Immunostimulatory Defective Viral Genomes from Respiratory Syncytial Virus Promote a Strong Innate Antiviral Response during Infection in Mice and Humans

Abstract

Human respiratory syncytial virus (RSV) is a major cause of severe respiratory illness in children and susceptible adults. RSV blocks the development of the innate antiviral immune response and can grow to high titers in the respiratory tract. Here we demonstrate that immunostimulatory defective viral genomes (iDVGs) that are naturally generated during RSV replication are strong inducers of the innate antiviral response to RSV in mice and humans. In mice, RSV iDVGs stimulated the expression of antiviral genes, restricted viral replication, and prevented weight loss and lung inflammation. In human cells, the antiviral response to RSV iDVGs was dominated by the expression of IFN-λ1 over IFN-β and was driven by rapid intranuclear accumulation of the transcription factor IRF1. RSV iDVGs were detected in respiratory secretions of hospitalized patients, and their amount positively correlated with the level of expression of antiviral genes in the samples. Infection of explanted human lung tissue from different donors revealed that most humans can respond to RSV iDVGs and that the rate of accumulation of iDVGs during infection directly correlates with the quality of the antiviral response. Taken together, our data establish iDVGs as primary triggers of robust antiviral responses to RSV and provide the first evidence for an important biological role for naturally occurring iDVGs during a paramyxovirus infection in humans.

Fig 1. RSV DVGs prevent viral pathogenesis in vivo.

(A) Hep2 cells were infected with RSV-LD or HD at a moi of 1.5 TCID₅₀/cell and DVGs were detected by PCR at the indicated times. Details of the PCR assay can be seen in S1 Fig and sequences of the amplicons labeled with a star can be found in S2 Fig Base pair size references are indicated in the gel. (B) Mice weight loss was monitored overtime. Error bars indicate standard deviation of data pooled from two independent experiments (n = 7–9 mice per group total; **p<0.01, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test. Variance was not significantly different between groups as per Bartlett’s test). (C) Representative H&E staining for lung sections from mock, RSV-LD, or RSV-HD-infected mice on day 2 post infection. Picture magnification: 10X; insert is a digital amplification. Red arrows indicate alveolar cellular infiltrate. (D) Pathology score for alveolar infiltration in the lung (n = 6 mice per group; **p<0.01, by two-tailed Mann Whitney test). (E) Differential counts from cytospins from mice bronchoalveolar lavage (BAL) on day 2 post infection (Mono: monocytes and macrophages, PMNs: polymorphonuclear cells; ***p<0.001 by two-way ANOVA with Bonferroni’s post hoc test, n = 5–8 mice per group). (F) Representative cytospin images (20X). (G) Representative flow cytometry plots from whole lung single cell suspensions on day 2 post infection. Plots are pre-gated in singlets, live, CD45⁺CD11b⁺ cells. (H) Quantification of different cell types in the lung of infected mice on day 2 post infection (****p<0.0001 by two-way ANOVA with Bonferroni’s post hoc test, n = 5–8 mice per group, Alv. Macs: alveolar macrophages). (I) Expression of pro-inflammatory genes in whole lung tissue on day 2, 5, and 8 post infection. (n = 3–5 mice per group, *p<0.05, **p<0.01 by one-way ANOVA with Bonferroni’s post hoc test).

Fig 2. RSV iDVGs rapidly induce an antiviral response that controls viral load in mice.

Balb/c mice were infected intranasally with 5 x 10⁶ TCID₅₀/mouse of RSV-LD or HD, or mock infected. Expression of (A) RSV G mRNA and (B) virus titer in whole lung homogenates at the indicated time points. (C) Ifnb1 mRNA and IFNβ protein in the bronchoalveolar lavage (BAL). (D) Expression of Ifnl2 in whole lung homogenates. Error bars indicate mean ± SEM of individual measurements in two pooled independent experiments each with triplicate technical measurements. (n = 7–9 mice per group total; *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Bonferroni’s post hoc test). Gene expression is shown as copy number relative to a house keeping gene expression index determined from the expression of Rps11 and α-tubulin.

Fig 3. RSV iDVGs promote a MAVS-mediated antiviral response during RSV infection.

