STAT2 Limits Host Species Specificity of Human Metapneumovirus

Figure 1. HMPV infection of and STAT2−/− mice. (A) Mice were infected with HMPV TN/94-49 and euthanized at day five of infection. Lung virus titers were determined by plaque assay. (B) HMPV TN/94-49 was serially passaged in STAT2−/− mice, and virus titers were measured from lung homogenate at day five of infection for various passages by plaque assay. *** p < 0.001, **** p < 0.0001, One-way ANOVA with Tukey post-hoc test. For (B), * p < 0.05, ** p < 0.01 compared to STAT2−/− P0, One-way ANOVA with Tukey post-hoc test. Data are representative of two independent experiments in A. Data from B are from one experiment, n = 3–5 mice/group or passage.

Figure 2. Nuclear localization of STAT1 and STAT2 is inhibited by HMPV. Human BEAS2b cells were infected with HMPV or mock for 20 h, and treated with IFN for 40 min. After IFN treatment, cells were fixed, stained for immunofluorescence, and images were captured at 40× magnification. The fluorescent intensities of the nuclear and cytosolic STAT1 or STAT2 fluorescence signals was quantified. (A) HMPV infection inhibits nuclear translocation of STAT1. (B) STAT2 nuclear localization is impaired during HMPV infection. Note reduction of STAT1/2 (red) signal in nuclei of HMPV-infected (green) cells. ** p < 0.01, **** p < 0.0001 compared to Mock + IFN, one-way ANOVA with Tukey post-hoc test. Images are representative of 2 independent experiments. Data in graphs are from two experiments.

Figure 3. HMPV reduces STAT1 and STAT2 activation in primate cells. (A) VeroE6 (primate) cells were infected with HMPV for 24 h, then treated with IFN for 30 min before cells were lysed for Western blotting against total and phosphorylated STAT1 and STAT2. (B,C) Fold change of total STAT1 (B) and STAT2 (C) expression normalized to value of mock-infected cells. (D) Fold change of pSTAT1 and pSTAT2 compared to mock-infected cells. (E) Relative fold change of pSTAT1 and pSTAT2 (fold change of pSTAT1/2 divided by fold change of STAT1/2). * p < 0.05, ** p < 0.01, one sample t test comparison to normalized mock value of 1. Data are representative of (A) or combined from (B–E) four independent experiments.

Figure 4. HMPV infection of murine cells leads to upregulation and phosphorylation of STAT1 and STAT2. (A) CMT64/61 (murine), left, and NIH3T3 (murine), right, cells were infected with HMPV for 24 h, then treated with IFN for 30 min before cells were lysed for Western blotting against total and phosphorylated STAT1 and STAT2. (B,C) Fold change of total STAT1 and STAT2 expression compared to mock-infected wells for CMT64/61 cells. (D) Fold change of pSTAT1 and pSTAT2 compared to mock-infected wells in CMT64/61 cells. (E,F) For NIH3T3 cells, fold change of STAT1 and STAT2 compared to mock-infected wells. (G) Fold change of pSTAT1 and pSTAT2 compared to mock-infected cells in NIH3T3 cells. * p < 0.05, one sample t test comparing samples to normalized mock value of 1. Data are representative of (A) or combined from four (B–D) or two (E–G) independent experiments.

Figure 5. HMPV does not require STAT1 or STAT2 to inhibit expression and phosphorylation of the other. STAT2-deficient U6A (A) and STAT1-deficient U3A (B) cells were infected with HMPV for 20 h at an MOI of 1 and 3, treated with IFN for 40 min, and lysed for Western blotting. (A,B) Expression and phosphorylation of STAT1 and STAT2 in U6A and U3A cells. (C) Fold change of STAT1/pSTAT1 protein levels in U6A cells. (D) Fold change of STAT2/pSTAT2 levels in U3A cells. * p < 0.05, ** p < 0.01, one sample t test to normalized mock value of 1. Data are representative of (A,B) or combined from (C,D) two independent experiments.

Figure 6. Expression of human STAT2 promotes STAT1 and STAT2 inhibition, while murine STAT2 inhibits STAT degradation. STAT2-deficient U6A cells were transfected with hSTAT2 or mSTAT2, then infected with HMPV. Cells were treated with IFN 16 h after infection for 40 min before cell lysis and protein harvesting for Western blotting. (A) STAT1 and STAT2 expression and phosphorylation in U6A cells in the presence of human or murine STAT2. (B–E) Quantification of band intensity as a measure of fold change in U6A cells for total STAT1 (B), pSTAT1 (C), total STAT2 (D), and pSTAT2 (E). * p < 0.05, *** p < 0.001, one sample t test against normalized mock value of 1. Data are representative of (A) or combined from (B–E) two independent experiments.

Figure 7. hSTAT2 KI mice have greater disease severity and inhibition of ISGs compared to WT mice after infection by HMPV, despite no increase in virus titer. hSTAT2 KI mice and C57BL/6 mice were intratracheally inoculated with HMPV at a low (2 × 106 pfu/mL) or high (5 × 106 pfu/mL) titer and weighed daily over the course of infection (A). (B) Lung specimens were taken at day five post-inoculation with HMPV at low titer, stained with H&E, and scored by a pathologist. Histology images were captured at 100×. Histological scoring was calculated by percentage of inflammation per field of view, with scores of 0, 1, 2 (mild/moderate) representing 0, 1–25, and 26–50%, and 3 or 4 (severe) representing 51–75 or 76–100%, respectively. The difference between WT and hSTAT2 KI mice in the frequency of mild/moderate and severe inflammation scores was statistically significant by Fisher’s exact test (p = 0.0024). (C) Virus titer kinetics in lung homogenate for low inoculum infection (left) or day five virus titers in lungs for high inoculum infection (right). (D) qPCR of the ISGs Mx1 and SOCS1 at 24 h post-infection, fold change normalized to mock infection. (E) protein concentration of IFNα in lung…

Figure 8. HMPV-infected hSTAT2 KI mice adopt a Th2-skewed cytokine profile. hSTAT2 KI mice and C57BL/6 mice were intratracheally inoculated with HMPV at 5 × 106 pfu/mL. At day five post-infection, lungs were homogenized and cytokines were measured by multiplex cytokine assay. (A) Measurement of prototypical Th1 cytokines IFNγ and TNFα in lung homogenates of hSTAT2 KI and C57BL/6 mice. (B) Concentration of Th2-related cytokines IL-4, IL-5, IL-13, Eotaxin, and IL-31 in murine lung homogenates. * p < 0.05, ** p < 0.01, unpaired t test. n = 5/group, one experiment.