Mice lacking the ISG15 E1 enzyme UbE1L demonstrate increased susceptibility to both mouse-adapted and non-mouse-adapted influenza B virus infection

Abstract

ISG15 functions as a critical antiviral molecule against influenza virus, with infection inducing both the conjugation of ISG15 to target proteins and production of free ISG15. Here, we report that mice lacking the ISG15 E1 enzyme UbE1L fail to form ISG15 conjugates. Both UbE1L(-/-) and ISG15(-/-) mice display increased susceptibility to influenza B virus infection, including non-mouse-adapted strains. Finally, we demonstrate that ISG15 controls influenza B virus infection through its action within radioresistant stromal cells and not bone marrow-derived cells. Thus, the conjugation of ISG15 to target proteins within stromal cells is critical to its activity against influenza virus.

FIG. 1.

UbE1L^−/− mice fail to produce ISG15 conjugates and are more susceptible to influenza B virus infection than WT mice. Mice were infected i.n. with B/Lee at 1 × 10⁶ PFU and analyzed for ISG15 conjugation, lethality, and viral load. (A) Lung lysates from either mock-treated or B/Lee-infected UbE1L^−/− (U), ISG15^−/− (15), or WT mice were analyzed for ISG15 expression by Western blotting on the indicated days postinfection. Parallel blots were probed with anti-β-actin as a loading control. The asterisk denotes a background band. The blot is representative of three independent experiments. (B) Sera from B/Lee-infected WT (n = 17), UbE1L^−/− (n = 10), and ISG15^−/− (n = 5) mice were analyzed by enzyme-linked immunosorbent assay as previously described (14) to detect released ISG15. The difference between WT and UbE1L^−/− mice was not statistically significant. *, P = 0.0129 (versus WT) and P = 0.0070 (versus UbE1L^−/−). (C) UbE1L^−/−, ISG15^−/−, and WT mice were infected as described above and monitored for lethality. The P value refers to the comparison between WT and UbE1L^−/− mice. P was <0.0001 for the comparison of WT and ISG15^−/− mice. There was no statistically significant difference between ISG15^−/− and UbE1L^−/− mice. (D) Lungs were harvested at 3 and 6 days postinfection from WT, ISG15^−/−, and UbE1L^−/− mice and analyzed for viral titer by standard plaque assay. **, P < 0.0001 for WT versus ISG15^−/− mice; +, P = 0.0149, and ++, P = 0.0011 for WT versus UbE1L^−/− mice. There was no statistical difference between ISG15^−/− and UbE1L^−/− mice. WT and ISG15^−/− survival rates (C) and titers (D) include historical data from reference .

FIG. 2.

ISG15^−/− and UbE1L^−/− mice are susceptible to non-mouse-adapted influenza B virus infection. (A and B) WT, ISG15^−/−, UbE1L^−/−, and IFNAR1^−/− mice were infected i.n. with 3 × 10⁵ PFU influenza B/Yamagata/88 virus and monitored for weight loss (A) and lethality (B). (A) +, less than 30% of mice remained. (B) Differences between WT mice and ISG15^−/−, UbE1L^−/−, and IFNAR1^−/− mice were statistically significant (P < 0.0001). The difference between ISG15^−/− and IFNAR1^−/− mice was significant (P = 0.0219). Differences between UbE1L^−/− and IFNAR1^−/− mice and between UbE1L^−/− and ISG15^−/− mice were not statistically significant. (C and D) WT, ISG15^−/−, UbE1L^−/−, and IFNAR1^−/− mice were infected with influenza B/Yamagata/88 virus (C) or B/Yamagata/73 virus (D) as described above, and their lungs were removed at 3 or 6 days postinfection and assayed for viral titer by standard plaque assay. The numbers of mice per group are in parentheses. +, P < 0.05; *, P < 0.005; **, P ≤ 0.0002 (WT mice versus the other groups).

FIG. 3.

The antiviral activity of ISG15 against influenza B virus is mediated by radioresistant stromal cells. Chimeric mice were generated by injecting B6 (Ly5.1) or ISG15^−/− (Ly5.2) bone marrow into lethally irradiated B6 (Ly5.1) or ISG15^−/− (Ly5.2) recipients. (A) Peripheral blood lymphocytes were obtained 6 weeks after bone marrow transfer and stained for expression of Ly5.1 to assess reconstitution. Representative histograms gating on the lymphocyte population are shown for each group. ISG15^−/− → B6 and B6 → ISG15^−/− mice displayed reconstitution rates between 95 and 98%. (B) Chimeric mice were infected i.n. with influenza virus B/Lee at 1 × 10⁶ PFU and monitored for signs of disease and lethality. On day 6 postinfection, the mice were sacrificed, and their lungs were assayed by plaque assay for viral load. The numbers in parentheses are numbers of mice per group. **, P < 0.05 compared to B6 → B6 mice or P < 0.005 compared to ISG15^−/− → B6 mice. The difference between B6 → B6 and ISG15^−/− → B6 mice was not statistically significant.