(A) Expression of RSV G and antiviral genes in HEp2 cells infected for 10 h with RSV-LD at a moi of 1 TCID₅₀/cell alone or in the presence of 20 pg/cell of purified defective particles (pDP) or UV-inactivated pDPs (UV). (B) Expression of RSV G and antiviral genes in wild type, Mavs^-/-, or Ifnar1^-/- MEFs infected with RSV-LD or HD for 10 h at a moi of 1 TCID₅₀/cell. Gene expression is shown as copy number relative to a house keeping gene expression index (hki) determined from the expression of ACTB (β-actin) and GAPDH (A) or Rps11 and α-tubulin (B). Error bars indicate mean ± SEM of three independent experiments each with triplicate technical measurements (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test).

Fig 4. RSV iDVGs stimulate an IRF1/IFNL1-mediated antiviral response.

A549 cells were infected with RSV-LD or RSV-HD at a moi of 1.5 TCID₅₀/cell. Expression of (A) RSV G and antiviral genes mRNA, and (B) protein level of IFNB1 and IFNL1/3 in the cultures supernatants at 24 h post infection (*p<0.05 by one way unpaired t-test). (C, D) Cells were lysed or fixed at 2, 6, 12, and 24 h post infection for western blot (WB) and IFA. (C) For WB, nuclear and cytosolic fractions were immunoblotted for IRF1, GAPDH, and Histone 3. (D) For IFA, cells were co-stained for IRF1 (green, left panel), RSV F + G proteins (red in merged panel), and nuclei (blue). (E) Quantification of nuclear IRF1 upon iDVGs stimulation at designated time points post RSV-HD infection based on IFA images. (F, G) D54 control cells and D54 cells overexpressing IRF1 (D54-IRF1) were infected with RSV-HD at a moi of 1.5 TCID₅₀/cell for 6 h. (F) IRF1 and IRF3 protein detected by WB from whole cell lysates. (G) Expression of RSV G and IFNL1 mRNA. (H, I) A549 cells were mock transfected (WT) or transfected with control siRNA (si-C), or IRF1 siRNA (si-1). After 40 h, the cells were mock infected or infected with RSV-HD at moi of 1.5 TCID₅₀/cell. (H) WB for IRF3 and IRF1 was performed to confirm specific knockdown of IRF1 protein. (I) Expression of RSV G and other antiviral genes at 10 h post infection. Gene expression is shown as copy number relative to a house keeping gene expression index determined from the expression of ACTB (β-actin) and GAPDH. All error bars indicate mean ± SEM of at least three independent experiments (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001 by two-way ANOVA with Bonferroni post hoc test).

Fig 5. iDVGs associate with high expression of antiviral genes in respiratory secretions from patients infected with RSV.

(A) Representative PCR results for gRSV and DVGs in human nasopharyngeal control samples infected with adenovirus (A1-A5) and samples infected with RSV (R1-R4). (B) Gene expression determined by RT-qPCR shown as copy number relative to house keeping genes (*p<0.05, **p<0.01, by two-tailed Mann Whitney test). (C) Samples were scored based on the intensity of the DVG amplicons (1–4, absent to highest intensity) and correlated with the level of expression of antiviral genes. (r = correlation coefficient, p<0.0001 for slope deviation from 0).

Fig 6. Host-intrinsic factors determine the response to iDVGs in the human lung.

(A) Images of precision cut lung slices from human lungs (hPCLS) infected for 24 h with 10⁶ RSV-GFP TCID₅₀/slice. Top: Overlay of bright field and fluorescence channels; Bottom: fluorescence channel alone. (B) Gene expression from hPCLS infected with 10⁷ TCID₅₀/slice of RSV-LD or HD for up to 5 days (n = 7–8, grey numbers indicate individual lung donor). Results show paired data, numbers correspond to different donors. (*p<0.05, **p<0.01 by one-tailed Wilcoxon matched-pairs signed rank test). (C) Ratio of gene expression in RSV-HD and RSV-LD infected tissue from different donors (P3-P9). Ratio>1: HD induced a higher gene expression than LD. (D) PCR for DVGs in hPCLS infected with 10⁶ TCID₅₀/slice of RSV-LD. (E) Gene expression from (D). Error bars indicate mean ± SEM of three slices from the same patient.