) indicate N-hRSV–RFP colocalization in untreated and vehicle-treated cells. Thin arrows indicate accumulation of N within the RFP-GALNT2+ compartment in BFA-treated cells. *P < 0.05; ***P < 0.001. n.s., nonsignificant.

Fig. 4.

The hRSV N protein prevents mature IS assembly by naïve CD4+ T cells. (A) Total internal reflection fluorescence (TIRF) microscopy analyses showing the frequencies of mature IS assembled by naïve CD4+ T cell stimulated on SLB containing 5 molec/μm2 of I-Ek-MCC, 200 molec/μm2 of Alexa Fluor 405-labeled ICAM-1, and 10 molec/μm2 of either hRSV N, hRSV M2-1, or CD58. Bilayers with no additional proteins (untreated) and preincubated with vehicle were used as additional controls. (B) Frequencies of mature ISs assembled by naïve T cells stimulated with SLB loaded with increasing densities of N-hRSV (none/vehicle and 2–60 molec/μm2). (C) Representative pictures of T cells stimulated with untreated (Upper) and N-hRSV-loaded (Lower) SLB. White arrows show cSMACs (ICAM-1 exclusion and TCR-central clustering). (D) TFI for TCR were measured as readout for centripetal TCRm accumulation at the T-cell−SLB interface. (E) MFIs were measured for ICAM-1 at interfaces formed by AND naïve CD4+ T cells and untreated or N-hRSV-loaded SLB. Mean values ± SEM are shown in A−E (n = 3). One-way ANOVA and Dunnett´s multiple comparison test were used for statistical analyses. *P < 0.05; **P < 0.005; ***P < 0.001. n.s., nonsignificant.

Fig. 5.

Impairment of IS assembly by hRSV N protein is associated with a reduction in TCR signaling. (A) TIRF microscopy analyses of tyrosine phosphorylation in naïve AND CD4+ T cells stimulated over SLB containing I-Ek, ICAM-1, and 10 molec/μm2 of CD58, hRSV N, or hRSV M2-1. Following fixation, T cells were permeabilized and stained with an anti-pTyr antibody (clone PY20) (n = 3). (B) MFIs for pTyr in naïve AND T cells stimulated with SLB containing increasing nucleoprotein densities (none/vehicle and 2–60 molec/μm2). (C) TFIs for I-Ek loaded with DR-640-labeled MCCp in the interface of T cells stimulated with untreated or N-loaded (10 molec/μm2) SLB. (D) Frequencies of mature ISs assembled by naïve CD4+ T cells stimulated with SLB containing 20 molec/μm2 of an Alexa Fluor 568-labeled anti-CD3ε antibody (clone 2C11), and none or 10 molec/μm2 of either hRSV N- or M2-1. (E) TFIs for anti-CD3ε at contacts of naïve T cells for the experiments shown in D. Mean fluorescent AUs ± SEM are shown from >100 cells (n = 3). *P < 0.05; ***P < 0.001. n.s., nonsignificant.

Fig. 6.

The hRSV N protein accumulates within cell contacts and cosegregates with the TCR. MFI for hRSV N at both contacts and cell-unbound bilayer from naïve (A) and blast (B) AND CD4+ T cells (n = 3). (C) TCR−N colocalization analyses at T-cell−bilayer interfaces formed over SLB loaded with 10 molec/μm2 of hRSV N (green) and either 20 (Upper) or 0 (Lower) molec/μm2 of I-Ek-MCC (magenta). Arrows show colocalizing pixels for nucleoprotein, TCR-β, and I-Ek (white pixels) or colocalization of N and TCR-β (yellow pixels). Image zooms are shown for each ROI highlighted in merge (n = 3).